No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A chromatin insulator mediates transgene homing and very long-range enhancer-promoter communication
Abstract
Insulator sequences help to organize the genome into discrete functional regions by preventing inappropriate cross-regulation. This is thought to be mediated in part through associations with other insulators located elsewhere in the genome. Enhancers that normally drive Drosophila even skipped (eve) expression are located closer to the TER94 transcription start site than to that of eve. We discovered that the region between these genes has enhancer-blocking activity, and that this insulator region also mediates homing of P-element transgenes to the eve-TER94 genomic neighborhood. Localization of these activities to within 0.6 kb failed to separate them. Importantly, homed transgenic promoters respond to endogenous eve enhancers from great distances, and this long-range communication depends on the homing/insulator region, which we call Homie. We also find that the eve promoter contributes to long-distance communication. However, even the basal hsp70 promoter can communicate with eve enhancers across distances of several megabases, when the communication is mediated by Homie. These studies show that, while Homie blocks enhancer-promoter communication at short range, it facilitates long-range communication between distant genomic regions, possibly by organizing a large chromosomal loop between endogenous and transgenic Homies.
... Construction of eZ (used for Figs. 2, 3, 7A, 7C and Suppl. Fig. 1) and eZ-eG (used for Figs. 4, 5, 6, vectors used for the LR interaction and enhancer blocking assays was described previously (FUJIOKA et al. 2009;FUJIOKA et al. 2016). These vectors carrying modified homie sequences were inserted into the attP site at -142kb relative to the eve locus (FUJIOKA et al. 2009). ...
... Fig. 1) and eZ-eG (used for Figs. 4, 5, 6, vectors used for the LR interaction and enhancer blocking assays was described previously (FUJIOKA et al. 2009;FUJIOKA et al. 2016). These vectors carrying modified homie sequences were inserted into the attP site at -142kb relative to the eve locus (FUJIOKA et al. 2009). Analysis of hebe enhancer activity (Suppl. ...
... The Drosophila even skipped (eve) gene is flanked by two insulators, nhomie (neighbor of homie) and homie (homing insulator at eve), at its 5'-and 3'-ends, respectively. These two elements define the eve TAD (topologically associated domain) (FUJIOKA et al. 2009;FUJIOKA et al. 2016;BING et al. 2024) (see map in Fig. 1A). The properties of the 3' insulator homie have been well-characterized. ...
Chromatin insulators are a major determinant of chromosome architecture. Specific architectures induced by insulators profoundly influence nuclear processes, including how enhancers interact with promoters over long distances and between homologous chromosomes. Insulators can pair with copies of themselves in trans , thereby facilitating homolog pairing. They can also pair with other insulators, sometimes with great specificity, inducing long-range chromosomal loops in cis . Contrary to their canonical function of enhancer blocking, these loops can bring distant enhancers and promoters together to activate gene expression, while at the same time blocking other interactions in cis . The details of these effects depend on the choice of pairing partner, and on the orientation specificity of pairing, implicating the 3-dimensional architecture as a major determinant of function. Here we dissect the homie insulator from the Drosophila even skipped ( eve ) locus, to understand its complex substructure. We test pairing function in cis based on homie -carrying transgenes interacting with endogenous eve . The assay is sensitive to both pairing strength and orientation specificity. Using this assay, we found that a consensus Su(Hw) binding site in homie is required for efficient long-range interaction, although some activity remains without it. This binding site also contributes to the canonical insulator activities of enhancer blocking and barrier function. Based on this and other results from our functional dissection, enhancer blocking and barrier activities appear to be partially separable. Overall, our results show the complexity inherent in insulator functions, which can be provided by an array of proteins with both shared and distinct properties.
... The complex regulatory region of the eve gene ( Figure 3A) is flanked by housekeeping genes, which are expressed in all cells [111,112]. The housekeeping gene TER94 is on one side of the regulatory region of the eve gene and is actively transcribed in all cells. ...
... A 368 bp insulator ( Figure 3A) was found immediately upstream of the core promoter of the TER94 gene [111,112]. The insulator efficiently blocks the activity of embryonic enhancers in model transgenic lines. ...
... The insulator efficiently blocks the activity of embryonic enhancers in model transgenic lines. When the insulator was inserted into the P-transposon, the construct was found to preferentially integrate into the genomic region near the eve locus [111,112]. This effect is called homing and is explained as follows. ...
In higher eukaryotes, the regulation of developmental gene expression is determined by enhancers, which are often located at a large distance from the promoters they regulate. Therefore, the architecture of chromosomes and the mechanisms that determine the functional interaction between enhancers and promoters are of decisive importance in the development of organisms. Mammals and the model animal Drosophila have homologous key architectural proteins and similar mechanisms in the organization of chromosome architecture. This review describes the current progress in understanding the mechanisms of the formation and regulation of long-range interactions between enhancers and promoters at three well-studied key regulatory loci in Drosophila.
... Recent studies have shown that insulators are multi-functional elements, performing several important tasks [1]. They block both enhancer-promoter (E-P) interactions [2][3][4][5][6][7] and the spreading of repressive histone modifications along the chromatin fiber [8][9][10][11][12]. At the same time, they form relatively stable interactions with other insulators, sometimes nearby and sometimes at great distances, thus contributing to chromosome organization [7,[13][14][15][16][17][18][19][20]. ...
... They block both enhancer-promoter (E-P) interactions [2][3][4][5][6][7] and the spreading of repressive histone modifications along the chromatin fiber [8][9][10][11][12]. At the same time, they form relatively stable interactions with other insulators, sometimes nearby and sometimes at great distances, thus contributing to chromosome organization [7,[13][14][15][16][17][18][19][20]. When distant regions are brought together, novel E-P interactions can occur that contribute significantly to patterns of gene expression. ...
... Here, we focus on the two insulators that flank the eve locus, homie and nhomie [7,12,19]. We show that the stripe imbalance seen at the pseudo-locus when an insulator is removed is caused by the capturing of eve enhancer activity by a flanking P-element promoter [60][61][62][63]. ...
Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. Here, we describe a novel mechanism of functional separation whereby an insulator prevents gene repression. When the homie insulator is deleted from the end of a Drosophila even skipped (eve) locus, a flanking P-element promoter is activated in a partial eve pattern, causing expression driven by enhancers in the 3' region to be repressed. The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even when homie is in place. Furthermore, when the flanking promoter is turned around, repression is minimal. Transcriptional read-through that does not produce anti-sense RNA can still repress expression, ruling out RNAi as the mechanism in this case. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses eve promoter-driven expression. We also show that enhancer-promoter specificity and processivity of transcription can have decisive effects on the consequences of insulator removal. First, a core heat shock 70 promoter that is not activated well by eve enhancers did not cause read-through sufficient to repress the eve promoter. Second, these transcripts are less processive than those initiated at the P-promoter, measured by how far they extend through the eve locus, and so are less disruptive. These results highlight the importance of considering transcriptional read-through when assessing the effects of insulators on gene expression.
... Notably, at such distance the chromatin fiber can display fast random movements, which creates an entropic hurdle for specific long-range chromatin interactions and thus a kinetic barrier for the establishment of a productive pre-initiation complex. We therefore included in our reporter cassette the 368 bp insulator element homie ( Supplementary Fig. 1a) 21,22 , which facilitates the formation of a stable loop by self-pairing with the endogenous homie element 23 located at the 3' end of the eve locus 21,22 . In fixed embryos containing our reporter cassette, we observe sporadic expression (~15%) of the reporter gene, solely within the limits of the endogenous eve stripes ( Supplementary Fig. 1b), which suggests that the reporter is specifically activated by the eve enhancers 142 kb away 21 . ...
... Notably, at such distance the chromatin fiber can display fast random movements, which creates an entropic hurdle for specific long-range chromatin interactions and thus a kinetic barrier for the establishment of a productive pre-initiation complex. We therefore included in our reporter cassette the 368 bp insulator element homie ( Supplementary Fig. 1a) 21,22 , which facilitates the formation of a stable loop by self-pairing with the endogenous homie element 23 located at the 3' end of the eve locus 21,22 . In fixed embryos containing our reporter cassette, we observe sporadic expression (~15%) of the reporter gene, solely within the limits of the endogenous eve stripes ( Supplementary Fig. 1b), which suggests that the reporter is specifically activated by the eve enhancers 142 kb away 21 . ...
... We therefore included in our reporter cassette the 368 bp insulator element homie ( Supplementary Fig. 1a) 21,22 , which facilitates the formation of a stable loop by self-pairing with the endogenous homie element 23 located at the 3' end of the eve locus 21,22 . In fixed embryos containing our reporter cassette, we observe sporadic expression (~15%) of the reporter gene, solely within the limits of the endogenous eve stripes ( Supplementary Fig. 1b), which suggests that the reporter is specifically activated by the eve enhancers 142 kb away 21 . ...
A long-standing question in gene regulation is how remote enhancers communicate with their target promoters, and specifically how chromatin topology dynamically relates to gene activation. Here, we combine genome editing and multi-color live imaging to simultaneously visualize physical enhancer-promoter interaction and transcription at the single-cell level in Drosophila embryos. By examining transcriptional activation of a reporter by the endogenous even-skipped enhancers, which are located 150 kb away, we identify three distinct topological conformation states and measure their transition kinetics. We show that sustained proximity of the enhancer to its target is required for activation. Transcription in turn affects the three-dimensional topology as it enhances the temporal stability of the proximal conformation and is associated with further spatial compaction. Furthermore, the facilitated long-range activation results in transcriptional competition at the locus, causing corresponding developmental defects. Our approach offers quantitative insight into the spatial and temporal determinants of long-range gene regulation and their implications for cellular fates.
... In the studies reported here, we have used insulators from the even skipped (eve) locus to address the questions posed above about the architecture of eukaryotic chromosomes. The eve domain spans 16 kb and is bordered upstream by the Nhomie (Neighbor of Homie, this study) insulator and downstream by Homie (Homing insulator at eve) [25,26]. eve encodes a homeodomain transcription factor that is required initially for segmentation, and subsequently in the development of the CNS, muscles, and anal plate [27,28]. ...
... The Homie insulator has two striking properties [26]. First it directs homing of otherwise randomly inserting transgenes to a~5 Mb region centered on the eve locus. ...
... Early stripe and later CNS expression are limited to 200 kb from eve, mesodermal expression has an intermediate distance dependence, while anal plate ring (APR) expression is seen at distances of several Mb. We showed previously that reporter expression at a site within the hebe gene 142 kb upstream of eve requires Homie [26]. Since other fly insulators mediate long-distance regulatory interactions by direct physical contact [22,33], we used high-resolution chromosome conformation capture (H3C) [34] to map contacts between transgenes at -142 kb and eve (see below). ...
The chromosomes of multicellular animals are organized into a series of topologically independent looped domains. This domain organization is critical for the proper utilization and propagation of the genetic information encoded by the chromosome. A special set of architectural elements, called boundaries or insulators, are responsible both for subdividing the chromatin into discrete domains and for determining the topological organization of these domains. Central to the architectural functions of insulators are homologous and heterologous insulator:insulator pairing interactions. The former (pairing between copies of the same insulator) dictates the process of homolog alignment and pairing in trans, while the latter (pairing between different insulators) defines the topology of looped domains in cis. To elucidate the principles governing these architectural functions, we use two insulators, Homie and Nhomie, that flank the Drosophila even skipped locus. We show that homologous insulator interactions in trans, between Homie on one homolog and Homie on the other, or between Nhomie on one homolog and Nhomie on the other, mediate transvection. Critically, these homologous insulator:insulator interactions are orientation-dependent. Consistent with a role in the alignment and pairing of homologs, self-pairing in trans is head-to-head. Head-to-head self-interactions in cis have been reported for other fly insulators, suggesting that this is a general principle of self-pairing. Homie and Nhomie not only pair with themselves, but with each other. Heterologous Homie-Nhomie interactions occur in cis, and we show that they serve to delimit a looped chromosomal domain that contains the even skipped transcription unit and its associated enhancers. The topology of this loop is defined by the heterologous pairing properties of Homie and Nhomie. Instead of being head-to-head, which would generate a circular loop, Homie-Nhomie pairing is head-to-tail. Head-to-tail pairing in cis generates a stem-loop, a configuration much like that observed in classical lampbrush chromosomes. These pairing principles provide a mechanistic underpinning for the observed topologies within and between chromosomes.
... A further indication of functional redundancy comes from genome wide ChIP experiments [21][22][23][24][25][26]. Thus, for the homie insulator downstream of the evenskipped (eve) gene [27] ModEncode lists Su(Hw) dCTCF, GAF and BEAF [25]. Also given the complexity of the cis-elements and trans-acting factors associated with typical insulators, it is not altogether surprising that RNAi knockdowns of insulator proteins in flies often show only minimal effects on insulator activity (e.g., the spread of Polycomb silencing [22,26]). ...
... The first evidence for insulator dependent long distance interactions came from genetic assays with transgenes located at distant sites containing the BX-C Mcp [74,75] or the gypsy su(Hw) insulators [76]. Other insulators, like Fab-7 and homie, are also able to mediate long distance genetic interactions [27,77]. Moreover, recent studies show the genetic interactions in these experiments involve direct physical contact between insulators in the distant transgene inserts [78]. ...
... comm.) recently discovered an even more striking example of insulator dependent enhancer-promoter interactions in the eve locus. As noted above, the homie insulator is downstream of eve between the stripe enhancers 1, 4, 5, and 6 and the neighboring TER94 gene [27]. Deletion experiments show that in addition to ensuring that eve and TER94 function autonomously, homie is also require by these four stripe enhancers to activate eve stripe expression. ...
Insulators play a central role in subdividing the chromosome into a series of discrete topologically independent domains and in ensuring that enhancers and silencers contact their appropriate target genes. In this review we first discuss the general characteristics of insulator elements and their associated protein factors. A growing collection of insulator proteins have been identified including a family of proteins whose expression is developmentally regulated. We next consider several unexpected discoveries that require us to completely rethink how insulators function (and how they can best be assayed). These discoveries also require a reevaluation of how insulators might restrict or orchestrate (by preventing or promoting) interactions between regulatory elements and their target genes. We conclude by connecting these new insights into the mechanisms of insulator action to dynamic changes in the three-dimensional topology of the chromatin fiber and the generation of specific patterns of gene activity during development and differentiation.
... The even skipped (eve) locus is a well-defined Pc domain based on genome-wide analysis [13][14][15][16][17], and is regulated by PcG genes [50][51][52][53][54]. An insulator flanks its well-characterized regulatory region, which includes the eve PRE at its 39 end [51,55]. Thus, this insulator is in a position to separate both positive and negative eve regulatory elements from the constitutively expressed neighboring gene TER94, and/or to prevent ectopic activation of eve by TER94 enhancers. ...
... In addition to enhancer blocking, it causes homing of P-element transgenes to the endogenous eve neighborhood, for which it was nicknamed Homie (Homing insulator at eve). Furthermore, from within a several megabase region flanking endogenous eve, it causes long-range interactions of transgenic promoters with endogenous eve enhancers [55]. Genome-wide analysis showed that most known insulator proteins bind to the Homie region [27,33]. ...
... This transgene extends from 26.4 to +11.3 kb relative to the eve TSS, from the 59-most enhancer of eve to the 3 rd exon of TER94. In addition to all of the eve enhancers, this region contains a characterized PRE [51] located just upstream (on the eve side) of Homie [55]. On the other side of Homie is the TER94 promoter and TSS, which are sufficient for ubiquitous expression, augmented by enhancers in the TER94 introns (data not shown). ...
Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is "replaced" by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.
... Bithorax complex [12]. The most well-studied interaction is between the Neighbor of Homie (Nhomie) insulator and the Homing insulator at eve (Homie), which are located at the boundaries of 16 kb even skipped (eve) regulatory region [13][14][15]. Nhomie and Homie interact with each other and can also support a super-long-distance interaction between the transgene and the endogenous eve locus, which allows endogenous enhancers for the activation of the reporter gene promoter in the transgene [14][15][16]. ...
... The most well-studied interaction is between the Neighbor of Homie (Nhomie) insulator and the Homing insulator at eve (Homie), which are located at the boundaries of 16 kb even skipped (eve) regulatory region [13][14][15]. Nhomie and Homie interact with each other and can also support a super-long-distance interaction between the transgene and the endogenous eve locus, which allows endogenous enhancers for the activation of the reporter gene promoter in the transgene [14][15][16]. The group of architectural proteins responsible for the functions of known insulators and involved in maintaining specific long-distance interactions has been well-studied in Drosophila [17]. ...
Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems. Drosophila became the first model organism in which architectural proteins that determine the activity of insulators were described. In Drosophila, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and attP-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in cis and trans only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer–promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the D. melanogaster model.
... Later in 387 development, both reporters are expressed in a repeating pattern along the dorsal midline ( Fig. 388 6E). This expression is driven by the hebe gene enhancers located just beyond the gfp-reporter 389 (Fujioka et al., 2009). 390 ...
... 813The -142 kb attP landing site was described previously(Fujioka et al., 2009). The -142 814 kb site contains two attP target sites for phiC31 recombinase-mediated cassette exchange 815 (RMCE) (Bateman et al., 2006) and mini-white as a marker. ...
Two different models have been proposed to explain how the endpoints of chromatin looped domains ("TADs") in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop (and an unanchored loop). In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the "loop-extrusion" and the "boundary-pairing" models. Our findings are incompatible with the loop-extrusion model and instead suggest that endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head, or head-to-tail, with varying degrees of specificity. How the partners find each other is not clear but is unlikely to require a loop extrusion mechanism.
... These elements were first found in Drosophila melanogaster (14)(15)(16) but later identified in several developmental genes of flies and vertebrates (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). On the basis of transgenic experiments in Drosophila, it was proposed that chromatin insulators bias chromatin folding by interacting with each other (28,29). ...
... For additional statistics and the list of elements, see Fig. 3 and table S3. This catalog includes Fub, 62D, 1A2, SF1, and Homie insulator elements identified by genetic assays (19,22,39,44,55,(59)(60)(61). Approximately one-third of the insulators from our catalog (from 28.99% of the elements defined at 15% FDR to 30.67% defined at 5% FDR) reside within 2 kb from their nearest TAD border. ...
Drosophila insulators were the first DNA elements found to regulate gene expression by delimiting chromatin contacts. We still do not know how many of them exist and what impact they have on the Drosophila genome folding. Contrary to vertebrates, there is no evidence that fly insulators block cohesin-mediated chromatin loop extrusion. Therefore, their mechanism of action remains uncertain. To bridge these gaps, we mapped chromatin contacts in Drosophila cells lacking the key insulator proteins CTCF and Cp190. With this approach, we found hundreds of insulator elements. Their study indicates that Drosophila insulators play a minor role in the overall genome folding but affect chromatin contacts locally at many loci. Our observations argue that Cp190 promotes cobinding of other insulator proteins and that the model, where Drosophila insulators block chromatin contacts by forming loops, needs revision. Our insulator catalog provides an important resource to study mechanisms of genome folding.
... These elements were first discovered in Drosophila melanogaster (14)(15)(16) but later identified in several developmental genes of flies and vertebrates (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Based on transgenic experiments in Drosophila, it was proposed that chromatin insulators bias chromatin folding by interacting with each other (28,29). ...
... For additional statistics and the list of elements, see Figure 3 and Table S3. This catalogue includes Fub, 62D, 1A2, SF1 and Homie insulator elements identified by genetic assays (19,22,39,44,55,(59)(60)(61). Approximately one third of the insulators from our catalogue (from 28.99% of the elements defined at 15% FDR to 30.67%defined at 5% FDR) reside within 2kb from their nearest TAD border. ...
Drosophila insulators were the first DNA elements discovered to regulate gene expression by delimiting chromatin contacts. Remarkably, it is still unclear how many of them exist in the Drosophila genome and whether they have a pervasive impact on the genome folding. Contrary to vertebrates, there is no evidence that fly insulators block cohesin-mediated chromatin loop extrusion. Therefore, their mechanism of action remains an open question. To bridge these gaps, we mapped genomic contacts, transcriptomes and binding landscapes of insulator associated proteins in Drosophila cells deficient for CTCF and Cp190. With this approach, we discovered hundreds of chromatin insulator elements. Their study indicates that Drosophila insulators play a minor role in shaping the overall chromosome folding patterns but impact chromatin contacts locally at many individual loci. Our observations argue that Cp190 promotes co-binding of other insulator proteins and that the model, where Drosophila insulators block chromatin contacts by forming loops, needs revision. The extended catalogue of insulator elements presented here provides a significant new resource to study mechanisms that shape the folding of eukaryotic genomes.
... Two insulators, Neighbor of Homie (Nhomie) and Homing insulator at eve (Homie), were identified at the 16-kb boundaries of the regulatory region of the even-skipped (eve) gene [134][135][136]. Nhomie and Homie interact with each other and can also maintain superlong-distance interactions between the transgene and the endogenous eve locus, which allows endogenous enhancers to activate the reporter gene promoter in the transgene [135,136]. ...
... Two insulators, Neighbor of Homie (Nhomie) and Homing insulator at eve (Homie), were identified at the 16-kb boundaries of the regulatory region of the even-skipped (eve) gene [134][135][136]. Nhomie and Homie interact with each other and can also maintain superlong-distance interactions between the transgene and the endogenous eve locus, which allows endogenous enhancers to activate the reporter gene promoter in the transgene [135,136]. ...
In higher eukaryotes, enhancers determine the activation of developmental gene transcription in specific cell types and stages of embryogenesis. Enhancers transform the signals produced by various transcription factors within a given cell, activating the transcription of the targeted genes. Often, developmental genes can be associated with dozens of enhancers, some of which are located at large distances from the promoters that they regulate. Currently, the mechanisms underlying specific distance interactions between enhancers and promoters remain poorly understood. This review briefly describes the properties of enhancers and discusses the mechanisms of distance interactions and potential proteins involved in this process.
... A well-characterized pair-rule gene even-skipped (eve) is also located within a clear TAD structure ( Figure 2B). Previous genetic studies showed that the eve TAD is shaped by pairing of well-characterized boundary elements, Nhomie and Homie (Fujioka et al., 2009(Fujioka et al., , 2013, both of which contain high level of CTCF and Rad21 binding ( Figure 2C). This interaction is thought to be strictly orientation dependent because inversion of Nhomie or Homie disrupts long-range interaction in cis and trans (Fujioka et al., 2009(Fujioka et al., , 2016. ...
... Previous genetic studies showed that the eve TAD is shaped by pairing of well-characterized boundary elements, Nhomie and Homie (Fujioka et al., 2009(Fujioka et al., , 2013, both of which contain high level of CTCF and Rad21 binding ( Figure 2C). This interaction is thought to be strictly orientation dependent because inversion of Nhomie or Homie disrupts long-range interaction in cis and trans (Fujioka et al., 2009(Fujioka et al., , 2016. We have also examined orientation dependence of Homie elements by analyzing trans-homolog enhancer-promoter interaction or transvection (Lim et al., 2018b). ...
Formation of self-associating loop domains is a fundamental organizational feature of metazoan genomes. Here, we employed quantitative live-imaging methods to visualize impacts of higher-order chromosome topology on enhancer-promoter communication in developing Drosophila embryos. Evidence is provided that distal enhancers effectively produce transcriptional bursting from target promoters over distances when they are flanked with boundary elements. Importantly, neither inversion nor deletion of a boundary element abrogates this “enhancer-assisting activity,” suggesting that they can facilitate intra-domain enhancer-promoter interaction and production of transcriptional bursting independently of topologically associating domain (TAD) formation. In contrast, domain-skipping activity of distal enhancers was lost after disruption of topological domains. This observation raises a possibility that intra-domain and inter-domain enhancer-promoter interactions are differentially regulated by chromosome topology.
... While it appears that enhancers are intrinsically capable of coactivating multiple promoters when tested (Fukaya et al., 2016), specificity of regulatory interactions seems to be tightly regulated by the local genome configuration to prevent promiscuous transcriptional activation. For example, in the Drosophila even-skipped (eve) locus, stripe enhancers and eve transcription unit are all embedded in a single TAD that is bordered by two insulators, Homie and Nhomie (Figure 4a, left; Fujioka, Sun, & Jaynes, 2013;Fujioka, Wu, & Jaynes, 2009;Cubenas-Potts et al., 2017). When a synthetic enhancer-less lacZ reporter was placed in a remote location outside of the eve TAD, the stripe enhancers do not activate lacZ (Fujioka et al., 2009). ...
... For example, in the Drosophila even-skipped (eve) locus, stripe enhancers and eve transcription unit are all embedded in a single TAD that is bordered by two insulators, Homie and Nhomie (Figure 4a, left; Fujioka, Sun, & Jaynes, 2013;Fujioka, Wu, & Jaynes, 2009;Cubenas-Potts et al., 2017). When a synthetic enhancer-less lacZ reporter was placed in a remote location outside of the eve TAD, the stripe enhancers do not activate lacZ (Fujioka et al., 2009). However, when re-organization of genome structure was induced by using the pairing of endogenous Homie and synthetic Homie, the stripe enhancers start to co-activate both the endogenous eve and synthetic lacZ simultaneously (Figure 4a, right; Chen et al., 2018). ...
Transcriptional enhancers are regulatory DNAs that instruct when and where genes should be transcribed in response to a variety of intrinsic and external signals. They contain a cluster of binding sites for sequence‐specific transcription factors and co‐activators to determine the spatiotemporal specificity of gene activities during development. Enhancers are often positioned in distal locations from their target promoters. In some cases, they work over a million base pairs or more. In the traditional view, enhancers have been thought to stably interact with promoters in a targeted manner. However, quantitative imaging studies provide a far more dynamic picture of enhancer action. Moreover, recent Hi‐C methods suggest that regulatory interactions are dynamically regulated by the higher‐order chromosome topology. In this review, we summarize the emerging findings in the field and propose that assembly of “transcription hubs” in the context of 3D genome structure plays an important role in transcriptional regulation. Enhancer DNAs play a central role in the spatiotemporal control of gene activities during development. In this review, we summarize the emerging findings in the field and propose that assembly of “transcription hubs” in the context of 3D genome topology is an important feature of enhancer function.
... Heterologous head-to-tail pairing generates stem-loops Among the most thoroughly studied heterologous partners are the eve boundaries homie and nhomie [65,74,75], which flank the $17 kb eve locus. nhomie is located $7 kb upstream of eve, while homie is $10 kb downstream (Fig. 4A). ...
... In flies, the choice of pairing partners depends on two factors, boundary matching and proximity. For example, homie and nhomie can interact with each other over distances of several Mb, "skipping over" many intervening boundaries [65,75]. Here, pairing preference plays the key role in partner choice. ...
Chromosomes in multicellular animals are subdivided into a series of looped domains. In addition to being the underlying principle for organizing the chromatin fiber, looping is critical for processes ranging from gene regulation to recombination and repair. The subdivision of chromosomes into looped domains depends upon a special class of architectural elements called boundaries or insulators. These elements are distributed throughout the genome and are ubiquitous building blocks of chromosomes. In this review, we focus on features of boundaries that are critical in determining the topology of the looped domains and their genetic properties. We highlight the properties of fly boundaries that are likely to have an important bearing on the organization of looped domains in vertebrates, and discuss the functional consequences of the observed similarities and differences.
... It has also been found that two identical insulators can support interactions between regulatory elements located in transgenes inserted at distances of up to several megabases from each other (30)(31)(32)(33)(34). The most striking example is the insulator named Homie that is located between the TER94 promoter and regulatory region of the eve gene (35). The presence of Homie in a transgene as far as 3.3 Mb away from the endogenous copy facilitates long-range communication between endogenous eve enhancers located near Homie and a promoter placed on the transgene (35,36). ...
... The most striking example is the insulator named Homie that is located between the TER94 promoter and regulatory region of the eve gene (35). The presence of Homie in a transgene as far as 3.3 Mb away from the endogenous copy facilitates long-range communication between endogenous eve enhancers located near Homie and a promoter placed on the transgene (35,36). These facts suggest that proteins bound to insulators can support very specific distant interactions through the cell cycle. ...
According to recent models, as yet poorly studied architectural proteins appear to be required for local regulation of enhancer–promoter
interactions, as well as for global chromosome organization. Transcription factors ZIPIC, Pita and Zw5 belong to the class
of chromatin insulator proteins and preferentially bind to promoters near the TSS and extensively colocalize with cohesin
and condensin complexes. ZIPIC, Pita and Zw5 are structurally similar in containing the N-terminal zinc finger-associated
domain (ZAD) and different numbers of C2H2-type zinc fingers at the C-terminus. Here we have shown that the ZAD domains of
ZIPIC, Pita and Zw5 form homodimers. In Drosophila transgenic lines, these proteins are able to support long-distance interaction between GAL4 activator and the reporter gene
promoter. However, no functional interaction between binding sites for different proteins has been revealed, suggesting that
such interactions are highly specific. ZIPIC facilitates long-distance stimulation of the reporter gene by GAL4 activator
in yeast model system. Many of the genomic binding sites of ZIPIC, Pita and Zw5 are located at the boundaries of topologically
associated domains (TADs). Thus, ZAD-containing zinc-finger proteins can be attributed to the class of architectural proteins.
... Interactions have been identified between sequences on the same chromosome (in cis) or on different chromosomes (in trans) (LIEBERMAN-AIDEN et al. 2009;DUAN et al. 2010;SEXTON et al. 2012; VAN DE WERKEN et al. 2012;NAGANO et al. 2013;ZHANG et al. 2013). Many long-distance interactions in cis underlie the activation of specific genes, and in some cases, sequences have been identified that facilitate interactions between a distal enhancer and a specific promoter target (ZHOU and LEVINE 1999;CALHOUN et al. 2002;CALHOUN and LEVINE 2003;LIN 2003;AKBARI et al. 2008;FUJIOKA et al. 2009;MAJUMDER et al. 2015). In contrast, the genetic impacts of trans-interactions between chromosomes are less clearly understood. ...
... An intriguing possibility is that the larger ct enhancer fragment could possess sequences that aid in targeting distal promoters, including promoters in trans, that are not present in the smaller fragment. Indeed, the ct enhancer is normally 80 kb away from its promoter target, and sequences that promote long-range enhancer-promoter interactions in cis and in trans have been characterized for other genes (HOPMANN et al. 1995;ZHOU and LEVINE 1999;CALHOUN et al. 2002;CALHOUN and LEVINE 2003;LIN 2003;AKBARI et al. 2008;FUJIOKA et al. 2009;MAJUMDER et al. 2015). The notion that variation in transvection could be partially dependent on factors beyond enhancer strength is supported by previous studies. ...
The interphase nucleus is organized such that genomic segments interact in cis, on the same chromosome, and in trans, between different chromosomes. In Drosophila and other Dipterans, extensive interactions are observed between homologous chromosomes, which can permit enhancers and promoters to communicate in trans. Enhancer action in trans has been observed for a handful of genes in Drosophila, but it is as yet unclear whether this is a general property of all enhancers or specific to a few. Here, we test a collection of well-characterized enhancers for the capacity to act in trans. Specifically, we tested 18 enhancers that are active in either the eye or wing disc of third instar Drosophila larvae, and, using two different assays, found evidence that each enhancer can act in trans. However, the degree to which trans-action was supported varied greatly between enhancers. Quantitative analysis of enhancer activity supports a model wherein an enhancer's strength of transcriptional activation is a major determinant of its ability to act in trans, but that additional factors may also contribute to an enhancer's trans-activity. In sum, our data suggest that a capacity to activate a promoter on a paired chromosome is common among Drosophila enhancers.
... It has also been found that two identical insulators can support interactions between regulatory elements located in transgenes inserted at distances up to several megabases from each other (Sigrist and Pirrotta, 1997;Muller et al., 1999;Kravchenko et al., 2005;Vazquez et al., 2006;Li et al., 2011. The most striking example is the insulator termed Homie that is located between the TER94 promoter and regulatory region of the eve gene (Fujioka et al., 2009). The presence of Homie in a transgene as far as 3.3 Mb away from the endogenous copy facilitates long-range communication between endogenous eve enhancers located near Homie and a promoter placed on the transgene (Fujioka et al., 2009(Fujioka et al., , 2013. ...
... The most striking example is the insulator termed Homie that is located between the TER94 promoter and regulatory region of the eve gene (Fujioka et al., 2009). The presence of Homie in a transgene as far as 3.3 Mb away from the endogenous copy facilitates long-range communication between endogenous eve enhancers located near Homie and a promoter placed on the transgene (Fujioka et al., 2009(Fujioka et al., , 2013. These facts suggest that proteins bound to insulators can support very specific distant interactions through the cell cycle. ...
Due to advances in genome-wide technologies, consistent distant interactions within chromosomes of higher eukaryotes have been revealed. In particular, it has been shown that enhancers can specifically and directly interact with promoters by looping out intervening sequences, which can be up to several hundred kilobases long. This review is focused on transcription factors that are supposed to be involved in long-range interactions. Available data are in agreement with the model that several known transcription factors and insulator proteins belong to an abundant but poorly studied class of proteins that are responsible for chromosomal architecture.
... To explore the contribution of individual Yan-bound regions to mesodermal eve expression we recombineered specific deletions into a functional genomic bacterial artificial chromosome (BAC) construct encompassing the entire 16.4-kb eve locus carrying an in-frame YFP tag ( Fig. 1A; Ludwig et al. 2011). As enhancer-blocking activity has previously been identified between the MHE and Ter94 gene (Fujioka et al. 2009), we reasoned that the D4 Yanbound element was more likely to contribute to Ter94 than to eve regulation and excluded it from our analysis. Transgenes were generated, and altered function was assessed by examining expression in the Eve-positive cardiogenic precursors. ...
... (A) Yan ChIP-chip patterns at eve. Yan binds to the proximal promoter (D1), a region that partially overlaps a cis-regulatory module responsible for neuronal and anal plate expression (D2), the previously characterized MHE (Halfon et al. 2000) (D3), and a region upstream of the TER94 locus (D4) that overlaps a previously characterized insulator and homing element (Fujioka et al. 2009). Red boxes depict engineered deletions. ...
Long-range integration of transcriptional inputs is critical for gene expression, yet the mechanisms remain poorly understood. We investigated the molecular determinants that confer fidelity to expression of the heart identity gene even-skipped (eve). Targeted deletion of regions bound by the repressor Yan defined two novel enhancers that contribute repressive inputs to stabilize tissue-specific output from a third enhancer. Deletion of any individual enhancer reduced Yan occupancy at the other elements, impacting eve expression, cell fate specification, and cardiac function. These long-range interactions may be stabilized by three-dimensional chromatin contacts that we detected between the elements. Our work provides a new paradigm for chromatin-level integration of general repressive inputs with specific patterning information to achieve robust gene expression.
... However, a class of DNA elements, dubbed chromatin insulators, seems to have evolved for their ability to constrain chromatin contacts across. These elements were discovered in Drosophila (65)(66)(67) and subsequently identified in several developmental genes of flies and vertebrates (68)(69)(70)(71)(72)(73)(74). At first, chromatin insulators were operationally defined as DNA elements that block the activation of a promoter by a transcriptional enhancer element when placed between the two. ...
Even when split into several chromosomes, DNA molecules that make up our genome are too long to fit into the cell nuclei unless massively folded. Such folding must accommodate the need for timely access to selected parts of the genome by transcription factors, RNA polymerases, and DNA replication machinery. Here, we review our current understanding of the genome folding inside the interphase nuclei. We consider the resulting genome architecture at three scales with a particular focus on the intermediate (meso) scale and summarize the insights gained from recent experimental observations and diverse computational models.
... Since then, this phenomenon, termed transvection, which involves pairing-dependent interallelic complementation, has been observed at multiple individual loci (Pirrotta, 1999;Wu and Morris, 1999;Duncan, 2002;Kennison and Southworth, 2002;McKee, 2004;Apte and Meller, 2012;Kassis, 2012;Blick et al., 2016;Joyce et al., 2016;Fukaya and Levine, 2017;Lim et al., 2018;Tian et al., 2019;Galouzis and Prud'homme, 2021). Homolog pairing can drive or silence gene expression through various regulatory elements including Polycomb response elements (PREs), insulators, enhancers, and promoters (Kassis et al., 1991;Fauvarque and Dura, 1993;Kassis, 1994;Gindhart and Kaufman, 1995;Kapoun and Kaufman, 1995;Geyer, 1997;Sigrist and Pirrotta, 1997;Fujioka et al., 1999;Muller et al., 1999;Zhou et al., 1999;Shimell et al., 2000;Duncan, 2002;Kennison and Southworth, 2002;Bantignies et al., 2003;Kravchenko et al., 2005;Vazquez et al., 2006;Fujioka et al., 2009;Li et al., 2011;Kassis, 2012;Blick et al., 2016;Fujioka et al., 2016;Joyce et al., 2016;Fukaya and Levine, 2017;Lim et al., 2018;Piwko et al., 2019;Galouzis and Prud'homme, 2021). Firstly, several specific factors were suggested to regulate pairing (Fritsch et al., 2006;Williams et al., 2007;Hartl et al., 2008), then comprehensive global screens were conducted to identify more factors implicated in somatic pairing (Bateman and Wu, 2008;Bateman et al., 2012b;Joyce et al., 2012). ...
Genome organization includes contacts both within a single chromosome and between distinct chromosomes. Thus, regulatory organization in the nucleus may include interplay of these two types of chromosomal interactions with genome activity. Emerging advances in omics and single-cell imaging technologies have allowed new insights into chromosomal contacts, including those of homologs and sister chromatids, and their significance to genome function. In this review, we highlight recent studies in this field and discuss their impact on understanding the principles of chromosome organization and associated functional implications in diverse cellular processes. Specifically, we describe the contributions of intra-chromosomal, inter-homolog, and inter-sister chromatid contacts to genome organization and gene expression.
... Two insulators, Nhomie (Neighbor of Homie) and Homie (Homing insulator at eve), were identified at the 16-kb boundaries of the regulatory region of the even-skipped (eve) gene (Fujioka et al. 1999;. Nhomie and Homie interact with each other and can also maintain super-long-distance interactions between the transgene and the endogenous eve locus, which allows endogenous enhancers to activate the reporter gene promoter in the transgene [122,123]. ...
In higher eukaryotes, enhancers determine the activation of developmental gene transcription in specific cell types and stages of embryogenesis. Enhancers transform the signals produced by various transcription factors within a given cell, activating the transcription of the targeted genes. Often, developmental genes can be associated with dozens of enhancers, some of which are located at large distances from the promoters that they regulate. Currently, the mechanisms that underly the specific distance interactions between enhancers and promoters remain unknown. This review describes the properties and activities of enhancers and discusses the mechanisms of distance interactions and potential proteins involved in this process.
... Many models of insulator function invoke physical contact between insulators to form "looped" chromatin 340 domains (Fujioka, Wu, & Jaynes, 2009;Kravchenko et al., 2005;Kyrchanova & Georgiev, 2014;Yang & Corces, 2012), and a substantial literature exists demonstrating that many insulator proteins are able to interact with each other and to self-associate (Blanton, Gaszner, & Schedl, 2003;Büchner et al., 2000;Gause, Morcillo, & Dorsett, 2001;Ghosh, Gerasimova, & Corces, 2001;Golovnin et al., 2007;Mohan et al., 2007;Pai et al., 2004;Vogelmann et al., 2014). In general, we do not observe looping interactions between domain boundaries in our Hi-C data. ...
Evidence has emerged in recent years linking insulators and the proteins that bind them to the higher order structure of animal chromatin, but the precise nature of this relationship and the manner by which insulators influence chromatin structure have remained elusive. Here we present high-resolution genome-wide chromatin conformation capture (Hi-C) data from early Drosophila melanogaster embryos that allow us to map three-dimensional interactions to 500 base pairs. We observe a complex, nested pattern of regions of chromatin self-association, and use a combination of computational and manual annotation to identify boundaries between these topological associated domains (TADs). We demonstrate that, when mapped at high resolution, boundaries resemble classical insulators: short (500 - 1000 bp) genomic regions that are sensitive to DNase digestion and strongly bound by known insulator proteins. Strikingly, we show that for regions where the banding pattern of polytene chromosomes has been mapped to genomic position at comparably high resolution, there is a perfect correspondence between polytene banding and our chromatin conformation maps, with boundary insulators forming the interband regions that separate compacted bands that correspond to TADs. We propose that this precise, high-resolution relationship between insulators and TADs on the one hand and polytene bands and interbands on the other extends across the genome, and suggest a model in which the decompaction of insulator regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.
... In this experimental system, a fluorescently labeled reporter gene, which additionally allows the instantaneous measurement of transcriptional activity, was integrated 142 kb upstream of the fluorescently marked endogenous eve enhancer. An insulator element homie [63,64] was placed proximal to the reporter, self-pairing with the endogenous homie downstream of the enhancer, and thereby facilitating enhancer-reporter contact. Only at alleles that display high physical proximity between transgene and enhancer, a fluorescent spot of nascent transcripts was observed. ...
The three‐dimensional organization of our genome is an important determinant for the transcriptional output of a gene in (patho)physiological contexts. The spatial organization of linear chromosomes within nucleus is dominantly inferred using two distinct approaches, chromosome conformation capture (3C) and DNA fluorescent in situ hybridization (DNA‐FISH). While 3C and its derivatives score genomic interaction frequencies based on proximity ligation events, DNA‐FISH methods measure physical distances between genomic loci. Despite these approaches probe different characteristics of chromosomal topologies, they provide a coherent picture of how chromosomes are organized in higher‐order structures encompassing chromosome territories, compartments and topologically associating domains. Yet, at the finer topological level of promoter‐enhancer communication, the imaging‐centered and the 3C methods give more divergent and sometimes seemingly paradoxical results. Here, we compare and contrast observations made applying visual DNA‐FISH and molecular 3C approaches. We emphasize that the 3C approach, due to its inherently competitive ligation step, measures only ‘relative’ proximities. A 3C interaction enriched between loci, therefore does not necessarily translates into a decrease in absolute spatial distance. Hence, we advocate caution when modelling chromosome conformations.
... In this work, we focus on the functions of ELBA and Insv in active chromatin regions because of their enrichment in close proximity to active promoters. However, we have detected enrichment of ELBA and Insv in several known elements that could mediate long-range interactions, such as the homienhomie 44 and scs and scs' loci 6,45 . Future studies will be needed to determine the roles of ELBA and Insv in chromatin organization. ...
The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units. The BEN-solo proteins—including Insensitive (Insv), Elba1 and Elba2—function in both transcriptional repression and chromatin insulation. Here, the authors investigate the role of these proteins in Drosophila embryos, finding that ELBA and Insv function as general insulators and partition active chromatin to ensure proper gene activation in Drosophila.
... 5,40 and reviewed by ref. 41 ), the observation that some insulator proteins and insulator elements promote transvection (refs. 16,19,42,43 , and reviewed by ref. 44 ), and the enrichment of architectural proteins at genomic sites involved in allelic interactions 27 . Indeed, despite a few discrepancies among different published ChIP-seq datasets, many insulator proteins were enriched at PnM boundaries, with strong correlations between the ChIP-seq peaks and PS for some (e.g., Nup98, with the highest correlation coefficient) and a weak anti-correlation for others (e.g., Su(Hw), with the weakest correlation coefficient) (Supplementary Table 3). ...
Trans-homolog interactions have been studied extensively in Drosophila, where homologs are paired in somatic cells and transvection is prevalent. Nevertheless, the detailed structure of pairing and its functional impact have not been thoroughly investigated. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, showing that homologs pair with varying precision genome-wide, in addition to establishing trans-homolog domains and compartments. We also elucidate the structure of pairing with unprecedented detail, observing significant variation across the genome and revealing at least two forms of pairing: tight pairing, spanning contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional implication genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing, including the disruption of some interaction peaks.
... On the one hand, there are clear examples of genomic loci that can both act as insulators and support long-distance regulation in Hox genes (see section 4.2.1.) and at the even-skipped (eve) locus [52]. At eve, two insulators have been characterized that are capable of highly specific pairing with themselves or with each other when they are placed in specific orientations on the same chromosome or on homologous chromosomes [53]. ...
Development is orchestrated by regulatory elements that turn genes ON or OFF in precise spatial and temporal patterns. Many safety mechanisms prevent inappropriate action of a regulatory element on the wrong gene promoter. In flies and mammals, dedicated DNA elements (insulators) recruit protein factors (insulator binding proteins, or IBPs) to shield promoters from regulatory elements. In mammals, a single IBP called CCCTC-binding factor (CTCF) is known, whereas genetic and biochemical analyses in Drosophila have identified a larger repertoire of IBPs. How insulators function at the molecular level is not fully understood, but it is currently thought that they fold chromosomes into conformations that affect regulatory element-promoter communication. Here, we review the discovery of insulators and describe their properties. We discuss recent genetic studies in flies and mice to address the question: Is gene insulation important for animal development? Comparing and contrasting observations in these two species reveal that they have different requirements for insulation, but that insulation is a conserved and critical gene regulation strategy.
... In this study, we focused more on the functions of Elba and Insv in active chromatin regions because of their enrichment in close proximity to active promoters. However, we detected enrichment of Elba and Insv in several known elements that could mediate long-range interactions, such as the homie-nhomie (Fujioka et al. 2009) and scs and scs' loci (Kellum and Schedl 1991;Blanton et al. 2003). Future studies will be needed to determine the roles of Elba and Insv in chromatin organization. ...
The Drosophila genome encodes four closely related proteins including Insensitive (Insv), Elba1, Elba2 and Elba3 that possess activities in both transcriptional repression and chromatin insulation. The first three proteins are BEN-solo factors, all with a DNA binding BEN domain. The fourth protein Elba3 bridges Elba1 and Elba2 to form a hetero-trimeric complex Elba. Here we report comprehensive investigation on the in vivo functions of these proteins in Drosophila embryos. Insv and Elba bind to many common and distinct genomic loci. Unexpectedly the adapter protein Elba3 can associate with chromatin and repress gene expression independent of Elba1 and Elba2. Our ChIP-nexus analyses show Insv binding to DNA in a symmetric configuration and Elba binding asymmetrical in vivo . In finding that the motifs of known insulator proteins are enriched in Elba and Insv ChIP peaks, we confirmed that Elba collaborates with other insulator proteins to regulate developmental patterning in embryos. To differentiate the insulator function of Elba and Insv from their repressor activity, we determined real-time transcription change in mutant embryos using precise nuclear run-on sequencing (PRO- seq) assay. elba mutation dampens expression difference between the two Elba-bound neighbor genes. Finally, transgenic reporters confirm insulation activity of Elba- and Insv-bound sites. Together, these findings define Elba and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators in the partition of transcription units.
... Haplotype-resolved Hi-C has been used to investigate cis interactions within mammalian genomes (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and diploid homolog pairing in yeast (36) and, in our companion paper (Erceg, AlHaj Abed, Golobordko et al. bioRxiv (37)), we developed a methodology for applying this approach to study homolog pairing in hybrid Drosophila embryos. In that study, we demonstrated pairing to be genome-wide and provided a framework in which to consider pairing 15 in terms of precision, proximity, and continuity. ...
Trans -homolog interactions encompass potent regulatory functions, which have been studied extensively in Drosophila, where homologs are paired in somatic cells and pairing-dependent gene regulation, or transvection, is well-documented. Nevertheless, the structure of pairing and whether its functional impact is genome-wide have eluded analysis. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, discovering that homologs pair relatively precisely genome-wide in addition to establishing trans -homolog domains and compartments. We also elucidated the structure of pairing with unprecedented detail, documenting significant variation across the genome. In particular, we characterized two forms: tight pairing, consisting of contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional role genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing.
One Sentence Summary
Haplotype-resolved Hi-C reveals structures of homolog pairing and global implications for gene activity in hybrid PnM cells.
... We hypothesize that CTCF promotes DNA repair by recruiting Rad51, and that together with Rad51, CTCF stabilizes the DSB ends and/or homologous DNA sequences with the local chromosome architectural network, which could prevent the ends of DSBs from drifting apart and could facilitate the pairing of homologous sequences necessary for repair. Such an activity is supported by a "homing" phenomenon of P elements in Drosophila whereby transposable elements carrying genomic sequences containing CTCF-binding sites tend to integrate back to the very genomic regions from which these sequences derive (39). ...
Significance
CTCF interacts with the genome through thousands of sites and organizes the genome into topological domains, but whether CTCF has direct functions in the maintenance of genome stability is not known. Here, we report that CTCF depletion increases chromosomal instability and activates the DNA damage response. We show that CTCF is recruited to sites of DNA lesions in a process that depends on DNA damage signaling and the DNA-binding domain of CTCF, and that CTCF participates in homologous recombination repair of DNA double-strand breaks by interacting with Rad51 and promoting Rad51 repair foci formation. Thus, CTCF maintains genome stability by participating in DNA repair, highlighting a potential link between genome organization and genome stability.
... The assembled protein factors are proposed to interact with similar complexes in the genome and to be physically recruited to a particular locus, near which the integration then takes place. It should be noted that, at least in some cases, homing might depend on the presence of insulators in the transgenes [101]. ...
Combinatorial expression of the genes in multicellular organisms leads to the development of different cell types. The important epigenetic regulators of higher eukaryotes are the Polycomb group (PcG) and Trithorax group (TrxG) proteins. These factors control the transcription of a large number of genes involved in various cellular processes. Dysregulation of PcG and TrxG systems leads to developmental abnormalities and cancer. This review focuses on the main characteristics and properties of the Drosophila PRE elements. Furthermore, we summarize the information on the protein components of the PcG and TrxG groups and their functional activities and discuss the main aspects of competition between the proteins of these classes as well as their possible mechanisms of action.
... In the late embryonic stage, polycomb proteins shut down eve gene expression completely (13). In Kc as well as in S2 cells, the eve gene is embedded in an H3K27me3 domain and insulated from the surrounding highly expressed genes by Homie/NHomie insulators ( Figure 6B) (13,(45)(46)(47). For specific targeting we used the CRISPR/Cas9 system with a catalytically inactive protein dCas9 (48). ...
Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila in-sulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of hete-rochromatin expansion. Here, we searched for enzymes , which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding. Depletion of CP190 reduces Gcn5 binding to chromatin. Binding dependency was further supported by Gcn5 mediated co-precipitation of CP190. Gcn5 is known to activate transcription by histone acetylation. We used the dCas9 system to target CP190 or Gcn5 to a Poly-comb repressed and H3K27me3 marked gene locus. Both, CP190 as well as Gcn5, activate this locus, thus supporting the model that CP190 recruits Gcn5 and thereby activates chromatin.
... In the late embryonic stage, polycomb proteins shut down eve gene expression completely (13). In Kc as well as in S2 cells, the eve gene is embedded in an H3K27me3 domain and insulated from the surrounding highly expressed genes by Homie/NHomie insulators ( Figure 6B) (13,(45)(46)(47). For specific targeting we used the CRISPR/Cas9 system with a catalytically inactive protein dCas9 (48). ...
Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila in-sulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of hete-rochromatin expansion. Here, we searched for enzymes , which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding. Depletion of CP190 reduces Gcn5 binding to chromatin. Binding dependency was further supported by Gcn5 mediated co-precipitation of CP190. Gcn5 is known to activate transcription by histone acetylation. We used the dCas9 system to target CP190 or Gcn5 to a Poly-comb repressed and H3K27me3 marked gene locus. Both, CP190 as well as Gcn5, activate this locus, thus supporting the model that CP190 recruits Gcn5 and thereby activates chromatin.
... The architectural functions depend upon physical interactions between insulators. The first indication that boundary elements interact with each other came from the discovery that insulators can facilitate regulatory interactions between transgenes inserted at distant sites [11][12][13][14][15][16]. Subsequent work confirmed that the long distance regulatory interactions involved direct physical contacts between boundaries [17,18]. ...
Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.
... These long distance regulatory interactions are dependent on the formation of stable and direct insulator:insulator physical connections. Long distance interactions have also been found for several non-BX-C boundaries, including the homie insulator from the even-skipped locus and the gypsy transposon su(Hw) insulator ( Fujioka et al., 2013( Fujioka et al., , 2009 ). ...
The parasegment-specific expression of the three Drosophila Bithorax complex homeotic genes is orchestrated by nine functionally autonomous regulatory domains. Functional autonomy depends upon special elements called boundaries or insulators that are located between each domain. The boundaries ensure the independent activity of each domain by blocking adventitious interactions with initiators, enhancers and silencers in the neighboring domains. However, this blocking activity poses a regulatory paradox-- the Bithorax boundaries are also able to insulate promoters from regulatory interactions with enhancers and silencers and six of the nine Bithorax regulatory domains are separated from their target genes by at least one boundary element. Here we consider several mechanisms that have been suggested for how the Bithorax regulatory domains are able to bypass intervening boundary elements and direct the appropriate parasegment-specific temporal and spatial expression of their target gene. HIGHLIGHT.
Copyright © 2015. Published by Elsevier Ireland Ltd.
... While the mechanisms behind homing remain elusive, it is worthwhile mentioning that the homing pigeon fragment spans the Fub boundary that separates the bxd/pbx regulatory domain from the iab-2 domain (see above; Bender and Lucas 2013). The idea of boundaries mediating homing is further substantiated by a more recent case of homing discovered at the eve locus by Fujioka and Jaynes (Fujioka et al. 2009). In this case, the homing fragment spans the homie boundary that insulates the eve locus from the next adjacent gene TER94 (Fujioka et al. 2013). ...
After nearly 30 years of effort, Ed Lewis published his 1978 landmark paper in which he described the analysis of a series of mutations that affect the identity of the segments that form along the anterior-posterior (AP) axis of the fly (Lewis 1978). The mutations behaved in a non-canonical fashion in complementation tests, forming what Ed Lewis called a "pseudo-allelic" series. Because of this, he never thought that the mutations represented segment-specific genes. As all of these mutations were grouped to a particular area of the Drosophila third chromosome, the locus became known of as the bithorax complex (BX-C). One of the key findings of Lewis' article was that it revealed for the first time, to a wide scientific audience, that there was a remarkable correlation between the order of the segment-specific mutations along the chromosome and the order of the segments they affected along the AP axis. In Ed Lewis' eyes, the mutants he discovered affected "segment-specific functions" that were sequentially activated along the chromosome as one moves from anterior to posterior along the body axis (the colinearity concept now cited in elementary biology textbooks). The nature of the "segment-specific functions" started to become clear when the BX-C was cloned through the pioneering chromosomal walk initiated in the mid 1980s by the Hogness and Bender laboratories (Bender et al. 1983a; Karch et al. 1985). Through this molecular biology effort, and along with genetic characterizations performed by Gines Morata's group in Madrid (Sanchez-Herrero et al. 1985) and Robert Whittle's in Sussex (Tiong et al. 1985), it soon became clear that the whole BX-C encoded only three protein-coding genes (Ubx, abd-A, and Abd-B). Later, immunostaining against the Ubx protein hinted that the segment-specific functions could, in fact, be cis-regulatory elements regulating the expression of the three protein-coding genes. In 1987, Peifer, Karch, and Bender proposed a comprehensive model of the functioning of the BX-C, in which the "segment-specific functions" appear as segment-specific enhancers regulating, Ubx, abd-A, or Abd-B (Peifer et al. 1987). Key to their model was that the segmental address of these enhancers was not an inherent ability of the enhancers themselves, but was determined by the chromosomal location in which they lay. In their view, the sequential activation of the segment-specific functions resulted from the sequential opening of chromatin domains along the chromosome as one moves from anterior to posterior. This model soon became known of as the open for business model. While the open for business model is quite easy to visualize at a conceptual level, molecular evidence to validate this model has been missing for almost 30 years. The recent publication describing the outstanding, joint effort from the Bender and Kingston laboratories now provides the missing proof to support this model (Bowman et al. 2014). The purpose of this article is to review the open for business model and take the reader through the genetic arguments that led to its elaboration.
... In contrast with many retrotransposons that interact with nucleosomes, the DNA transposon Hermes inserts preferentially in budding yeast into nucleosome-free regions of the genome (Gangadharan et al., 2010). The widely used P element DNA transposons in Drosophila show targeting (called "P element homing") by incorporating binding sites for various regulatory factors, including chromatin insulators (Bender and Hudson, 2000;Fujioka et al., 2009) and Polycomb group response elements (Kassis, 2002;Cheng et al., 2012). ...
Mobile DNA in the genome is subject to RNA-targeted epigenetic control. This control regulates the activity of transposons, retrotransposons and genomic proviruses. Many different life history experiences alter the activities of mobile DNA and the expression of genetic loci regulated by nearby insertions. The same experiences induce alterations in epigenetic formatting and lead to trans-generational modifications of genome expression and stability. These observations lead to the hypothesis that epigenetic formatting directed by non-coding RNA provides a molecular interface between life history events and genome alteration.
... Working in Drosophila allows us to build GRN models from known quantitative data on gene expression. In addition, with respect to the current project, the evolutionary significance of transposons has been directly studied in the Drosophila genome ( [43,44]), and comparative work within insects provides evidence for gene co-option and network complexification [45,46]. For instance, the transition from grasshoppers to flies appears to have occurred with a doubling of number of genes in the segmentation network (at least) over a short geological time span. ...
The co-evolution of species with their genomic parasites (transposons) is thought to be one of the primary ways of rewiring gene regulatory networks (GRNs). We develop a framework for conducting evolutionary computations (EC) using the transposon mechanism. We find that the selective pressure of transposons can speed evolutionary searches for solutions and lead to outgrowth of GRNs (through co-option of new genes to acquire insensitivity to the attacking transposons). We test the approach by finding GRNs which can solve a fundamental problem in developmental biology: how GRNs in early embryo development can robustly read maternal signaling gradients, despite continued attacks on the genome by transposons. We observed co-evolutionary oscillations in the abundance of particular GRNs and their transposons, reminiscent of predator-prey or host-parasite dynamics. In addition to modeling genomic evolution, we feel these oscillations may offer a new technique in EC for overcoming premature convergence.
... The availability of expression data helps to identify candidate target genes within the vicinity of a TF binding event by providing additional functional cues. Nevertheless, given that TF-target genes interactions could occur over long distances [22,89,90], it is still possible that seemingly isolated Zic3 binding events with no responsive genes within a set distance criteria might actually be regulating a target located further away. Until a more detailed understanding of the architecture of genome-wide interactions have been achieved, this possibility could not be ruled out. ...
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches - ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.
... As mentioned previously, many of these border sites can be further classified as "aligned" sites, which also feature Su(Hw) and BEAF-32 binding [5]. One such endogenous aligned site between the eve and TER94 loci, termed Homie, was previously identified as a sequence that promotes transgene homing [93], a phenomenon in which a transgene displays preferential insertion in the vicinity (~300 kb) of a particular locus [94,95]. Homing is dependent on a copy of Homie, but not the nearby PRE, being present in the transgene. ...
The control of complex, developmentally regulated loci and partitioning of the genome into active and silent domains is in part accomplished through the activity of DNA-protein complexes termed chromatin insulators. Together, the multiple, well-studied classes of insulators in Drosophila melanogaster appear to be generally functionally conserved. In this review, we discuss recent genomic-scale experiments and attempt to reconcile these newer findings in the context of previously defined insulator characteristics based on classical genetic analyses and transgenic approaches. Finally, we discuss the emerging understanding of mechanisms of chromatin insulator regulation.
... To verify, we used ChIP to biochemically measure chromatin removal during osmostress. Using S2 cells, we tested chromatin enrichment during stress at three types of Su(Hw) insulators: the gypsy insulator (Su(Hw), CP190, and Mod(mdg4)67.2), the homie super insulator (all known insulator proteins; Fujioka et al., 2009), and an endogenous intragenic insulator (3L:12247800) that binds only to Su(Hw). All stressed samples show an 50-80% decrease in the amount of chromatin-bound Su(Hw) compared with media-only controls, depending on the insulator. ...
Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol-p38 mitogen-activated protein kinase-independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization.
The prevailing view of metazoan gene regulation is that transcription is facilitated through the formation of static activator complexes at distal regulatory regions. Here, we employed quantitative single-cell live-imaging and computational analysis to provide evidence that the dynamic assembly and disassembly process of transcription factor (TF) clusters at enhancers is a major source of transcriptional bursting in developing Drosophila embryos. We further show that the regulatory connectivity between TF clustering and burst induction is highly regulated through intrinsically disordered regions (IDRs). Addition of a poly-glutamine tract to the maternal morphogen Bicoid demonstrated that extended IDR length leads to ectopic TF clustering and burst induction from its endogenous target genes, resulting in defects in body segmentation during embryogenesis. Moreover, we successfully visualized the presence of "shared" TF clusters during the co-activation of two distant genes, which provides a concrete molecular explanation for the newly proposed "topological operon" hypothesis in metazoan gene regulation.
A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified, and they remain poorly understood. Here, we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that we identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. Our results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and 104-105 in humans.
Homologous chromosomes colocalize to regulate gene expression in processes including genomic imprinting, X-inactivation, and transvection. In Drosophila, homologous chromosomes pair throughout development, promoting transvection. The "button" model of pairing proposes that specific regions along chromosomes pair with high affinity. Here, we identify buttons interspersed across the fly genome that pair with their homologous sequences, even when relocated to multiple positions in the genome. A majority of transgenes that span a full topologically associating domain (TAD) function as buttons, but not all buttons contain TADs. Additionally, buttons are enriched for insulator protein clusters. Fragments of buttons do not pair, suggesting that combinations of elements within a button are required for pairing. Pairing is necessary but not sufficient for transvection. Additionally, pairing and transvection are stronger in some cell types than in others, suggesting that pairing strength regulates transvection efficiency between cell types. Thus, buttons pair homologous chromosomes to facilitate cell-type-specific interchromosomal gene regulation.
A new study uses multicolor live imaging to simultaneously visualize enhancer–promoter interaction and transcription in Drosophila embryos.
How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. Here, we exploit the properties of transvection to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. We successfully visualized the activation of an MS2-tagged reporter gene by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability—but not the frequency—of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, we found that a shared enhancer co-activates separate PP7 and MS2 reporter genes in cis and in trans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. We propose that transvecting alleles form a trans-homolog “hub,” which serves as a scaffold for the accumulation of transcription complexes. Lim et al. explore a process called transvection in living Drosophila embryos, whereby enhancers on one homolog activate transcription units on the other homolog. They show that insulators facilitate transvection by stabilizing allele-allele pairing. Surprisingly, a shared enhancer coactivates a cis-linked PP7 reporter gene along with a trans-linked MS2 reporter contained on the other homolog. This coactivation is consistent with emerging evidence for transcription “hubs” containing clusters of RNA polymerase II and associated activators.
ELife digest
The DNA inside a cell is packaged into threaded structures called chromosomes. Early studies of chromosomes using insect larvae revealed a pattern of dark- and light-colored bands on the chromosomes that was unique to every region. For decades, it remained unclear if the bands had a specific role.
More advanced techniques have shown that chromosomes are organized into a series of compact domains that contain separate regions of genes. Each region can be turned on or off at different times, depending on the needs of different cells. This allows cells to specialize into different cell types and tissues. Until now, it was unclear how these different regions are formed and controlled, and how they relate to the chromosome bands.
Here, Stadler, Haines and Eisen used a specific technique to map the structure of chromosomes in early fly embryos, by using a chemical trap to capture closely located DNA pieces. The results showed that the chromosome domains corresponded to the banding patterns seen in the early studies. This suggest that light bands represent extended DNA that act as spacers between the dark gene regions.
This study adds to the view that the way the DNA is organized influences gene activity. Creating a high-resolution model of the chromosomes will help us to better understand how their structure can influence the activity of genes. In future, scientists may be able to identify diseases that are caused by errors in the chromosome organization.
To date, there has been an increasing number of drugs produced in mammalian
cell cultures. In order to enhance the expression level and stability of target
recombinant proteins in cell cultures, various regulatory elements with poorly
studied mechanisms of action are used. In this review, we summarize and discuss
the potential mechanisms of action of such regulatory elements.
Chromatin boundaries are architectural elements that determine the 3-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity The Fab-7 boundary from the Drosophila Bithorax complex (BX-C) is required for the parasegment specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific and we describe a novel ∼700kD complex, the LBC, that binds to Fab-7 sequences that have insulator function in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late but not early embryos and that it contains three insulator proteins, GAF, Mod(mdg4) and E(y)2. Its DNA bindings properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display little sequence similarities. Finally we show that mutations, which abrogate LBC binding in vitro inactivate the Fab-7 boundary in BX-C.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Of the numerous classes of elements involved in modulating eukaryotic chromosome structure and function, chromatin insulators arguably remain the most poorly understood in their contribution to these processes in vivo. Indeed, our view of chromatin insulators has evolved dramatically since their chromatin boundary and enhancer blocking properties were elucidated roughly a quarter of a century ago as a result of recent genome-wide, high-throughput methods better suited to probing the role of these elements in their native genomic contexts. The overall theme that has emerged from these studies is that chromatin insulators function as general facilitators of higher-order chromatin loop structures that exert both physical and functional constraints on the genome. In this review, we summarize the result of recent work that supports this idea as well as a number of other studies linking these elements to a diverse array of nuclear processes, suggesting that chromatin insulators exert master control over genome organization and behavior.
A very large cis-regulatory region of −300 kb is responsible for the complex patterns of expression of the three homeotic
genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has
revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. We report the analysis of two new Mcp mutant deletions (Mcp27 and Mcp8116) that allow us to localize sequences essential for boundary function to a − 0 .4 kb DNA segment. These essential sequences
closely coincide to a −0.3 kb nuclease hypersensitive region in chromatin. We also show that sequences contributing to the
Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure.
FlyBase (http://flybase.org) is a database of Drosophila genetic and genomic information. Gene Ontology (GO) terms are used to describe three attributes of wild-type gene products:
their molecular function, the biological processes in which they play a role, and their subcellular location. This article
describes recent changes to the FlyBase GO annotation strategy that are improving the quality of the GO annotation data. Many
of these changes stem from our participation in the GO Reference Genome Annotation Project—a multi-database collaboration
producing comprehensive GO annotation sets for 12 diverse species.
FLYSNPdb provides high-resolution single nucleotide polymorphism (SNP) data of Drosophila melanogaster. The database currently contains 27 367 polymorphisms, including >3700 indels (insertions/deletions), covering all major chromsomes. These SNPs are clustered into 2238 markers, which are evenly
distributed with an average density of one marker every 50.3 kb or 6.6 genes. SNPs were identified automatically, filtered
for high quality and partly manually curated. The database provides detailed information on the SNP data including molecular
and cytological locations (genome Releases 3–5), alleles of up to five commonly used laboratory stocks, flanking sequences,
SNP marker amplification primers, quality scores and genotyping assays. Data specific for a certain region, particular stocks
or a certain genome assembly version are easily retrievable through the interface of a publicly accessible website (http://flysnp.imp.ac.at/flysnpdb.php).
Some developmentally important genes can be regulated via two enhancers, one located nearby and the other, a "shadow" enhancer,
10 to 20 kilobases away.
CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation
of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in
the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific
methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation,
and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they
interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which
CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to
recruit and bind polycomb repressive complexes.
Vectors derived from the Drosophila P element transposon are widely used to make transgenic Drosophila. Insertion of most P-element-derived vectors is nonrandom, but they exhibit a broad specificity of target sites. During experiments to identify cis-acting regulatory elements of the Drosophila segmentation gene engrailed, we identified a fragment of engrailed DNA that, when included within a P-element vector, strikingly alters the specificity of target sites. P-element vectors that contain this fragment of engrailed regulatory DNA insert at a high frequency near genes expressed in stripes.
The SCL (tal-1, TCL5) gene is a member of the basic domain, helix-loop-helix (bHLH) class of putative transcription factors. We found that (i) the SCL promoter for exon Ia contains a potential recognition site for GATA-binding transcription factors, (ii) SCL mRNA is expressed in all erythroid tissues and cell lines examined, and (iii) SCL mRNA increases upon induced differentiation of murine erythroleukemia (MEL) cells, and inferred that SCL may play a physiologic role in erythroid differentiation. We used gel shift and transfection assays to demonstrate that the GATA motif in the SCL promoter binds GATA-1 (and GATA-2), and also mediates transcriptional transactivation. To identify a role for SCL in erythroid differentiation, we generated stable transfectants of MEL and K562 (a human chronic myelogenous leukemia cell line that can differentiate along the erythroid pathway) cells overexpressing wild-type, antisense or mutant SCL cDNA. Increasing the level of SCL expression in two independent MEL lines (F4-6 and C19, a 745 derivative) and K562 cells increased the rate of spontaneous (i.e. in the absence of inducer) erythroid differentiation. Conversely, induced differentiation was inhibited in MEL transfectants expressing either antisense SCL cDNA or a mutant SCL lacking the basic domain. Our experiments suggest that the SCL gene can be a target for the erythroid transcription factor GATA-1 and that the SCL gene product serves as a positive regulator of erythroid differentiation.
The bithorax complex specifies the identity of parasegments 5-14 of Drosophila. Although nine parasegment-specific functions, abx/bx, bxd/pbx and iab-2 to iab-8,9 have been identified, the whole bithorax complex appears to encode only three classes of proteins, Ubx, abd-A and Abd-B. Many observations suggest that the parasegment-specific functions act as positive cis-regulatory elements of Ubx, abd-A and Abd-B. We report the molecular genetics of a new gain-of-function mutation, Fab-7, which transforms parasegment 11 into parasegment 12. Induction of Abd-B mutations in cis (one of which removes the Abd-B homeobox) causes reversion of the dominant phenotype, demonstrating that Fab-7 misregulates Abd-B. A 4 kb deletion, 30 kb downstream from the Abd-B transcription unit, is solely responsible for the Fab-7 phenotype. We consider that the parasegment-specific functions lie in DNA domains that are sequentially and independently 'opened' along the chromosome. Once a domain is opened, the cis-regulatory sequences within it can carry out their function. We propose that the Fab-7 deletion removes a boundary separating the iab-6 and iab-7 cis-regulatory regions (the functions specific for parasegments 11 and 12) allowing the open configuration of iab-6 to invade iab-7 in parasegment 11. This is strongly supported by our finding that Fab-7 can be caused to revert by lesions not only in iab-7 but also in iab-6.
The pair-rule gene even-skipped (eve) plays a key role in the regulatory hierarchy governing segmentation in Drosophila. Here we describe the use of P-transformation and eve promoter fusions to identify cis elements that regulate the periodic seven-stripe eve pattern. A distal region of the eve promoter, located between -5.9 and -5.2 kb, controls autoregulation. Sequences from this region will induce striped expression of a heterologous hsp70 basal promoter in the presence, but not absence, of endogenous eve+ products. Autoregulatory activity was localized to a 200-bp region of the distal eve promoter. We also provide evidence that individual eve expression stripes are regulated by separate cis sequences. eve promoter sequences located between -4.7 and -3 kb upstream of the transcription start site are important for the initiation of stripe 3, whereas sequences between -1.7 and -0.4 kb are needed for stripes 2 and 7. It is possible that these latter regions are directly regulated by the products of gap genes.
Mutations at the suppressor of Hairy-wing [su(Hw), 3-54.8] locus reverse the phenotype of second-site mutations induced by the gypsy transposable element in Drosophila melanogaster. This gene encodes a protein with a predicted molecular weight of 109,000 that contains an acidic domain and 12 copies of the DNA-binding 'Zn finger' motif. The su(Hw) protein was overexpressed in Escherichia coli and Drosophila cells, and partially purified. It was shown to interact specifically in vitro with a 367-bp DNA fragment that contains 12 copies of the sequence PyPuTTGCATACCPy located in the 5'-untranslated region of gypsy, between the 5' long terminal repeat (LTR) and the first ATG initiation codon. This sequence shows striking homology to some mammalian transcriptional enhancer elements, supporting a role for the su(Hw) protein in the control of gypsy transcription. In addition, the su(Hw) protein is present at approximately 100-200 sites on Drosophila polytene chromosomes, suggesting that it also interacts in vivo with DNA and might be involved functionally in the regulation of normal cellular genes.
Hypomorphic alleles of the locus polyhomeotic (ph) produce multiple, homeotic-like transformations in adult flies that mimic dominant mutations in the Antennapedia and Bithorax complexes. Analysis of null alleles of ph has revealed a complex, embryonically lethal phenotype that includes cell death of the ventral epidermis and abnormalities in the patterns of expression of homeotic and segmentation genes. There is also a dramatic alteration in the pattern of axon pathways in the central nervous system, such that the wild-type array of segmentally repeated commissures and connectives is replaced by bundles of axons confined to the hemiganglia of origin. It is possible that this axonal phenotype is the result of loss of neuronal identity caused by abnormal homeotic and segmentation gene expression.
The homeo box gene even-skipped (eve) plays a key role in the regulation of the Drosophila segmentation pattern. eve- embryos lack segment borders and show altered activities of several segmentation genes, including fushi tarazu (ftz), engrailed (en), and wingless (wg). Here, we present evidence that eve influences its own expression in a tissue-specific manner. Each of four different eve mutations disrupts the normal eve expression pattern, and null mutations cause a premature loss of eve products in ectodermal, but not mesodermal, tissues. Molecular characterization of eve mutations indicates that disruptions of the eve pattern are not due to alterations in the eve promoter but, instead, involve abnormal eve proteins. Two different eve mutations cause single amino acid substitutions within the homeo box, and we discuss the implications of these changes with regard to homeo box gene function. We also present evidence that eve+ gene activity is not only required for the activation of the odd-numbered en stripes but also for the correct positioning of each ftz stripe. We present a model for the loss of en expression in eve- embryos, based on the concentration-dependent regulation of the ftz pattern by eve+ products.
The infra-abdominal (iab) elements in the bithorax complex of Drosophila melanogaster regulate the transcription of the homeotic genes abdominal-A (abd-A) and Abdominal-B (Abd-B) in cis. Here we describe two unusual aspects of regulation by the iab elements, revealed by an analysis of an unexpected complementation between mutations in the Abd-B transcription unit and these regulatory regions. First, we find that iab-6 and iab-7 can regulate Abd-B in trans. This iab trans regulation is insensitive to chromosomal rearrangements that disrupt transvection effects at the nearby Ubx locus. In addition, we show that a transposed Abd-B transcription unit and promoter on the Y chromosome can be activated by iab elements located on the third chromosome. These results suggest that the iab regions can regulate their target promoter located at a distant site in the genome in a manner that is much less dependent on homologue pairing than other transvection effects. The iab regulatory regions may have a very strong affinity for the target promoter, allowing them to interact with each other despite the inhibitory effects of chromosomal rearrangements. Second, by generating abd-A mutations on rearrangement chromosomes that break in the iab-7 region, we show that these breaks induce the iab elements to switch their target promoter from Abd-B to abd-A. These two unusual aspects of iab regulation are related by the iab-7 breakpoint chromosomes that prevent iab elements from acting on Abd-B and allow them to act on abd-A. We propose that the iab-7 breaks prevent both iab trans regulation and target specificity by disrupting a mechanism that targets the iab regions to the Abd-B promoter.
The Abdominal-B (Abd-B) gene of the bithorax complex (BX-C) of Drosophila controls the identities of the fifth through seventh abdominal segments and segments in the genitalia (more precisely, parasegments 10-14). Here we focus on iab-5, iab-6 and iab-7, regulatory regions of Abd-B that control expression in the fifth, sixth and seventh abdominal segments (parasegments 10-12). By analysis of partial BX-C deficiencies, we show that these regions are able to promote fifth and sixth abdominal segment identities in the absence of an Abd-B gene in cis. We establish that this ability does not result from cis-regulation of the adjacent abd-A or Ubx genes of the BX-C but rather occurs because the iab-5,6,7 region is able to interact with Abd-B in trans. We demonstrate that this interaction is proximity dependent and is, therefore, a case of what E. B. Lewis has called transvection. Interactions of this type are presumably facilitated by the synapsis of homologues that occurs in somatic cells of Dipterans. Although transvection has been detected in a number of Drosophila genes, transvection of the iab-5,6,7 region is exceptional in two ways. First, interaction in trans with Abd-B does not require that homologues share homologous sequences within, or for some distance to either side of, the BX-C. This is the first case of transvection shown to be independent of local synapsis. A second unusual feature of iab-5,6,7 transvection is that it is remarkably difficult to disrupt by heterozygosity for chromosome rearrangements. The lack of requirement for local synapsis and the tenacity of trans-interaction argue that the iab-5,6,7 region can locate and interact with Abd-B over considerable distance. This is consistent with the normal role of iab-5,6,7, which must act over some 20-60 kb to influence its regulatory target in cis at the Abd-B promoter. Evidence is presented that trans-action of iab-5,6,7 requires, and may be mediated by, the region between distal iab-7 and Abd-B. Also, we show that iab-5,6,7 transvection is independent of the allelic state of zeste, a gene that influences several other cases of transvection. The long-range nature of interactions in trans between iab-5,6,7 and Abd-B suggests that similar interactions could operate effectively in organisms lacking extensive somatic pairing. Transvection may, therefore, be of more general significance than previously suspected.
Previous studies on the regulation of the segmentation gene even-skipped (eve) have centered on the transcription of stripe 2. Here, we characterize another enhancer module contained within the complex eve promoter that directs expression of stripes 3 and 7. This enhancer is approximately 500 bp in length and maps approximately 3.3 kb upstream of the transcription start site. The stripe 3 + 7 enhancer appears to be regulated by one or more ubiquitously distributed activators, including components of a JAK-Stat pathway. The two-stripe pattern results via multiple tiers of repressors which delimit this ubiquitous activation. The zinc finger repressor hunchback appears to be responsible for establishing the anterior border of stripe 3 and the posterior border of stripe 7. knirps, a member of the nuclear receptor family of transcription factors, appears to establish the posterior border of stripe 3 and the anterior border of stripe 7. Activator and repressor proteins bind in vitro to several sites within the enhancer. These findings suggest a general model for the regulation of segmentation stripes, whereby enhancers integrate positional information provided by broadly distributed activators and spatially restricted repressors.
Boundary elements are thought to define the peripheries of chromatin domains and to restrict enhancer-promoter interactions
to their target genes within their domains. We previously characterized a cDNA encoding the BEAF-32A protein (32A), which
binds with high affinity to the scs' boundary element from the Drosophila melanogaster 87A7 hsp70 locus. Here, we report a
second protein, BEAF-32B, that differs from 32A only in its amino terminus. Unlike 32A, it has the same DNA binding specificity
as the complete BEAF activity affinity purified from Drosophila. We characterize three domains in these proteins. Heterocomplex
formation is mediated by their identical carboxy-terminal domains, and DNA binding is mediated by their unique amino-terminal
domains. The identical middle domains of 32A and 32B are dispensable for the functions described here, although they may be
important for boundary element function. 32A and 32B apparently form trimers, and the ratio of 32A to 32B varies at different
loci on polytene chromosomes as judged by immunofluorescence. The scs' element contains a high- and low-affinity binding site
for BEAF. We observed that interaction with the low-affinity site is facilitated by binding to the high-affinity site some
200 bp distant.
Parasegmental (PS)-specific expression of the homeotic genes of the bithorax-complex (BX-C) appears to depend upon the subdivision of the complex into a series of functionally independent cis-regulatory domains. Fab-7 is a regulatory element that lies between iab-6 and iab-7 (the PS11- and PS12-specific cis-regulatory domains, respectively). Deletion of Fab-7 causes ectopic expression of iab-7 in PS11 (where normally only iab-6 is active). Two models have been proposed to account for the dominant Fab-7 phenotype. The first considers that Fab-7 functions as a boundary element that insulates iab-6 and iab-7. The second model envisages that Fab-7 contains a silencer element that keeps iab-7 repressed in parasegments anterior to PS12. Using a P-element inserted in the middle of the Fab-7 region (the bit transposon), we have generated an extensive collection of new Fab-7 mutations that allow us to subdivide Fab-7 into a boundary element and a Polycomb-respond element (PRE). The boundary lies within 1 kb of DNA on the proximal side of the bit transposon (towards iab-6). Deletions removing this element alone cause a complex gain- and loss-of-function phenotype in PS11; in some groups of cells, both iab-6 and iab-7 are active, while in others both iab-6 and iab-7 are inactive. Thus, deletion of the boundary allows activating as well as repressing activities to travel between iab-6 and iab-7. We also provide evidences that the boundary region contains an enhancer blocker element. The Polycomb-response element lies within 0.5 kb of DNA immediately distal to the boundary (towards iab-7). Deletions removing the PRE alone do not typically cause any visible phenotype as homozygotes. Interestingly, weak ectopic activation of iab-7 is observed in hemizygous PRE deletions, suggesting that the mechanisms that keep iab-7 repressed in the absence of this element may depend upon chromosome pairing. These results help to reconcile the previously contradictory models on Fab-7 function and to shed light on how a chromatin domain boundary and a nearby PRE concur in the setting up of the appropriate PS-specific expression of the Abd-B gene of the BX-C.
Transcriptional silencing by the Polycomb Group of genes maintains the position-specific repression of homeotic genes throughout Drosophila development. The Polycomb Group of genes characterized to date encode chromatin-associated proteins that have been suggested to form heterochromatin-like structures. By studying the expression of reporter genes, we have identified a 725 bp fragment, called MCP725, in the homeotic gene Abdominal-B, that accurately maintains position-specific silencing during proliferation of imaginal cells. Silencing by MCP725 requires the Polycomb and the Polycomblike genes, indicating that it contains a Polycomb response element To investigate the mechanisms of transcriptional silencing by MCP725, we have studied its temporal requirements by removing MCP725 from the transgene at various times during development. We have discovered that excision of MCP725 during larval stages leads to loss of silencing. Our findings indicate that the silencer is required for the maintenance of the repressed state throughout cell proliferation. They also suggest that propagation of the silenced state does not occur merely by templating of a heterochromatin structure by virtue of protein-protein interactions. Rather, they suggest that silencers play an active role in the maintenance of the position-specific repression throughout development.
The Abd-B gene, one of the three homeotic genes in the Drosophila bithorax complex (BX-C), is required for the proper identity of the fifth through the eighth abdominal segments (corresponding to parasegments 10-14) of the fruitfly. The morphological difference between these four segments is due to the differential expression of Abd-B, which is achieved by the action of the parasegment-specific cis-regulatory regions infra-abdominal-5 (iab-5), -6, -7 and -8. The dominant gain-of-function mutation Frontabdominal-7 (Fab-7) removes a boundary separating two of these cis-regulatory regions, iab-6 and iab-7. As a consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in Fab-7 flies. Here we report that point mutations of the Abd-B gene in trans suppress the Fab-7 phenotype in a pairing-dependent manner and thus represent a type of transvection. We show that the observed suppression is the result of trans-regulation of the defective Abd-B gene by the ectopically activated iab-7. Unlike previously demonstrated cases of trans-regulation in the Abd-B locus, trans-suppression of Fab-7 is sensitive to heterozygosity for chromosomal rearrangements that disturb homologous pairing at the nearby Ubx locus. However, in contrast to Ubx, the transvection we observed in the Abd-B locus is insensitive to the allelic status of zeste. Analysis of different deletion alleles of Abd-B that enhance trans-regulation suggests that an extensive upstream region, different from the sequences required for transcription initiation, mediates interactions between the iab cis-regulatory regions and the proximal Abd-B promoter. Moreover, we find that the amount of DNA deleted in the upstream region is roughly proportional to the strength of trans-interaction, suggesting that this region consists of numerous discrete elements that cooperate in tethering the iab regulatory domains to Abd-B. Possible implications of the tethering complex for the regulation of Abd-B are discussed. In addition, we present evidence that the tenacity of trans-interactions in the Abd-B gene may vary, depending upon the tissue and stage of development.
Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. A transgenic embryo assay was used to obtain evidence that the Drosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. The eve promoter contains an optimal TATA element and a GAGA sequence. The analysis of various chimeric promoters provides evidence that TATA is essential for promoter competition, whereas GAGA mediates enhancer blocking. The Trithorax-like (Trl) protein interacts with GAGA, and mutations in trl attenuate eve promoter insulator activity. We suggest that Trl-GAGA increases the stability of enhancer-promoter interactions by creating an open chromatin configuration at the core promoter.
P element insertion is essentially random at the scale of the genome. However, P elements containing regulatory sequences from Drosophila engrailed and polyhomeotic genes and from the Bithorax and Antennapedia complexes show some insertional specificity by frequently inserting near the parent gene (homing) and/or near genes containing Polycomb group response elements (preferential insertion). This phenomenon is thought to be mediated by Polycomb group proteins. In this report, we describe a case of homing of P elements containing regulatory sequences of the linotte gene. This homing occurs with high frequency (up to 20% of the lines) and high precision (inserted into a region of <1 kilobase). We present evidence showing that it is not mediated by Polycomb group proteins but by a new, as yet unknown, mechanism. We also suggest that P element homing could be a more frequent phenomenon than generally assumed and that it could become a powerful tool of Drosophila reverse genetics, for which there is no other described gene targeting technique.
Alternative promoters within the same gene are a general phenomenon in gene expression. Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between non-coding RNA and the major promoter, direct interaction of the non-coding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation and RNA immunoprecipitation, we show that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Regulatory DNA from a diverse group of Drosophila genes causes silencing of the linked reporter gene mini-white in the P-element vector CaSpeR. This silencing can occur in flies heterozygous for the P-element construct but is often enhanced in flies homozygous for the construct. In Drosophila, somatic chromosomes are paired and this pairing is important for the enhancement of silencing in most cases. Thus, this type of silencing has been called pairing-sensitive silencing. element required for the activity of the Polycomb group of trancriptional Many of the DNA fragments that cause pairing-sensitive silencing are regulatory repressors (Polycomb group response elements, PREs). However, some PREs do not appear to cause pairing-sensitive silencing, and some fragments of DNA that cause pairing-sensitive silencing do not appear to act as PREs. I suggest that many PREs are composite elements of sites important for silencing and sites important for “pairing” or bringing together distant DNA elements. Both activities may be required for PRE function. In a related phenomenon, fragments of DNA included within P-element vectors can cause those transposons to insert in the genome near the parent gene of the included DNA (transposon homing). I suggest that DNA fragments that cause transposon homing or pairing-sensitive silencing are bound by protein complexes that can interact to bring together distant DNA fragments.
We have cloned a Drosophila homolog of the membrane fusion protein CDC48/p97. The open reading frame of the Drosophila homolog encodes an 801 amino acid long protein (TER94), which shows high similarity to the known CDC48/p97 sequences. The chromosomal position of TER94 is 46 C/D. TER94 is expressed in embryo, in pupae and in imago, but is suppressed in larva. In the imago, the immunoreactivity was exclusively present in the head and in the gonads of both sexes. In the head the most striking staining was observed in the entire neuropil of the mushroom body and in the antennal glomeruli. Besides TER94, sex-specific forms were also detected in the gonads of the imago: p47 in the ovaries and p98 in the testis. TER94/p47 staining was observed in the nurse cells and often in the oöcytes, while TER94/p98 staining was present in the sperm bundles. On the basis of its distribution we suggest that TER94 functions in the protein transport utilizing endoplasmic reticulum and Golgi derived vesicles.
The Abd-B Hox gene contains an extended 3' cis-regulatory region that is subdivided into a series of separate lab domains. The lab-7 domain activates Abd-B in parasegment 12 (ps12), whereas lab-8 controls expression in ps13. iab-7 is flanked by two insulators, Fab-7 and Fab-8, which are thought to prevent regulatory factors, such as Polycomb silencers, from influencing neighboring iab domains. This organization poses a potential paradox, since insulator DNAs can work in a dominant fashion to block enhancer-promoter interactions over long distances. Here, we present evidence for a novel cis-regulatory sequence located within lab-7, the promoter targeting sequence (PTS), which permits distal enhancers to overcome the blocking effects of Fab-8 and the heterologous su(Hw) insulator. We propose that the PTS converts dominant, long-range insulators into local regulatory elements that separate neighboring lab domains.
INTRODUCTION
The Drosophila melanogaster P-transposable element is a powerful and widely used research tool. Sequences flanking the P-element can be recovered and the site of insertion can be mapped to the nucleotide, to connect the genetic and physical maps and facilitate molecular analysis of the gene of interest. The Berkeley Drosophila Genome Project (BDGP) has assembled a well-characterized collection of lethal mutations induced by single P-element insertions generated by a number of laboratories. The genomic DNA sequences adjacent to these insertions have been recovered by either plasmid rescue or inverse polymerase chain reaction (PCR). The combination of a complete genomic DNA sequence and relatively fast and easy molecular methods for mapping P-element insertion sites to the nucleotide enhances the use of P-elements as tools in Drosophila research. This protocol provides detailed procedures for isolating DNA flanking P-element insertions.
Enhancers are often located many tens of kilobases away from the promoter they regulate, sometimes residing closer to the promoter of a neighboring gene. How do they know which gene to activate? We have used homing P[en] constructs to study the enhancer-promoter communication at the engrailed locus. Here we show that engrailed enhancers can act over large distances, even skipping over other transcription units, choosing the engrailed promoter over those of neighboring genes. This specificity is achieved in at least three ways. First, early acting engrailed stripe enhancers exhibit promoter specificity. Second, a proximal promoter-tethering element is required for the action of the imaginal disc enhancer(s). Our data suggest that there are two partially redundant promoter-tethering elements. Third, the long-distance action of engrailed enhancers requires a combination of the engrailed promoter and sequences within or closely linked to the promoter proximal Polycomb-group response elements. These data show that multiple mechanisms ensure proper enhancer-promoter communication at the Drosophila engrailed locus.
Although epigenetic maintenance of either the active or repressed transcriptional state often involves overlapping regulatory elements, the underlying basis of this is not known. Epigenetic and pairing-sensitive silencing are related properties of Polycomb-group proteins, whereas their activities are generally opposed by the trithorax group. Both groups modify chromatin structure, but how their opposing activities are targeted to allow differential maintenance remains a mystery. Here, we identify a strong pairing-sensitive silencing (PSS) element at the 3' border of the Drosophila even skipped (eve) locus. This element can maintain repression during embryonic as well as adult eye development. Transgenic dissection revealed that silencing activity depends on a binding site for the Polycomb-group protein Pleiohomeotic (Pho) and on pho gene function. Binding sites for the trithorax-group protein GAGA factor also contribute, whereas sites for the known Polycomb response element binding factors Zeste and Dsp1 are dispensible. Normally, eve expression in the nervous system is maintained throughout larval stages. An enhancer that functions fully in embryos does not maintain expression, but the adjacent PSS element confers maintenance. This positive activity also depends on pho gene activity and on Pho binding. Thus, a DNA-binding complex requiring Pho is differentially regulated to facilitate epigenetic transcriptional memory of both the active and the repressed state.
Polycomb group (PcG) proteins form conserved regulatory complexes that modify chromatin to repress transcription. Here, we report genome-wide binding profiles of PhoRC, the Drosophila PcG protein complex containing the DNA-binding factor Pho/dYY1 and dSfmbt. PhoRC constitutively occupies short Polycomb response elements (PREs) of a large set of developmental regulator genes in both embryos and larvae. The majority of these PREs are co-occupied by the PcG complexes PRC1 and PRC2. Analysis of PcG mutants shows that the PcG system represses genes required for anteroposterior, dorsoventral, and proximodistal patterning of imaginal discs and that it also represses cell cycle regulator genes. Many of these genes are regulated in a dynamic manner, and our results suggest that the PcG system restricts signaling-mediated activation of target genes to appropriate cells. Analysis of cell cycle regulators indicates that the PcG system also dynamically modulates the expression levels of certain genes, providing a possible explanation for the tumor phenotype of PcG mutants.
Enhancer-blocking insulators are DNA elements that disrupt the communication between a regulatory sequence, such as an enhancer or a silencer, and a promoter. Insulators participate in both transcriptional regulation and global nuclear organization, two features of chromatin that are thought to be maintained from one generation to the next through epigenetic mechanisms. Furthermore, there are many regulatory mechanisms in place that enhance or hinder insulator activity. These modes of regulation could be used to establish cell-type-specific insulator activity that is epigenetically inherited along a cell and/or organismal lineage. This review will discuss the evidence for epigenetic inheritance and regulation of insulator function.
Eukaryotic cells store their genome inside a nucleus, a dedicated organelle shielded by a double lipid membrane. Pores in these membranes allow the exchange of molecules between the nucleus and cytoplasm. Inside the mammalian cell nucleus, roughly 2 m of DNA, divided over several tens of chromosomes is packed. In addition, protein and RNA molecules functioning in DNA-metabolic processes such as transcription, replication, repair and the processing of RNA fill the nuclear space. While many of the nuclear proteins freely diffuse and display a more or less homogeneous distribution across the nuclear interior, some appear to preferentially cluster and form foci or bodies. A non-random structure is also observed for DNA: increasing evidence shows that selected parts of the genome preferentially contact each other, sometimes even at specific sites in the nucleus. Currently a lot of research is dedicated to understanding the functional significance of nuclear architecture, in particular with respect to the regulation of gene expression. Here we will evaluate evidence implying that the folding of DNA is important for transcriptional control in mammals and we will discuss novel high-throughput techniques expected to further boost our knowledge on nuclear organisation.
Expression of the yellow (y) gene of Drosophila melanogaster is controlled by a series of tissue-specific transcriptional enhancers located in the 5' region and intron of the gene. Insertion of the gypsy retrotransposon in the y2 allele at -700 bp from the start of transcription results in a spatially restricted phenotype: Mutant tissues are those in which yellow expression is controlled by enhancers located upstream from the insertion site, but all other structures whose enhancers are downstream of the insertion site are normally pigmented. This observation can be reproduced by inserting just a 430-bp fragment containing the suppressor of Hairy-wing [su(Hw)]-binding region of gypsy into the same position where this element is inserted in y2, suggesting that the su(Hw)-binding region is sufficient to confer the mutant phenotype. Insertion of this sequence into various positions in the y gene gives rise to phenotypes that can be rationalized assuming that the presence of the su(Hw) protein inhibits the action of those tissue-specific enhancers that are located more distally from the su(Hw)-binding region with respect to the promoter. These results are discussed in light of current models that explain long-range effects of enhancers on gene expression.
In an effort to determine how crude gradients of transcriptional activators and repressors specify sharp stripes of gene expression in the early embryo, we have conducted a detailed study of even-skipped (eve) stripe 2. A combination of promoter fusions and P-transformation assays were used to show that a 480 bp region of the eve promoter is both necessary and sufficient to direct a stripe of LacZ expression within the limits of the endogenous eve stripe 2. The maternal morphogen bicoid (bcd) and the gap proteins hunchback (hb), Kruppel (Kr) and giant (gt) all bind with high affinity to closely linked sites within this small promoter element. Activation appears to depend on cooperative interactions among bcd and hb proteins, since disrupting single binding sites cause catastrophic reductions in expression. gt is directly involved in the formation of the anterior border, although additional repressors may participate in this process. Forming the posterior border of the stripe involves a delicate balance between limiting amounts of the bcd activator and the Kr repressor. We propose that the clustering of activator and repressor binding sites in the stripe 2 element is required to bring these weakly interacting regulatory factors into close apposition so that they can function both cooperatively and synergistically to control transcription.
Chromosomes of higher eukaryotes are thought to be organized into a series of discrete and topologically independent higher-order
domains. In addition to providing a mechanism for chromatin compaction, these higher-order domains are thought to define independent
units of gene activity. Implicit in most models for the folding of the chromatin fiber are special nucleoprotein structures,
the domain boundaries, which serve to delimit each higher-order chromosomal domain. We have used an "enhancer-blocking assay"
to test putative domain boundaries for boundary function in vivo. This assay is based on the notion that in delimiting independent
units of gene activity, domain boundaries should be able to restrict the scope of activity of enhancer elements to genes which
reside within the same domain. In this case, interposing a boundary between an enhancer and a promoter should block the action
of the enhancer. In the experiments reported here, we have used the yolk protein-1 enhancer element and an hsp70 promoter:lacZ
fusion gene to test putative boundary DNA segments for enhancer-blocking activity. We have found that several scs-like elements
are capable of blocking the action of the yp-1 enhancer when placed between it and the hsp70 promoter. In contrast, a MAR/SAR
DNA segment and another spacer DNA segment had no apparent effect on enhancer activity.
The Drosophila engrailed gene is expressed in the cells of the posterior developmental compartments. To investigate how the engrailed gene is regulated, chimeric genes consisting of fragments of the engrailed promoter and Escherichia coli lacZ were incorporated into the Drosophila germ line by P-element-mediated recombination. Fusion constructs with 7.5 kb of 5'-flanking sequence contain sufficient information to promote expression in most of the embryonic, larval, and imaginal posterior compartments; transformants with smaller fragments of the 5' region do not. Remarkably, of 20 independent transformants with constructs containing more than 1 kb of 5'-flanking DNA, 7 integrated in or around the engrailed locus. These strains inactivate engrailed function to varying degrees, and some express lacZ with a position- and temporal-specific program that is indistinguishable from the normal engrailed gene. Presumably, in these strains, lacZ is expressed in the context of the engrailed promoter.
We have transferred the site-specific recombination system of the yeast 2 micron plasmid, the FLP recombinase and its recombination targets (FRTs), into the genome of Drosophila. Flies were transformed with an FLP gene under the control of hsp70 regulatory sequences and with a white gene flanked by FRTs. The heat-induced recombinase catalyzes recombination between FRTs, causing loss of white (seen somatically as white patches in the eye) and, less frequently, gain of white (seen as dark-red patches). Loss and gain frequencies vary with the severity of the heat shock, and patterns of mosaicism vary with the developmental stage at which the heat shock is applied. The recombinase is also active in the germline, producing white-eyed and dark-red-eyed progeny.
We have identified the regulatory sequences required for the periodic expression of the Drosophila pair rule gene even skipped (eve). We find that the gradually changing pattern of periodic eve expression during early embryogenesis is directed by two distinct regulatory programs. Initially, eve expression in individual stripes is established by different regulatory elements, each of which responds to nonperiodic spatial cues provided, at least in part, by the gap genes. Later, coordinate expression of eve in all seven stripes is directed by a single regulatory region that responds to periodic cues provided by primary pair rule genes, including eve itself. As a consequence of this two-step regulatory program, eve functions both in the establishment of the periodic pattern of gene expression and in the subsequent specification of parasegmental boundaries.
We have analyzed the molecular structure of phenotypic revertants of gypsy-induced mutations to understand the molecular mechanisms by which this retrotransposon causes mutant phenotypes in Drosophila melanogaster. The independent partial revertants analyzed are caused by the insertion of different transposons into the same region of gypsy. One partial revertant of the yellow allele y2 arose as a consequence of the insertion of the jockey mobile element into gypsy sequences, whereas a second incomplete revertant is due to the insertion of the hobo transposon. In addition, a previously isolated partial revertant of the Hairy-wing allele Hw1 resulted from the integration of the BS transposable element into the same gypsy sequences. The region affected by the insertion of the three transposons contains 12 copies of a repeated motif that shows striking homology to mammalian transcriptional enhancers. Our results suggest that these sequences, which might be involved in the transcriptional control of the gypsy element, are also responsible for the induction of mutant phenotypes by this retrotransposon.
The Drosophila melanogaster transposon gypsy is the cause of numerous spontaneous mutations, most of which are suppressible by mutations in the suppressor of Hairy wing [su(Hw)] locus. We have examined the phenotype of four revertants of the gypsy element-induced mutation bithoraxoid1 (bxd1) and determined the molecular basis of these reversions. All four revertants have undergone deletions within the gypsy element. The altered gypsy element from one of the partial revertants has been cloned. It has a deletion of only 109 base pairs near the 5' end of the gypsy transcription unit. Similar deletion gypsy elements exist elsewhere in the Drosophila genome. We discuss a mechanism by which the 109-base segment might affect the bxd phenotype.
A method is presented for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle. This procedure of inverse PCR (IPCR) has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements. In this paper we show the feasibility of IPCR by amplifying the sequences that flank an IS1 element in the genome of a natural isolate of Escherichia coli.
The chromatin fiber of eukaryotic chromosomes is thought to be organized into a series of discrete domains or loops. To learn more about these large-scale structures, we have examined the sequence and chromatin organization of the DNA segments surrounding the two hsp 70 genes at the Drosophila melanogaster cytogenetic locus 87A7. These studies indicate that this heat shock locus is flanked on both the proximal and distal sides by novel chromatin structures, which we have called, respectively, scs and scs' (specialized chromatin structures). Each structure is defined by two sets of closely spaced nuclease-hypersensitive sites arranged around a central nuclease-resistant segment. Our findings suggest that these two structures define the proximal and distal boundaries of the 87A7 chromomere and, hence, may be one of the first examples of anchor points for the organization of eukaryotic chromosomes into a series of discrete higher order domains. Moreover, these structures may provide focal points both for the decondensation of the chromomere when the hsp 70 genes are induced by heat shock and for the subsequent rewinding and condensation of the chromomere during recovery from heat shock.
Exogenous DNA sequences were introduced into the Drosophila germ line. A rosy transposon (ry1), constructed by inserting a
chromosomal DNA fragment containing the wild-type rosy gene into a P transposable element, transformed germ line cells in
20 to 50 percent of the injected rosy mutant embryos. Transformants contained one or two copies of chromosomally integrated,
intact ry1 that were stably inherited in subsequent generations. These transformed flies had wild-type eye color indicating
that the visible genetic defect in the host strain could be fully and permanently corrected by the transferred gene. To demonstrate
the generality of this approach, a DNA segment that does not confer a recognizable phenotype on recipients was also transferred
into germ line chromosomes.
Certain spontaneous mutations of Drosophila melanogaster are suppressed by su(Hw), the suppressor of Hairy-wing (3R-54.8). We find that mutations suppressible by su(Hw) result from insertions of a mobile element at the affected loci. The element, named gypsy, is approximately 7.3 kilobases long and includes 0.5-kilobase direct terminal repeats. It was first identified in DNA cloned from the bithorax chromosomal region of several Drosophila stocks carrying suppressible mutations of the bithorax complex. Cloned gypsy DNA was used as a probe to test for the association of gypsy with suppressible mutations at various other loci by hybridization in situ. Gypsy was found to be associated with 19 suppressible alleles at 10 different loci: yellow, Hairy-wing, scute, diminutive, cut, lozenge, forked, Beadex, hairy, and the bithorax complex. It was found with wild-type or nonsuppressible mutations at any of these loci. Gypsy DNA was also used as a probe to clone the element and adjacent unique DNA from the loci of some suppressible mutations. This confirmed the presence of the full-length element and also provided cloned DNA from the previously uncloned loci scute and cut. The suppressor of Hairy-wing is generally recessive and behaves as a null mutation. Thus, the disruption of normal gene function caused by the inserted gypsy element appears to require some product of the wild-type suppressor gene, su(Hw)+.
We have purified two proteins from Drosophila that bind to the scs' boundary element of the hsp70 domain at locus 87A7. Their palindromic binding sites (CGATA-TATCG) symmetrically abut the previously mapped hypersensitive site of scs'. We have cloned a cDNA for one of these proteins, BEAF-32 (boundary element-associated factor of 32 kDa). It encodes a novel protein that is bound to scs' but not scs in vivo. Immunostaining localizes BEAF to hundreds of interbands and many puff boundaries on polytene chromosomes, suggesting that a chromosomal domain consists of a band (or puff) and part of the flanking interbands. Enhancer blocking assays implicate the palindromic binding site in boundary function. The lack of enhancer blocking in transiently transfected cells suggests an involvement of chromatin, nuclear structure, or both in boundary function.
Fab-7 deletions in the bithorax complex have a novel gain-of-function phenotype, typically transforming parasegment 11 (PS11) into PS12 identity. Genetic analysis indicates that removal of the Fab-7 element results in the fusion of the iab-6 (PS11) and iab-7 (PS12) cis-regulatory domains into a single regulatory domain that inappropriately regulates Abdominal-B in PS11. This has led to the hypothesis that Fab-7 is a chromatin domain boundary that normally functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains. We use several different enhancer blocking assays to demonstrate that Fab-7 has the insulating properties expected of a domain boundary. We define a minimal fragment of Fab-7 sufficient for enhancer blocking, and demonstrate that it is completely distinct from an adjacent Polycomb-dependent silencer. We compare Fab-7 to the su(Hw) insulator element, and show that Fab-7 enhancer blocking activity is intermediate between that of five and twelve reiterated binding sites for the Su(Hw) protein. These results support the model that Fab-7 functions as a domain boundary within the context of the bithorax complex, making Fab-7 one of the first boundary elements that is known to have an essential function in vivo.
Insulators are naturally occurring DNA sequences that protect transgenes from genomic position effects, thereby establishing independent functional domains within the chromosome. Recent studies have focused on the identification of the cis and trans requirements for insulator activity. These experiments demonstrate that insulators contain multiple components that cooperate to confer their unique properties. Additionally, they suggest that the mechanism of insulation is related to that of enhancer function. Two models of insulator can be considered: a domain boundary and a transcriptional decoy model.
The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is 'carved out' by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to contribute to efficient functional rescue.
The even-skipped (eve) gene of Drosophila melanogaster is a crucial member of the pair-rule class of segmentation genes. We report here the characterization of a 16-kb region sufficient for all known aspects of eve expression and the rescue of an eve null mutation. We began by examining 45 kb surrounding the eve coding sequence for DNaseI hypersensitive sites and other transcription units. We find that the previously identified eve regulatory elements, those for early stripes 2, 3, and 7 and the late element, do not generate prominent hypersensitive sites. However, strong, constitutive DNaseI hypersensitive sites flank a 16-kb region, within which one developmentally regulated site is found at the eve promoter region. P-element transformation of this 16-kb domain into eve mutants rescues them to adult viability. This 16-kb domain contains regulatory elements for all known features of eve expression: the seven major blastoderm stripes, minor stripe expression during germ band extension, and later expression in the lateral mesodermal muscle precursor cells, in the central nervous system, adjacent to the invaginating proctodeum, and in a ring around the anal pad. We have begun a preliminary dissection of the 16-kb domain into its constituent regulatory elements. Other major findings include the following: (1) There is a second element for late stripe expression adjacent to the traditional late element. (2) A stripe element 3' of the gene interacts with the late element to give rise to the minor stripes seen in the even-numbered parasegments. (3) Expression in the proctodeum and anal pad is driven by sequences both 5' and 3' of the gene. (4) Expression in different sites in the central nervous system is driven by separable elements widely dispersed throughout 8 kb 3' of the gene.