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Mediator Requirement for Both Recruitment and Postrecruitment Steps in Transcription Initiation
Abstract
Mediator complexes are required for activators to stimulate Pol II preinitiation complex assembly on an associated promoter. We show here that for the mouse Egr1 gene, controlled largely by MAP kinase phosphorylation of the ELK1 transcription factor, the MED23 Mediator subunit that interacts with phospho-ELK1 is also required to stimulate Pol II initiation at a step subsequent to preinitiation complex assembly. In Med23-/- cells, histone acetylation, methylation, and chromatin remodeling complex association at the Egr1 promoter were equivalent to that of wild-type cells, yet Egr1 induction was greatly reduced. MAP kinase activation stimulated Pol II and GTF promoter binding. However, the difference in factor binding between wild-type and mutant cells was much less than the difference in transcription, and Pol II remained localized to the promoter in mutant cells. These results indicate that an interaction with MED23 stimulates initiation by promoter bound Pol II in addition to Pol II and GTF recruitment.
... Whereas preinitiation complexes are dissociated by 0.25 M KCl, after synthesis of the first 10 bases, elongation complexes become stable to 0.6 M KCl (Cai and Luse 1987). Consistent with the in vitro results, when yeast or mammalian cells are treated with 0.5 M NaCl, Pol II dissociated from promoter regions but not downstream positions within several genes (Proft and Struhl 2004;Wang et al. 2005). To investigate the dynamics of Pol II at pause sites in human cells, we revisited the question of how blocking recruitment to promoters with high salt affects Pol II occupancy genome-wide using anti-Pol II ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing). ...
... In contrast to 5 ′ pause sites, Pol II within gene bodies was resistant to hypertonic conditions even after treatment for 30 min (Fig. 1I), consistent with the resistance of elongation complexes to high salt in vivo and in vitro (Cai and Luse 1987;Proft and Struhl 2004;Wang et al. 2005). High-salt treatment did not eliminate Pol II occupancy from the 5 ′ ends of all genes, however. ...
... Moreover, this is an active process that requires cell viability (Fig. 2F). In contrast, elongating Pol II situated within genes throughout the genome is resistant to hypertonic shock (Figs. 1, 2; Supplemental Fig. S1; Proft and Struhl 2004;Wang et al. 2005), and transcription often extends long distances past poly(A) sites (Fig. 6A, right), in agreement with previous work, suggesting a termination defect under hypertonic conditions (Vilborg et al. 2015). ...
Paused RNA polymerase II (Pol II) that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused Pol II is a stable or dynamic target remains unresolved. We report that most 5' paused Pol II throughout the genome is turned over within 2 min. This process is revealed under hypertonic conditions that prevent Pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation, suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel preinitiated state of Pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that Pol II occupancy near 5' ends is governed by a cycle of ongoing assembly of preinitiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of Pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause is by escape from premature termination.
... Enhancers are composed of combinations of protein binding sites, which recruit sequence-specific TFs. These TFs, in turn, recruit non-DNA-binding cofactors, which regulate transcription through a variety of mechanisms, including direct recruitment of RNA polymerase II and the basal transcription machinery, either directly or via Mediator, a large multiunit complex that promotes transcription via assembly of the basal transcription machinery Szutorisz et al., 2005;Wang et al., 2005). Enhancers also influence their local chromatin environment via epigenetic changes-for example, by recruiting ATP-dependent nucleosome remodeling complexes or histone acetyltransferases (HATs) or deacetylases (HDACs)-resulting in changes in chromatin structure that stimulate (or inhibit) transcription . ...
... Dev Dyn 229, 162-175. Wang, G., Balamotis, M.A., Stevens, J.L., Yamaguchi, Y., Handa, H., and Berk, A.J. (2005). Mediator requirement for both recruitment and postrecruitment steps in transcription initiation. ...
... (Szutorisz et al, 2005). Alternatively, enhancer binding transcription factors can recruit Mediator, the large multi-unit complex that promotes transcription via assembly of the basal transcription machinery at a genes promoter Wang et al, 2005). Enhancers are also capable of influencing their local chromatin environment via their specific protein interactions. ...
Enhancers, a subclass of cis-regulatory sequences control gene expression and patterning during development. As enhancers are often located at considerable genomic distances from their target genes, distal enhancer promoter interactions are a crucial part of transcriptional regulation, yet surprisingly little is known about how these interactions are facilitated. To better understand this essential enhancer function, we are characterizing the ability of, sparkling, the dPax2 cone cell-specific enhancer, ability to activate gene expression from both a promoter proximal, and distal position. Using this approach, we have identified a sequence within spa required when the enhancer is placed at a distance from the promoter, but is dispensable in a promoter proximal position. As this DNA sequence appears to convey long-range enhancer activity, we refer to it as the ???remote control??? element or RCE. Subsequent analysis has analyzed the distance at which sparkling requires the RCE to function and demonstrated that the RCE can function as a separable unit from the enhancer that can be moved to new locations and still facilitate sparkling action. Furthermore experimental observations support a role for the RCE in facilitating the ability of the enhancer to communicate with specific promoters. This work as also shown that additional regulator sequences, both outside of, and from within the enhancer can also support long-range enhancer activity. This study reveled that the regulatory sequences within in spa can be rearranged globally, but some local restrictions must be obeyed. We have also attempted to identify proteins that interact with the spa enhancer and have found that the Six family protein, Sine Oculis, can interact in vitro with at least two regions of the enhancer capable of conveying long distance gene regulation - the RCE and region 4. This work has contributed to the study of gene regulation by identifying the first sequence with an enhancer to regulate long-range gene transcription without requiring specific promoter elements as well. The knowledge gained from this work can ultimately aid in identifying novel enhancers, designing tissue/temporal specific synthetic enhancers, and when broadly applied a can give a clearer view of basic transcriptional mechanisms, signal transduction, and development.
... To better understand the dynamic changes in IEGs induced by the MED23 R617Q mutation, we performed serum stimuli assays, and again, we found significantly higher levels of the IEGs FOS and JUN in the MED23/R617Q mutant cells than in the WT cells in either the steady state or under serum induction ( Figure 2E). As we previously found, the ternary complex factor ELK1 transcriptionally activates the expression of many IEGs, especially EGR1, which is dependent on the presence of the Mediator subunit MED23 (36,37). Paradoxically, when ELK1 transcriptional activity was reduced in the MED23/R617Q mutant cells, IEGs were upregulated. ...
... Our previous research has established that MED23 deficiency resulted in the defective ELK1 activation in response to MAPK signaling, which further led to the defective serum induction of IEGs EGR1 and FOS (36,37). However, in this study we observed the barely-changed EGR1 and the upregulated FOS expression in MED23/R617Q cells, which is consistent with the unchanged EGR1 and the upregulated FOS in the MED23/R617Q patient's skin fibroblasts (23). ...
Transcriptional Mediator controls diverse gene programs for various developmental and pathological processes. The human Mediator MED23/R617Q mutation was reported in a familial intellectual disability (ID) disorder, although the underlying mechanisms remain poorly understood. Constructed by gene editing, the Med23/R617Q knock-in mutant mice exhibited embryonic lethality due to the largely reduced Med23/R617Q protein level, but the R617Q mutation in HEK293T cells didn't change its expression and incorporation into Mediator Complex. RNA-seq revealed that MED23/R617Q mutation disturbed gene expression, related to neural development, learning and memory. Specifically, R617Q mutation reduced the MED23-dependent activities of ELK1 and E1A, but in contrast, upregulated the MAPK/ELK1-driven early immediate genes (IEGs) JUN and FOS. ChIP-seq and Hi-C revealed that the MED23 R617Q mutation reprogramed a subset of enhancers and local chromatin interactions, which correlated well with the corresponding gene expression. Importantly, the enhancers and chromatin interactions surrounding IEGs were unchanged by the R617Q mutation, but DACH1, an upstream repressor of IEGs, showed reduced enhancer-promoter interactions and decreased expression in mutant cells, thus relieving its inhibition to the intellectual-related IEGs. Overall, unraveling the MED23-DACH1-IEG axis provides a mechanistic explanation for the effects of the MED23/R617Q mutation on gene dysregulation and inherited ID.
... Experiments with cells derived from knockout mice revealed that expression of the mediator of RNA polymerase II transcription (Mediator) subunit 23 (Med23) is essential for stimulus-induced biosynthesis of the transcription factor Egr-1 [49,50]. The Mediator multiprotein complex consists of 20-30 subunits that function as a bridge between DNA-bound transcription factors, RNA polymerase II and general transcription factors. ...
... Med23 bound specifically to phosphorylated Elk-1 [49], thus connecting Elk-1 phosphorylation with gene transcription. These data were corroborated in chromatin immunoprecipitation experiments showing that serum-induced phosphorylation of Elk-1 upregulates Mediator binding to the Egr-1 promoter region in ES cells, while this interaction is almost abolished in ES cells lacking Med23 [50]. A hydrophobic region within the transcriptional activation domain of Elk-1, including the residues F378 and W379, functions as docking site for Med23. ...
Elk-1 is a transcription factor that binds together with a dimer of the serum response factor (SRF) to the serum-response element (SRE), a genetic element that connects cellular stimulation with gene transcription. Elk-1 plays an important role in the regulation of cellular proliferation and apoptosis, thymocyte development, glucose homeostasis and brain function. The biological function of Elk-1 relies essentially on the interaction with other proteins. Elk-1 binds to SRF and generates a functional ternary complex that is required to activate SRE-mediated gene transcription. Elk-1 is kept in an inactive state under basal conditions via binding of a SUMO-histone deacetylase complex. Phosphorylation by extracellular signal-regulated protein kinase, c-Jun N-terminal protein kinase or p38 upregulates the transcriptional activity of Elk-1, mediated by binding to the mediator of RNA polymerase II transcription (Mediator) and the transcriptional coactivator p300. Strong and extended phosphorylation of Elk-1 attenuates Mediator and p300 recruitment and allows the binding of the mSin3A-histone deacetylase corepressor complex. The subsequent dephosphorylation of Elk-1, catalyzed by the protein phosphatase calcineurin, facilitates the re-SUMOylation of Elk-1, transforming Elk-1 back to a transcriptionally inactive state. Thus, numerous protein–protein interactions control the activation cycle of Elk-1 and are essential for its biological function.
... SRF often acts in concert with another transcription factor, Elk-1, which is directly phosphorylated by MAPK/ERK (Gualdrini et al., 2016;Marais et al., 1993;Sgambato et al., 1998;Sharrocks, 1995;Xia et al., 1996). Elk-1 has been shown to facilitate Pol2 recruitment via interactions with the same Mediator subunit MED23 (Allen and Taatjes, 2015;Besnard et al., 2011;Wang et al., 2005) that we found selectively pre-bound to rapid PRG promoters and rapid enhancers. A third function for MAPK/ERK signaling that could apply to either model is phosphorylation of histone tails either directly, via its downstream kinase MSK (Josefowicz et al., 2016), or via interactions with Elk-1 (Esnault et al., 2017). ...
... This apparent discrepancy could be a consequence of eRNA being important for H3K27ac maintenance rather than initiation. Indeed, in other contexts histone acetylation has been shown to accumulate despite blocking eRNA transcription, Pol2 recruitment, or initiation of transcription (Hah et al., 2013;Kaikkonen et al., 2013;Wang et al., 2005). These, as well as other analyses (Zhu et al., 2013) also suggest that eRNA transcription may be a better marker for enhancer activation than H3K27ac, more accurately reflecting an the extent to which an enhancer is activating transcription at a nearby promoter. ...
Brief and sustained neuronal activity patterns can have opposite effects on synaptic strength that both require activity-regulated gene (ARG) expression. However, whether distinct patterns of activity induce different sets of ARGs is unknown. In genome-scale experiments, we reveal that a neuron’s activity-pattern history can be predicted from the ARGs it expresses. Surprisingly, brief activity selectively induces a small subset of the ARG program that that corresponds precisely to the first of three temporal waves of genes induced by sustained activity. These first-wave genes are distinguished by an open chromatin state, proximity to rapidly activated enhancers, and a requirement for MAPK/ERK signaling for their induction. MAPK/ERK mediates rapid RNA polymerase recruitment to promoters, as well as enhancer RNA induction but not histone acetylation at enhancers. Thus, the same mechanisms that establish the multi-wave temporal structure of ARG induction also enable different sets of genes to be induced by distinct activity patterns.
... The Mediator subunit 23 (Med23, also known as sur2) was originally identified as a genetic suppressor of activated let-60 ras mutation in Caenorhabditis elegans (Singh and Han, 1995). Med23 functions in the mitogen-activated protein kinase (MAPK) signaling pathway by interacting with the ternary complex factor Elk1, and data from embryonic fibroblasts have indicated that MAPK-activated interaction of Med23-Elk1 is required for hormone-induced adipogenesis through controlling transcription of immediate early gene Egr2 (Stevens et al., 2002;Wang et al., 2005Wang et al., , 2009. Important roles of Med23 have also been reported in other biological processes including osteoblast differentiation , myogenesis (Yin et al., 2012), lung carcinogenesis (Yang et al., 2012;Yao et al., 2015), glucose and lipid metabolism , and T-cell activation (Sun et al., 2014). ...
... In the down-regulated genes of Med23-deficient NSCs, the expression of early response genes (such as Egr1, Egr2, Egr4) that are normally induced by serum growth factor (such as SFRP) activation, are greatly attenuated. These results are consistent with our previous study that Egr1 is the most severely affected by loss of MED23 (Wang et al., 2005). However, Med23-deficient embryonic stem cells display enhanced neural differentiation via BMP signaling (Sun et al., 2014), which is not significantly altered in Med23-deficient hippocampal NSCs. ...
Mammalian Mediator (Med) is a key regulator of gene expression by linking transcription factors to RNA polymerase II (Pol II) transcription machineries. The Mediator subunit 23 (Med23) is a member of the conserved Med protein complex and plays essential roles in diverse biological processes including adipogenesis, carcinogenesis, osteoblast differentiation, and T-cell activation. However, its potential functions in the nervous system remain unknown. We report here that Med23 is required for adult hippocampal neurogenesis in mouse. Deletion of Med23 in adult hippocampal neural stem cells (NSCs) was achieved in Nestin-CreER:Med23flox/flox mice by oral administration of tamoxifen. We found an increased number of proliferating NSCs shown by pulse BrdU-labeling and immunostaining of MCM2 and Ki67, which is possibly due to a reduction in cell cycle length, with unchanged GFAP+/Sox2+ NSCs and Tbr2+ progenitors. On the other hand, neuroblasts and immature neurons indicated by NeuroD and DCX were decreased in number in the dentate gyrus (DG) of Med23-deficient mice. In addition, these mice also displayed defective dendritic morphogenesis, as well as a deficiency in spatial and contextual fear memory. Gene ontology (GO) analysis of hippocampal NSCs revealed an enrichment in genes involved in cell proliferation, Pol II-associated transcription, Notch signaling pathway and apoptosis. These results demonstrate that Med23 plays roles in regulating adult brain neurogenesis and functions.
... Rekrutierung von Elongationsfaktoren und mRNA-prozessierenden Proteinen wie dem Superelongationskomplex SEC durch Med26 (Takahashi et al., 2011) Phosphorylierung der C-terminalen Domäne der RNA-Polymerase II und damit positive Regulierung der Elongation durch Cdk8 (Donner et al., 2010) Reaktivierung der pausierenden Pol II nach Transkriptionsstart durch Med23 (Wang et al., 2005) In der Bäckerhefe konnte eine Stimulation der Elongation durch den Mediator anhand seiner Fähigkeit, die Transkription durch die +1 Nukelosomen zu ermöglichen gezeigt werden. So zeigen genetische Analysen, dass Untereinheiten des Mediators mit der Histonmethyltransferase Set2 interagieren (Dettmann et al., 2010) welche für die Methylierungen am Histone 3 (K36) innerhalb codierender Regionen verantwortlich ist (Krogan et al., 2003). ...
... Das Kinasemodul ist in menschlichen Zellen an manchen Promotoren auch während der Transkription am Mediator assoziiert (Wang et al., 2005). Auch in Hefe gibt es Hinweise darauf, dass das Kinasemodul auch an der Transkriptionsaktivierung beteiligt ist (Larschan and Winston, 2005;Shahi et al., 2010). ...
Eukaryotic messenger RNA (mRNA) synthesis catalyzed by the RNA Polymerase II is the central and critical process for the regulation of gene expression. Several decades of research unearthed many details about this essential process of high complexity and dynamic. The mediator complex turned out to be crucial for the regulation of Pol II mediated transcription, especially the process of initiation. It functions as an interface between the general transcription machinery and multiple DNA binding transcriptional regulators. Binding these regulators via its tail module and binding the polymerase II via its head module, the mediator forms a bridge between upstream activating sequences and the core promotor and initiates the assembling of the Pre-Initiation complex consisting of the polymerase II and the general transcription factors. However, particularly the last years of research suggest the mediator complex within many other functions including transcription elongation, gene looping and chromatin remodeling. Considering the facts, that the mediator (a) consist of 25 subunits, which are partially flexible associated, (b) shows a flexible intrinsic structure and (c) is highly and dynamically phosphorylated it becomes easy to imagineplausible that the mediator complex meets all this functions, by serving as a transcriptional platform. In context of this thesis, and it was possible to “illustrate” the mediator within its versatile tasks and functions by presenting the most comprehensive analysis of the Mediator complex interactome to date. By optimizing the conditions of cell lysis and co-immunoprecipitation it was possible to preserve even transient and labile protein-protein interactions. The use of metabolic labeling (15N) in the control experiment, allowed us to distinguish between specific and non-specific captured proteins. In combination with high performance mass spectrometry, more than 400 proteins and even complete protein complexes interacting with the mediator complex could be identified, naming RNA-Polymerase II, all general transcription factors the SAGA complex, chromatin remodeling complexes and highly acetylated histones. Furthermore, many candidates where identified playing a role in co-transcriptional processes of mRNA, such as splicing, mRNA-decapping, mRNA transport and decay. This analysis not only confirmed several interactions , already can be found in the literature, but furthermore provide clear evidence, that mediator complex interacts not only with the RNA-Polymerase II, but also with the RNA Polymerase I and III. Next to the high numbers of potential known and unknown interacting proteins, it could be shown, that the interactome is highly dynamic and sensitive to detergent.
... SRF is required in vivo for the transcription of rPRGs (Ramanan et al., 2005) and often acts in concert with Elk-1, which is directly phosphorylated by MAPK/ERK ( Figure S6A; Marais et al., 1993;Sgambato et al., 1998;Xia et al., 1996). Elk-1 facilitates Pol2 recruitment via interactions with the Mediator subunit MED23 (Allen and Taatjes, 2015;Wang et al., 2005). We present correlative evidence that rPRGs may be regulated by SRF, Elk-1, MEF2, and MED23 and by activation of nearby rapid enhancers, but further work is required to causally link these mechanisms to rPRG induction. ...
... H3K27ac is a commonly used mark for enhancer activity (Creyghton et al., 2010;Rada-Iglesias et al., 2011), but we find that H3K27ac accumulates at enhancers even in the presence of MAPK/ERK inhibition, which blocks eRNA (and mRNA) induction. In other contexts, histone acetylation has been shown to accumulate despite blocking eRNA transcription, Pol2 recruitment, or initiation of transcription (Hah et al., 2013;Kaikkonen et al., 2013;Wang et al., 2005). These and other experiments (Zhu et al., 2013) suggest that eRNA transcription may be a better marker for enhancer activation than H3K27ac, more accurately reflecting the extent to which an enhancer is activating transcription at a nearby promoter. ...
A vast number of different neuronal activity patterns could each induce a different set of activity-regulated genes. Mapping this coupling between activity pattern and gene induction would allow inference of a neuron's activity-pattern history from its gene expression and improve our understanding of activity-pattern-dependent synaptic plasticity. In genome-scale experiments comparing brief and sustained activity patterns, we reveal that activity-duration history can be inferred from gene expression profiles. Brief activity selectively induces a small subset of the activity-regulated gene program that corresponds to the first of three temporal waves of genes induced by sustained activity. Induction of these first-wave genes is mechanistically distinct from that of the later waves because it requires MAPK/ERK signaling but does not require de novo translation. Thus, the same mechanisms that establish the multi-wave temporal structure of gene induction also enable different gene sets to be induced by different activity durations. Tyssowski et al. report that different durations of neuronal activity induce different gene expression profiles, enabling inference of past neuronal activity from gene expression data. Furthermore, they show that MAPK/ERK signaling partially establishes this activity-pattern-to-gene-induction coupling.
... 56,84 In line with close physical and functional associations between Mediator and Pol II, Mediator mutations or downregulation of Mediator subunits were shown to strongly compromise Pol II recruitment and transcription in yeast, mouse and human cells. 20,27,53,56,57,84,[110][111][112] A direct Mediator-Pol II interaction was shown to be generally required for Pol II recruitment and transcription. 21 Using in vivo photo-cross-linking strategy, this work identified Rpb3 Pol II subunit that crosslinked to Med17 Mediator head module subunit among the 80 pairwise Pol II-Mediator contacts tested. ...
... The best examples of such a role come from the Mediator studies in metazoan systems. 48,112,119,120 Mediator has been proposed to regulate promoter proximal pausing of Pol II, even though the exact mechanisms remain to be understood. For example, a model for Mediator function in Pol II transition to productive elongation has been proposed. ...
Mediator is a multisubunit complex conserved in eukaryotes that plays an essential coregulator role in RNA polymerase (Pol) II transcription. Despite intensive studies of the Mediator complex, the molecular mechanisms of its function in vivo remain to be fully defined. In this review, we will discuss the different aspects of Mediator function starting with its interactions with specific transcription factors, its recruitment to chromatin and how, as a coregulator, it contributes to the assembly of transcription machinery components within the preinitiation complex (PIC) in vivo and beyond the PIC formation.
... S1) (6)(7)(8)(9)(10)(11). Two MAP kinase-docking sites, the D-box and the Phe-Gln-Phe-Pro (FQFP) motif, control phosphorylation of these sites (12)(13)(14)(15). Multisite phosphorylation triggers transcriptional activation by TCFs, facilitating their interaction with the Mediator transcriptional co-activator complex (16)(17)(18)(19) but the kinetics with which the different sites are phosphorylated, and whether they serve distinct functions, remain unclear. ...
... Phosphorylation of Elk-1 promotes transcriptional activation by facilitating its MED23dependent interaction with the Mediator complex (16)(17)(18). We therefore investigated whether the different transcriptional activities of the Elk-1 mutants reflected alterations in Mediator binding. ...
A function for multisite phosphorylation
Many transcription factors are regulated by phosphorylation on multiple residues. Mylona et al. analyzed multisite phosphorylation in the transcription factor Elk-1 and showed that it may protect against excessive activation (see the Perspective by Whitmarsh and Davis). Phosphorylation by the kinase ERK2 occurred at eight sites, but the sites were phosphorylated at different rates. Those that were phosphorylated more quickly promoted transcriptional activation. Those that were phosphorylated more slowly dampened excessive activation by ERK2s without needing a phosphatase or any other negative regulatory component.
Science , this issue p. 233 ; see also p. 179
... Moreover, we established the transcriptome profile of DSACs using RNA-seq, which revealed significant upregulation of histone modification related genes, including H2BC4, H2BC5, H2AJ, H2AC6, H2BC12, H2BC2, H3C4, HDAC7 and HDAC10. Meanwhile, Mediator subnit, MED23, was included in the upregulated DEGs as well, which interacts with activators to stimulate transcription initiation [46]. Taken together, these findings may explain global chromatin-accessibility increasements observed in DSACs. ...
Background
Growing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying function of MX1, an effector gene of I-IFN, in DS-associated transcriptional regulation and phenotypic modulation.
Methods
We performed assay for transposase-accessible chromatin with high-throughout sequencing (ATAC-seq) to explore the difference of chromatin accessibility between DS derived amniocytes (DSACs) and controls. We then combined the annotated differentially expressed genes (DEGs) and enriched transcriptional factors (TFs) targeting the promoter region from ATAC-seq results with the DEGs in RNA-seq, to identify key genes and pathways involved in alterations of biological processes and pathways in DS.
Results
Binding motif analysis showed a significant increase in chromatin accessibility of genes related to neural cell function, among others, in DSACs, which is primarily regulated by members of the activator protein-1 (AP-1) transcriptional factor family. Further studies indicated that MX Dynamin Like GTPase 1 (MX1), defined as one of the key effector genes of I-IFN, is a critical upstream regulator. Its overexpression induced expression of AP-1 TFs and mediated inflammatory response, thus leading to decreased cellular viability of DS cells. Moreover, treatment with specific AP-1 inhibitor T-5224 improved DS-associated phenotypes in DSACs.
Conclusions
This study demonstrates that MX1-mediated AP-1 activation is partially responsible for cellular dysfunction of DS. T-5224 effectively ameliorated DS-associated phenotypes in DSACs, suggesting it as a potential treatment option for DS patients.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40659-023-00474-x.
... Certains facteurs d'élongation ont été identifiés comme des substrats du module kinase comme par exemple NELF (Knuesel et al., 2009;Tsai et al., 2013), et l'inhibition de CDK8 augmente la pause de la Pol II, suggérant son rôle anti-pause (Steinparzer et al., 2019). De plus, la déplétion de MED23 dans des cellules murines embryonnaires empêche son recrutement sur certaines promoteurs et empêche la Pol II de sortir de son état de pause (Wang et al., 2005). Dans un système in vitro dépourvu de P-TEFb, le Médiateur est également capable de lever l'inhibition transcriptionnelle médiée par DSIF (Malik et al., 2007), et c'est aussi le cas concernant l'inhibition médiée par Gdown1, une protéine inhibitrice de la transcription stimulant un état pausé de la Pol II (Jishage et al., 2012). ...
Dans les cellules eucaryotes, l'ADN est compacté sous forme de chromatine et tous les processus qui y sont liés y compris la transcription se déroulent dans ce contexte chromatinien. Le Médiateur de la régulation transcriptionnelle est un complexe multiprotéique essentiel et conservé qui intègre les signaux de régulation des facteurs de transcription et les transmet à la machinerie transcriptionnelle. Etant donné son rôle essentiel dans la transcription des gènes, des mutations qui l'affectent sont impliquées dans un large spectre de maladies comme le cancer. Bien que le Médiateur ait fait l'objet de nombreuses études, on ne sait pas comment il coordonne ses fonctions avec les autres corégulateurs transcriptionnels. Nous avons observé que plusieurs de ses sous-unités sont en contact avec le remodeleur de la chromatine RSC, très abondant dans la cellule et essentiel pour sa viabilité. L'importance de ce complexe est soulignée par la grande implication de son homologue humain PBAF dans les pathologies cancéreuses : 20 % des cancers portent une mutation dans au moins une sous-unité de cette famille de remodeleurs. Dans ce projet, nous avons étudié les mécanismes d'action du Médiateur en relation avec RSC dans la transcription. Nous avons caractérisé une interaction physique et fonctionnelle entre les deux complexes, en utilisant principalement la levure comme modèle d'étude. Ce projet représente un enjeu majeur dans le domaine de la transcription génique et nous permet de mieux comprendre ce processus cellulaire fondamental, impliqué dans l'établissement et le maintien de nombreuses maladies grave.
... Although it is well known that ERK1/2 phosphorylates a set of transcription factors, predominantly the ETS-related family members, the precise molecular mechanisms that lead to transcriptional induction have not been elucidated. Previous studies have implicated the transcriptional coactivators CBP/p300 or components of the Mediator complex in MAPK signaling (Janknecht and Nordheim 1996;Pandey et al. 2005;Wang et al. 2005;Jun et al. 2010;Galbraith et al. 2013). However, these studies are generally limited to the analysis of a single or a small number of MAPK-responsive genes in a specific cell line (Jun et al. 2010;Galbraith et al. 2013). ...
Activating mutations in the mitogen-activated protein kinase (MAPK) cascade, also known as the RAS-MEK-ex-tracellular signal-related kinase (ERK1/2) pathway, are an underlying cause of >70% of human cancers. While great strides have been made toward elucidating the cytoplasmic components of MAPK signaling, the key downstream coactivators that coordinate the transcriptional response have not been fully illustrated. Here, we demonstrate that the MAPK transcriptional response in human cells is funneled through Integrator, an RNA polymerase II-associated complex. Integrator depletion diminishes ERK1/2 transcriptional responsiveness and cellular growth in human cancers harboring activating mutations in MAPK signaling. Pharmacological inhibition of the MAPK pathway abrogates the stimulus-dependent recruitment of Integrator at immediate early genes and their enhancers. Following epidermal growth factor (EGF) stimulation, activated ERK1/2 is recruited to immediate early genes and phosphor-ylates INTS11, the catalytic subunit of Integrator. Importantly, in contrast to the broad effects of Integrator knockdown on MAPK responsiveness, depletion of a number of critical subunits of the coactivator complex Mediator alters only a few MAPK-responsive genes. Finally, human cancers with activating mutations in the MAPK cascade, rendered resistant to targeted therapies, display diminished growth following depletion of Integrator. We propose Integrator as a crucial transcriptional coactivator in MAPK signaling, which could serve as a downstream therapeutic target for cancer treatment.
... TF-Mediator binding coincides with conformational changes in the Mediator complex (FiG. 2a), which can be observed even at low resolution 13,33 , and TF-Mediator binding correlates with changes in Pol II activity 41,97 . Consequently, TF-directed structural shifts in Mediator may contribute to Mediator-dependent activation of Pol II transcription. ...
The Mediator complex, which in humans is 1.4 MDa in size and includes 26 subunits, controls many aspects of RNA polymerase II (Pol II) function. Apart from its size, a defining feature of Mediator is its intrinsic disorder and conformational flexibility, which contributes to its ability to undergo phase separation and to interact with a myriad of regulatory factors. In this Review, we discuss Mediator structure and function, with emphasis on recent cryogenic electron microscopy data of the 4.0-MDa transcription preinitiation complex. We further discuss how Mediator and sequence-specific DNA-binding transcription factors enable enhancer-dependent regulation of Pol II function at distal gene promoters, through the formation of molecular condensates (or transcription hubs) and chromatin loops. Mediator regulation of Pol II reinitiation is also discussed, in the context of transcription bursting. We propose a working model for Mediator function that combines experimental results and theoretical considerations related to enhancer–promoter interactions, which reconciles contradictory data regarding whether enhancer–promoter communication is direct or indirect. We conclude with a discussion of Mediator’s potential as a therapeutic target and of future research directions. The Mediator complex is an important regulator of RNA polymerase II. This Review discusses recent structural insights into Mediator function and proposes a model that reconciles contradictory data on whether enhancer–promoter communication during transcription is direct or indirect.
... A total of 1 × 10 7 cells were prepared for the ChIP assay. The ChIP protocol was performed following the methods reported 56 . Quantification of all ChIP samples was performed by quantitative PCR (qPCR) using a TaKaRa SYBR Premix Ex Taq kit and an ABI 7500 Fast system. ...
Cancer stemness represents a major source of development and progression of colorectal cancer (CRC). c-Met critically contributes to CRC stemness, but how c-Met is activated in CRC remains elusive. We previously identified the lipolytic factor ABHD5 as an important tumour suppressor gene in CRC. Here, we show that loss of ABHD5 promotes c-Met activation to sustain CRC stemness in a non-canonical manner. Mechanistically, we demonstrate that ABHD5 interacts in the cytoplasm with the core subunit of the SET1A methyltransferase complex, DPY30, thereby inhibiting the nuclear translocation of DPY30 and activity of SET1A. In the absence of ABHD5, DPY30 translocates to the nucleus and supports SET1A-mediated methylation of YAP and histone H3, which sequesters YAP in the nucleus and increases chromatin accessibility to synergistically promote YAP-induced transcription of c-Met, thus promoting the stemness of CRC cells. This study reveals a novel role of ABHD5 in regulating histone/non-histone methylation and CRC stemness.
... The rest of the two subunits are separated into a Y-shaped base at the bottom of the Saddle. Thus, MED23 and MED24 form a rigid framework of the Tail, in line with their critical roles in Mediator complex assembly (53,54). ...
A complete PIC-Mediator structure
As a critical transcription coactivator, the multisubunit Mediator complex binds RNA polymerase II (Pol II), facilitates preinitiation complex (PIC) assembly, and stimulates transcription and phosphorylation of the Pol II C-terminal domain (CTD). However, how these critical transcriptional events are coordinated by Mediator is not fully understood. Chen et al. determined the structures of human Mediator and Mediator-bound PIC in distinct conformational states, the latter of which represents a complete PIC-Mediator complex assembled on the 14-subunit transcription factor IID (TFIID). The structures show that Mediator undergoes reorganization during PIC-Mediator assembly, sandwiches and facilitates phosphorylation of Pol II CTD, and works with TFIID to organize TFIIH in PIC for transcription initiation.
Science , abg0635, this issue p. eabg0635
... Med23 has been shown to be important for gene expression and tissue development in C. elegans, Drosophila, zebrafish and mouse embryos (Singh and Han, 1995;Stevens et al., 2002;Kim and Lis, 2005;Wang et al., 2005Wang et al., , 2009Yin and Wang, 2014;Liu et al., 2016), and interestingly, mutations in MED23 in humans have been linked to intellectual disability, microcephaly and cardiovascular anomalies together with overall growth retardation defects (Lüneburg et al., 2014;Lionel et al., 2016;Hashemi-Gorji et al., 2019). Our work on Med23 sn/sn embryos has uncovered a previously unexplored role for Med23-containing Mediator in craniofacial and cranial ganglia development and it is tempting to speculate that a similar effect on WNT signaling and neuronal development may contribute to the pathogenesis of the neurological phenotypes observed in humans. ...
Development of the vertebrate head is a complex and dynamic process, which requires integration of all three germ layers and their derivatives. Of special importance are ectoderm-derived cells that form the cranial placodes, which then differentiate into the cranial ganglia and sensory organs. Critical to a fully functioning head, defects in cranial placode and sensory organ development can result in congenital craniofacial anomalies. In a forward genetic screen aimed at identifying novel regulators of craniofacial development, we discovered an embryonically lethal mouse mutant, snouty, which exhibits malformation of the facial prominences, cranial nerves and vasculature. The snouty mutation was mapped to a single nucleotide change in a ubiquitously expressed gene, Med23, which encodes a subunit of the global transcription co-factor complex, Mediator. Phenotypic analyses revealed that the craniofacial anomalies, particularly of the cranial ganglia, were caused by a failure in the proper specification of cranial placode neuronal precursors. Molecular analyses determined that defects in cranial placode neuronal differentiation in Med23sn/sn mutants were associated with elevated WNT/β-catenin signaling, which can be partially rescued through combined Lrp6 and Wise loss-of-function. Our work therefore reveals a surprisingly tissue specific role for the ubiquitously expressed mediator complex protein Med23 in placode differentiation during cranial ganglia development. This highlights the importance of coupling general transcription to the regulation of WNT signaling during embryogenesis.
... In this line, it has been shown that mediator helps in the recruitment of RNA Pol II (Asturias et al., 1999;Bernecky et al., 2011;Davis et al., 2002) and facilitates the assembly of the pre-initiation complex (Baek et al., 2006;Baek et al., 2002;Esnault et al., 2008;Jiang et al., 1998). Besides, Mediator has been implicated in the pausing and release of RNA Pol II and the elongation of transcription (Kremer et al., 2012;Malik et al., 2002;Meyer et al., 2010;Wang et al., 2005) and, through its interaction with the histone acetyltransferase p300, it is thought to induce transcription through the regulation of the chromatin state (Acevedo and Kraus, 2003). Moreover, mediator has a pivotal role in loop formation and maintenance. ...
Immunoglobulin (Ig) class switch recombination (CSR) takes place at the immunoglobulin heavy chain (IgH) constant locus upon B cell activation and results in a change of the isotype expressed. CSR is triggered by activation-induced cytidine deaminase (AID) and is dependent on inducible long-rangeenhancer/promoter looping and on germline transcription, which are controlled by the Eμ enhancer and the 3' regulatory region (3'RR) super-enhancer. Here, we characterize the role on switch transcription and recombination of g1E, a region located downstream of the Cg1 gene that bears marksof active enhancers and that interacts dynamically with both IgH enhancers upon B cell activation. We show that g1E deletion reduces CSR efficiency to IgA in CH12 cells and affects germline transcription and CSR in an isotype-specific manner in mice. On the other hand, whether transcription precedes orfollows looping to induce CSR is still unknown. To address this question, we targeted a transcriptional induction system based on the CRISPR/Cas9 technology to the Cg1 promoter in a background deficient for transcription and looping to study whether CSR could be restored.
... A representative experiment is shown. Petesch and Lis, 2012), a subunit of the mediator complex, MED23, which has been associated with Pol II transcriptional pause release depending on BRD4 (Wang et al., 2005;Lu et al., 2016), AFF4 and ELL2, which are component of the multi-subunit SEC He et al., 2010;Lu et al., 2016), and the Pol II associated factor 1 (PAF1), which is a component of the PAF1 multi-subunit complex (PAF1C) (Van Oss et al., 2017). Strikingly, all of these factors exhibit increased chromatin levels in SIRT6 KO compared to WT ESCs ( Figure 3H). ...
Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.
... Latent, nuclear ELK-1 acquires activity upon phosphorylation by MAPKs and deSUMOylation by PIASx (15)(16)(17)(18). Target gene activation by phospho-ELK-1 involves recruitment of active ERK to chromatin (19), phosphorylation of mediator subunits including MED14 (20) and, uniquely for ELK-1 among the TCFs, functional reliance on MED23 (21,22). In proliferating human ES cells (hESCs), ELK-1 also locates to the promoters of differentiation genes independently of ERK and is associated with their repression (2). ...
ELK-1 is a transcription factor involved in ERK-induced cellular proliferation. Here, we show that its transcriptional activity is modulated by ubiquitination at lysine 35 (K35). The level of ubiquitinated ELK-1 rises in mitogen-deprived cells and falls upon mitogen stimulation or oncogene expression. Ectopic expression of USP17, a cell cycle-dependent deubiquitinase, decreases ELK-1 ubiquitination and up-regulates ELK-1 target-genes with a concomitant increase in cyclin D1 expression. In contrast, USP17 depletion attenuates ELK-1-dependent gene expression and slows cell proliferation. The reduced rate of proliferation upon USP17 depletion appears to be a direct effect of ELK-1 ubiquitination because it is rescued by an ELK-1(K35R) mutant refractory to ubiquitination. Overall, our results show that ubiquitination of ELK-1 at K35, and its reversal by USP17, are important mechanisms in the regulation of nuclear ERK signalling and cellular proliferation. Our findings will be relevant for tumours that exhibit elevated USP17 expression and suggest a new target for intervention.
... As a key subunit of the transcriptional mediator complex, Med23 (also known as Sur2) plays an important role in orchestrating the transcriptional profiles in various cell types [10][11][12][13][14][15] . It has been reported that Med23 regulates the mitogen-activated protein kinase signaling pathway through mediating the responses of several immediate early genes (IEG) to serum mitogens 16,17 . Moreover, Med23 has important roles in diverse biological processes including adipogenesis, brain development, cell differentiation and carcinogenesis 12,14,18,19 . ...
In response to myeloablative stresses, HSCs are rapidly activated to replenish myeloid progenitors, while maintaining full potential of self-renewal to ensure life-long hematopoiesis. However, the key factors that orchestrate HSC activities during physiological stresses remain largely unknown. Here we report that Med23 controls the myeloid potential of activated HSCs. Ablation of Med23 in hematopoietic system leads to lymphocytopenia. Med23-deficient HSCs undergo myeloid-biased differentiation and lose the self-renewal capacity. Interestingly, Med23-deficient HSCs are much easier to be activated in response to physiological stresses. Mechanistically, Med23 plays essential roles in maintaining stemness genes expression and suppressing myeloid lineage genes expression. Med23 is downregulated in HSCs and Med23 deletion results in better survival under myeloablative stress. Altogether, our findings identify Med23 as a gatekeeper of myeloid potential of HSCs, thus providing unique insights into the relationship among Med23-mediated transcriptional regulations, the myeloid potential of HSCs and HSC activation upon stresses.
... Although it is well known that ERK1/2 phosphorylates a set of transcription factors, predominantly the ETS-related family members, the precise molecular mechanisms that lead to transcriptional induction have not been elucidated. Previous studies have implicated the transcriptional coactivators CBP/p300 or components of the Mediator complex in MAPK signaling (Janknecht and Nordheim 1996;Pandey et al. 2005;Wang et al. 2005;Jun et al. 2010;Galbraith et al. 2013). However, these studies are generally limited to the analysis of a single or a small number of MAPK-responsive genes in a specific cell line (Jun et al. 2010;Galbraith et al. 2013). ...
Activating mutations in the mitogen-activated protein kinase (MAPK) cascade, also known as the RAS–MEK–ex-tracellular signal-related kinase (ERK1/2) pathway, are an underlying cause of >70% of human cancers. While great strides have been made toward elucidating the cytoplasmic components of MAPK signaling, the key downstream coactivators that coordinate the transcriptional response have not been fully illustrated. Here, we demonstrate that the MAPK transcriptional response in human cells is funneled through Integrator, an RNA polymerase II-associated complex. Integrator depletion diminishes ERK1/2 transcriptional responsiveness and cellular growth in human cancers harboring activating mutations in MAPK signaling. Pharmacological inhibition of the MAPK pathway abrogates the stimulus-dependent recruitment of Integrator at immediate early genes and their enhancers. Following epidermal growth factor (EGF) stimulation, activated ERK1/2 is recruited to immediate early genes and phosphor-ylates INTS11, the catalytic subunit of Integrator. Importantly, in contrast to the broad effects of Integrator knockdown on MAPK responsiveness, depletion of a number of critical subunits of the coactivator complex Mediator alters only a few MAPK-responsive genes. Finally, human cancers with activating mutations in the MAPK cascade, rendered resistant to targeted therapies, display diminished growth following depletion of Integrator. We propose Integrator as a crucial transcriptional coactivator in MAPK signaling, which could serve as a downstream therapeutic target for cancer treatment.
... Depends on the cellular context the mediator complex shuttle between two different states: The Mediator core complex and Mediator core-kinase complex in which the former favors transcription (the mediator core associates with RNAP II) and the latter represses transcription (the kinase module-bound Mediator complex dissociates from RNAP II). The first studies on the mediator suggested that it is mainly linked to the initiation step of transcription [205,206], however, later on the mediator has be linked to different step in the transcription [207][208][209][210][211]. Specific functions in signaling pathways which are important during development and in response to biotic and abiotic stress have been reported for several plant mediator complex subunits [212][213][214][215][216][217]. For instance, MED21 has been proposed to function in defense against necrotrophic pathogens through interaction with HUB1 [218]. ...
In plants, different stimuli, both internal and external, activate production of reactive oxygen species (ROS). Photosynthesis is considered as high rate redox-metabolic process with rapid transients including light/photon capture, electron fluxes, and redox potentials that can generate ROS; thus, regulatory systems are required to minimize ROS production. Despite their potential for causing harmful oxidations, it is now accepted that redox homeostasis mechanisms that maintain the intracellular reducing environment make it possible to use ROS as powerful signaling molecules within and between cells. Redox and ROS information from the chloroplasts is a fine-tuning mechanism both inside the chloroplast and as retrograde signal to the cytosol and nucleus to control processes such as gene expression/transcription and translation. Wide repertoires of downstream target genes expression (activation/repression) is regulated by transcription factors. In many cases, transcription factors function through various mechanisms that affect their subcellular localization and or activity. Some post-translational modifications (PTMs) known to regulate the functional state of transcription factors are phosphorylation, acetylation, and SUMOylation, ubiquitylation and disulfide formation. Recently, oxPTMs, targeted in redox proteomics, can provide the bases to study redox regulation of low abundant nuclear proteins. This review summarizes the recent advances on how cellular redox status can regulate transcription factor activity, the implications of this regulation for plant growth and development, and by which plants respond to environmental/abiotic stresses.
... By using a MNP assay, we found that the EGR1 site in the promoter of the gene is covered by nucleosome N À 2. Nevertheless, as shown in Fig. 4, soon after TPA induction a remodelling event results in the downstream sliding of N À 2, which allows the accessibility of the product of the gene to its target sequence, thus initiating the events that eventually led to the repression of the gene (Riffo-Campos et al. 2015). Nucleosome N À 1 is also remodelled upon transcription, and results partially evicted (Fig. 4), probably to facilitate the assembly of the mediator complex, that binds the Egr1 promoter between the TSS and À400 (Wang et al. 2005). ...
The organisation of chromatin is first discussed to conclude that nucleosomes play both structural and transcription-regulatory roles. The presence of nucleosomes makes difficult the access of transcriptional factors to their target sequences and the action of RNA polymerases. The histone post-translational modifications and nucleosome remodelling are first discussed, from a historical point of view, as mechanisms to remove the obstacles imposed by chromatin structure to transcription. Instead of reviewing the state of the art of the whole field, this review is centred on some open questions. First, some "non-classical" histone modifications, such as short-chain acylations other than acetylation, are considered to conclude that their relationship with the concentration of metabolic intermediaries might make of them a sensor of the physiological state of the cells. Then attention is paid to the interest of studying chromatin organisation and epigenetic marks at a single nucleosome level as a complement to genome-wide approaches. Finally, as a consequence of the above questions, the review focuses on the presence of multiple histone post-translational modifications on a single nucleosome. The methods to detect them and their meaning, with special emphasis on bivalent marks, are discussed.
... Finally, all Mediator gene knockout mice attempted to date showed embryonic lethality (reviewed in Yin and Wang, 2014), highlighting the key role of Mediator in gene expression in mammals. Interestingly, however, embryonic stem cells or fibroblasts derived from Tail subunit knockout embryos do generally survive (Ito et al., 2002;Stevens et al., 2002;Wang et al., 2005), while those from Head or Middle subunit knockouts either die (Tudor et al., 1999) or show severe proliferation defects (Risley et al., 2010). These data suggest that, like in yeast, the Head and Middle domains in higher eukaryotes are ubiquitously required for gene expression while the Tail domain plays more specialized regulatory roles, notably in the establishment of developmental programs. ...
Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head, and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked, allowing detection of transient Mediator forms. We found that although all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, although Tail is required for Mediator recruitment to UAS, Tailless Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles.
... However, the contribution of each subunit to the function of the complex remains largely unexplored. Mediator plays a role in various fundamental processes, such as transcription initiation (Wang et al., 2005), pause release (Galli et al., 2015), elongation (Takahashi et al., 2011), and chromatin (legend continued on next page) architecture (Kagey et al., 2010;Ørom et al., 2010). The kinase module reversibly interacts with the Mediator core through MED13, causing a structural shift and further modulation of its function (Davis et al., 2013;Knuesel et al., 2009a). ...
... However, the contribution of each subunit to the function of the complex remains largely unexplored. Mediator plays a role in various fundamental processes, such as transcription initiation (Wang et al., 2005), pause release (Galli et al., 2015), elongation (Takahashi et al., 2011), and chromatin (legend continued on next page) architecture (Kagey et al., 2010;Ørom et al., 2010). The kinase module reversibly interacts with the Mediator core through MED13, causing a structural shift and further modulation of its function (Davis et al., 2013;Knuesel et al., 2009a). ...
Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.
... PARP1 in FOS regulation functions through directly enhancing ERK-mediated ELK1 phosphorylation (Cohen-Armon et al., 2007). Once the chromatin remodeling (modification) steps are completed, Mediator can be added by undergoing a phosphorylation-dependent interaction with ELK1, and finally RNA polymerase activity can increase at the FOS promoter (Wang et al., 2005). This has been shown for the EGR1 promoter, and it has been indicated that the process for the FOS promoter is similar (O'Donnell et al., 2012). ...
... PARP1 in FOS regulation functions through directly enhancing ERK-mediated ELK1 phosphorylation (Cohen-Armon et al., 2007). Once the chromatin remodeling (modification) steps are completed, Mediator can be added by undergoing a phosphorylation-dependent interaction with ELK1, and finally RNA polymerase activity can increase at the FOS promoter (Wang et al., 2005). This has been shown for the EGR1 promoter, and it has been indicated that the process for the FOS promoter is similar (O'Donnell et al., 2012). ...
... ChIP analysis was performed as previously described [52]. Briefly, EpH4 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. ...
Cyclin Y family can enhance Wnt/β-catenin signaling in mitosis. Their physiological roles in mammalian development are yet unknown. Here we show that Cyclin Y-like 1 (Ccnyl1) and Cyclin Y (Ccny) have overlapping function and are crucial for mouse embryonic development and mammary stem/progenitor cell functions. Double knockout of Ccnys results in embryonic lethality at E16.5. In pubertal development, mammary terminal end buds robustly express Ccnyl1. Depletion of Ccnys leads to reduction of Lrp6 phosphorylation, hampering β-catenin activities and abolishing mammary stem/progenitor cell expansion in vitro. In lineage tracing experiments, Ccnys-deficient mammary cells lose their competitiveness and cease to contribute to mammary development. In transplantation assays, Ccnys-deficient mammary cells fail to reconstitute, whereas constitutively active β-catenin restores their regeneration abilities. Together, our results demonstrate the physiological significance of Ccnys-mediated mitotic Wnt signaling in embryonic development and mammary stem/progenitor cells, and reveal insights in the molecular mechanisms orchestrating cell cycle progression and maintenance of stem cell properties.
... ChIP analysis was performed as previously described [52]. Briefly, EpH4 cells were cross-linked with 1% formaldehyde for 10 min at room temperature. ...
... Once the chromatin remodeling (modification) steps are completed, Mediator can be added by undergoing a phosphorylation-dependent interaction with ELK1, and finally RNA polymerase activity can increase at the FOS promoter (Wang et al., 2005). This has been shown for the EGR1 promoter, and it has been indicated that the process for the FOS promoter is similar (O'Donnell et al., 2012). ...
Immediate-early genes (IEGs) can be activated and transcribed within minutes after stimulation, without the need for de novo protein synthesis, and they are stimulated in response to both cell-extrinsic and cell-intrinsic signals. Extracellular signals are transduced from the cell surface, through receptors activating a chain of proteins in the cell, in particular extracellular-signal-regulated kinases (ERKs), mitogen-activated protein kinases (MAPKs) and members of the RhoA-actin pathway. These communicate through a signaling cascade by adding phosphate groups to neighboring proteins, and this will eventually activate and translocate TFs to the nucleus and thereby induce gene expression. The gene activation also involves proximal and distal enhancers that interact with promoters to simulate gene expression. The immediate-early genes have essential biological roles, in particular in stress response, like the immune system, and in differentiation. Therefore they also have important roles in various diseases, including cancer development. In this paper we summarize some recent advances on key aspects of the activation and regulation of immediate-early genes.
... The Mediator complex encompassing more than 25 subunits in eukaryotes, acts as an interface between transcriptional activators/repressors and RNA pol II and thus can have both inducing as well as repressing effect on transcription [17][18][19] . The role of these Mediator subunits in transcription has been established in the formation of pre-initiation complex, transcription initiation and elongation, splicing, gene looping and transcription termination [19][20][21][22][23][24][25][26][27][28][29][30] . Abundance of intrinsic disorder regions (IDRs) in MED subunits and the modular arrangement consisting of Head, Middle, Tail and Kinase modules makes Mediator a versatile regulator, as it can interact with various factors and attain diverse configurations for regulating multiple processes in eukaryotes 18,31,32 . ...
The present study deployed a Mediator (MED) genes-mediated integrated genomic strategy for understanding the complex genetic architecture of grain size/weight quantitative trait in rice. The targeted multiplex amplicon resequencing of 55 MED genes annotated from whole rice genome in 384 accessions discovered 3971 SNPs, which were structurally and functionally annotated in diverse coding and non-coding sequence-components of genes. Association analysis, using the genotyping information of 3971 SNPs in a structured population of 384 accessions (with 50–100 kb linkage disequilibrium decay), detected 10 MED gene-derived SNPs significantly associated (46% combined phenotypic variation explained) with grain length, width and weight in rice. Of these, one strong grain weight-associated non-synonymous SNP (G/A)-carrying OsMED4_2 gene was validated successfully in low- and high-grain weight parental accessions and homozygous individuals of a rice mapping population. The seed-specific expression, including differential up/down-regulation of three grain size/weight-associated MED genes (including OsMED4_2) in six low and high-grain weight rice accessions was evident. Altogether, combinatorial genomic approach involving haplotype-based association analysis delineated diverse functionally relevant natural SNP-allelic variants in 10 MED genes, including three potential novel SNP haplotypes in an OsMED4_2 gene governing grain size/weight differentiation in rice. These molecular tags have potential to accelerate genomics-assisted crop improvement in rice.
... MED23 is a subunit of the mediator complex, a key regulator of gene expression, and is required for Sp1 and ELK1-dependent transcriptional activation in response to activated Ras signaling. [24][25][26][27] MED1 and MED17 directly interact with p53, 27 suggesting a possible connection between p53 and MED23 via the mediator complex. Also, MED23 interacts directly with the transcription factor ESX/ELF3, 27 which is downstream of AKT1 in the PI3K pathway. ...
The central challenges in tumor sequencing studies is to identify driver genes and pathways, investigate their functional relationships, and nominate drug targets. The efficiency of these analyses, particularly for infrequently mutated genes, is compromised when subjects carry different combinations of driver mutations. Mutual exclusivity analysis helps address these challenges. To identify mutually exclusive gene sets (MEGS), we developed a powerful and flexible analytic framework based on a likelihood ratio test and a model selection procedure. Extensive simulations demonstrated that our method outperformed existing methods for both statistical power and the capability of identifying the exact MEGS, particularly for highly imbalanced MEGS. Our method can be used for de novo discovery, for pathway-guided searches, or for expanding established small MEGS. We applied our method to the whole-exome sequencing data for 13 cancer types from The Cancer Genome Atlas (TCGA). We identified multiple previously unreported non-pairwise MEGS in multiple cancer types. For acute myeloid leukemia, we identified a MEGS with five genes (FLT3, IDH2, NRAS, KIT, and TP53) and a MEGS (NPM1, TP53, and RUNX1) whose mutation status was strongly associated with survival (p = 6.7 × 10(-4)). For breast cancer, we identified a significant MEGS consisting of TP53 and four infrequently mutated genes (ARID1A, AKT1, MED23, and TBL1XR1), providing support for their role as cancer drivers.
... In the beginning, Mediator complex was thought to be involved only in the initiation step of transcription as evident by its interaction with the components of the transcription initiation complex (Mittler et al., 2001;Baek et al., 2002;Cantin et al., 2003;Johnson and Carey, 2003;Wang et al., 2005). However, in last few years, Mediator has been reported to be involved in the regulation of many other steps of transcription like promoter escape (Malik et al., 2007;Cheng et al., 2012;Jishage et al., 2012), elongation (Takahashi et al., 2011;Conaway and Conaway, 2013;Galbraith et al., 2013), termination Ansari, 2011, 2013), as well as in other co-transcriptional RNA processing events (Kim et al., 2011;Huang et al., 2012;Oya et al., 2013). ...
Class II gene loci of eukaryotes are transcribed by RNA Polymerase II, which functions in coordination with several other proteins like transcription factors, general transcription factors, and cofactors. Recently, Mediator complex, a multi-subunit, megadalton size protein complex has gained lots of attention as an important component of RNA pol II transcriptional machinery because of its essentiality in the regulation of most of the class II genes. Like yeast and other metazoans, plants also possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Recent times have experienced a fl urry of scientifi c papers containing the functional information of individual Mediator subunits in plants, although many were reported earlier without consideration of their association with the Mediator complex. Among its diverse functional aspects, several reports have established the Mediator complex as an important integrative hub of different biotic and abiotic stress signaling pathways, which have been discussed in this chapter from the functional genomics perspectives. Although reports are emerging in support of its inclusion as a component of the basic transcriptional machinery, the gene selective roles of the individual Mediator subunits are proven and indisputably accepted.
... In the beginning, Mediator complex was thought to be involved only in the initiation step of transcription as evident by its interaction with the components of the transcription initiation complex (Mittler et al., 2001;Baek et al., 2002;Cantin et al., 2003;Johnson and Carey, 2003;Wang et al., 2005). However, in last few years, Mediator has been reported to be involved in the regulation of many other steps of transcription like promoter escape (Malik et al., 2007;Cheng et al., 2012;Jishage et al., 2012), elongation (Takahashi et al., 2011;Conaway and Conaway, 2013;Galbraith et al., 2013), termination Ansari, 2011, 2013), as well as in other co-transcriptional RNA processing events (Kim et al., 2011;Huang et al., 2012;Oya et al., 2013). ...
Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes.
... Though of these six transcripts, three encode transcription factors (Crsp3/ Med23) or co-activators (Sertad3, and Zfp592) and were significantly down-regulated in AKAP13-ΔPKD1 hearts. The protein encoded by Crsp3/Med23 gene is involved in the regulated transcription of nearly all RNA polymerase II-dependent genes [23][24][25], and Sertad3 functions as an activator of cell growth [26]. These genes have not been previously identified as down-stream targets of PKD1 though evidence presented here suggests that AKAP13-mediated PKD1 anchoring is necessary for the basal expression of these genes and may have a role in homeostatic transcriptional regulation. ...
In the heart, scaffolding proteins such as A-Kinase Anchoring Proteins (AKAPs) play a crucial role in normal cellular function by serving as a signaling hub for multiple protein kinases including protein kinase D1 (PKD1). Under cardiac hypertrophic conditions AKAP13 anchored PKD1 activates the transcription factor MEF2 leading to subsequent fetal gene activation and hypertrophic response. We used an expression microarray to identify the global transcriptional response in the hearts of wild-type mice expressing the native form of AKAP13 compared to a gene-trap mouse model expressing a truncated form of AKAP13 that is unable to bind PKD1 (AKAP13-ΔPKD1). Microarray analysis showed that AKAP13-ΔPKD1 mice broadly failed to exhibit the transcriptional profile normally associated with compensatory cardiac hypertrophy following trans-aortic constriction (TAC). The identified differentially expressed genes in WT and AKAP13-ΔPKD1 hearts are vital for the compensatory hypertrophic response to pressure-overload and include myofilament, apoptotic, and cell growth/differentiation genes in addition to genes not previously identified as affected by AKAP13-anchored PKD1. Our results show that AKAP13-PKD1 signaling is critical for transcriptional regulation of key contractile, cell death, and metabolic pathways during the development of compensatory hypertrophy in vivo.
... We therefore developed an RNF20/40 ChIP assay to analyze binding of RNF20/40 to the MED23-targeted Egr1 gene locus using a mixture of antibodies specific to RNF20 and RNF40. As indicated in Fig 2I, MED23 deficiency reduced the recruitment of RNF20/40 by approximately threefold at the Egr1 promoter region, which coincided with our previous finding that MED23 deficiency reduces Mediator recruitment to a similar degree at the Egr1 gene promoter (Wang et al, 2005). ...
The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation.
© 2015 The Authors.
... Since specific MED subunits are associated with signal-activated transcription factors and RNA polymerase II (Pol II) [20] and function as conduits and integrators for channeling different signaling pathways, such as, the nuclear hormone receptor pathway (via MED1) [21], the TGF-β-signaling pathway (via MED12 or MED15) [22,23], the Wnt-signaling pathway (via MED12) [24], and the Ras-MAPK signaling pathway (via MED23) [25][26][27]. It was proposed that these signaling pathways can induce EMT [28][29][30][31][32], which is known to be associated with the metastasis, invasion, proliferation, and chemoresistance of epithelial cancer [29,33,34]. ...
MED30 is an essential member of the mediator complex that forms a hub between transcriptional activators and RNA polymerase II. However, the expressions and roles of MED30 have been poorly characterized in cancer. In this study, we examined the functional roles of MED30 during gastric cancer progression. It was found that MED30 was overexpressed in gastric cancer tissues and cell lines. Moreover, MED30 overexpression increased the proliferation, migration, and invasion of gastric cancer cells, whereas MED30 knockdown inhibited these effects. Furthermore the knockdown significantly inhibited tumorigenicity in SCID mice. MED30 also promoted the expressions of genes related to epithelial-mesenchymal transition and induced a fibroblast-like morphology. This study shows MED30 has pathophysiological roles in the proliferation, migration, and invasion of gastric cancer cells and suggests that MED30 should be viewed as a potent therapeutic target for malignant gastric carcinoma.
... In metazoans, the Pol II enzyme can pause after transcribing 30-60 nucleotides 87,88 ; these 'paused' Pol II complexes are widely observed at promoter-proximal sites throughout the genome, which establishes paused Pol II complexes as common regulatory intermediates [89][90][91] . Although Mediator appears to regulate Pol II pausing and/or pause release, the molecular mechanisms remain unclear 41,[92][93][94] . For example, it is not known whether Mediator remains bound to paused Pol II or whether paused Pol II complexes dissociate from Mediator (FIG. ...
The RNA polymerase II (Pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator - a large, conformationally flexible protein complex with a variable subunit composition (for example, a four-subunit cyclin-dependent kinase 8 module can reversibly associate with it). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes that are important for transcription, including the organization of chromatin architecture and the regulation of Pol II pre-initiation, initiation, re-initiation, pausing and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions seem to be specific to metazoans, which is indicative of more diverse regulatory requirements.
Introduction:
We previously reported a phenomenon called exercise hypertrophic preconditioning (EHP), the underlying mechanisms of which need further clarification.
Objectives:
We aimed to investigate whether circular RNAs (circRNAs) are involved in EHP.
Methods:
CircRNA sequencing of myocardial tissue was performed in male C57BL/6 mice with EHP and sedentary. Bioinformatics analysis and Sanger sequencing were used to screen hub circRNA expression and to detect full-length circRNAs, respectively. Loss-of-function analyses were conducted to assess the effects of circ-Ddx60 (c-Ddx) on EHP. After 21 days of swimming training or resting, mice underwent transverse aortic constriction (TAC) or sham surgery. Echocardiography, invasive hemodynamic measurement and histological analysis were used to evaluate cardiac remodeling and function. The presence of interaction between c-Ddx and proteins was investigated using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS).
Results:
In this study, we identified a novel circRNA, named c-Ddx that was preferentially expressed in myocardial tissue and significantly up-regulated in EHP mice. Silencing of c-Ddx attenuated the antihypertrophic effect of EHP and worsened heart failure in mice that underwent TAC. ChIRP-MS and molecular docking analysis validated the combination of c-Ddx and eukaryotic elongation factor 2 (eEF2). Mechanistically, c-Ddx silencing inhibited the increase of phosphorylation of eEF2 and its upstream AMP-activated protein kinase (AMPK) induced by EHP.
Conclusions:
C-Ddx contributes to the antihypertrophic memory of EHP by binding and activating eEF2, which would provide opportunity to search new therapeutic targets for pathological hypertrophy of heart.
Turning non-inflamed (cold) tumors into inflamed (hot) tumors is important for maximizing the effect of immune checkpoint inhibitors (ICIs) against malignancies. We showed that lactate, a product of the Warburg effect, inhibited the efficacy of ICIs and suppressed IL-12 p40 expression in dendritic cells (DCs) through reducing NF-κB p65, p50 and c-Rel DNA-binding activity to the IL-12 p40 promoter. Additionally, lactate promoted the expression of early growth response protein 1 (EGR1), whose expression was increased in human invasive melanoma compared with non-invasive melanoma. We also found that EGR1 interacts with serum response factor (SRF) and represses the expression of CD80 in DCs. These findings suggest that lactate and its induced EGR1 are key factors that turn hot tumors into cold tumors and may represent targets in cancer treatment with ICIs.
Although we have made significant progress, we still possess a limited understanding of how genomic and epigenomic information directs gene expression programs through sequence-specific transcription factors (TFs). Extensive research has settled on three general classes of TF targets in metazoans: promoter accessibility via chromatin regulation (e.g., SAGA), assembly of the general transcription factors on promoter DNA (e.g., TFIID) , and recruitment of RNA polymerase (Pol) II (e.g., Mediator) to establish transcription pre-initiation complex (PIC). Here we discuss TFs and their targets. We also place this in the context of our current work with Saccharomyces (yeast), where we find that promoters typically lack an architecture that supports TF function. Moreover, yeast promoters that support TF binding also display interactions with cofactors like SAGA and Mediator, but not TFIID. It is unknown to what extent all genes in metazoans require TFs and their cofactors.
Cyclin-dependent kinase 8 (CDK8) plays an momentous role in transcription regulation by forming kinase module or transcription factor phosphorylation. A large number of evidences have identified CDK8 as an important factor in cancer occurrence and development. In addition, CDK8 also participates in the regulation of cancer cell stress response to radiotherapy and chemotherapy, assists tumor cell invasion, metastasis, and drug resistance. Therefore, CDK8 is regarded as a promising target for cancer therapy. Most studies in recent years supported the role of CDK8 as a carcinogen, however, under certain conditions, CDK8 exists as a tumor suppressor. The functional diversity of CDK8 and its exceptional role in different types of cancer have aroused great interest from scientists but even more controversy during the discovery of CDK8 inhibitors. In addition, CDK8 appears to be an effective target for inflammation diseases and immune system disorders. Therefore, we summarized the research results of CDK8, involving physiological/pathogenic mechanisms and the development status of compounds targeting CDK8, provide a reference for the feasibility evaluation of CDK8 as a therapeutic target, and guidance for researchers who are involved in this field for the first time.
The synthesis of messenger RNA is a highly regulated process. During transcription initiation, a large number of proteins are recruited to gene promoter, including the RNA polymerase II, general transcription factors, co-activators, chromatin remodellers and the Mediator complex. Some DNA repair factors from the NER pathway are also recruited. Using cells derived from patients bearing mutations in either MED12 gene or XPC gene, we studied the roles of such proteins in transcription. MED12 patients are mostly characterised by intellectual disability and developmental delay. We showed that MED12 is implicated in the transcription regulation of immediate early genes like JUN, known for its role in neurological development and neuronal plasticity. JUN expression is markedly altered by MED12 mutations. We also showed that the position of the mutation influences this alteration, bringing possible explanation for inter-patients symptom variability. Meanwhile, XPC patients are mostly characterized by photosensitivity. We showed that XPC protein, which engages one of the NER pathways, is implicated in chromatin post-translational modification. Together with E2F1, it helps the recruitment of GCN5 acetyl-transferase to promoter of a certain set of genes. On the promoter, GCN5 notably cooperates with TFIIH to modify the chromatin environment during transcription initiation. In addition to help the comprehension of the transcription mechanisms, these results bring knew insight into the aetiology of mutations associated diseases.
In this review, we attempt to discuss emerging themes in the regulation of neuronal activity-regulated genes, focusing primarily on an important subset of immediate-early genes. We first discuss earlier studies that have illuminated the role of cis-acting elements within the promoters of immediate-early genes, the corresponding transcription factors that bind these elements, and the roles of major activity-regulated signaling pathways. However, our emphasis is on new studies that have revealed an important role for epigenetic and topological mechanisms, including enhancer-promoter interactions, enhancer RNAs (eRNAs), and activity-induced DNA breaks, that have emerged as important factors that govern the temporal dynamics of activity-induced gene transcription.
Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.
The Hippo-YAP signaling pathway has been established as a crucial regulator of tissue growth and tumorigenesis but the mechanism for transcriptional control remains unclear. In their study, Galli et al. show that YAP/TAZ binding is restricted to a small number of gene enhancers and that YAP/TAZ regulates transcriptional elongation by recruiting the Mediator complex.
The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.
The chain elongation portion of enzymatic RNA synthesis has been studied by employing a ternary complex containing RNA polymerase, a DNA template, and product RNA. Ternary complexes were isolated by passage through a gel exclusion column and were shown to carry out RNA chain elongation under conditions that preclude RNA chain initiation or termination. The properties of the elongation reaction have been studied by employing ternary complexes formed with synthetic DNA templates. A simple ping-pong kinetic model was derived and was shown to fit the data obtained with alternating copolymer templates. The model is also able to account for the inhibition observed at high concentrations of nucleoside triphosphates. A general rate equation can be devised for RNA chain elongation with DNA templates of complex sequence if one assumes that the Michaelis constants for the substrates are independent of the DNA sequence. This assumption appears to hold for the various synthetic DNA templates that we have examined but has not been adequately tested for more complex templates.
We have prepared three types of RNA polymerase II transcription complexes: a preinitiation complex (complex 0), a complex which has synthesized two phosphodiester bonds (complex 2), and a complex which has synthesized 10-13 bonds (complex 10). We have studied the differential response of these complexes to a variety of disruptions: detergent (Sarkosyl), high levels of KCl, extended incubation at 25 degrees C, proteolysis, and digestion with DNase I. Complex 0 is extremely stable at 25 degrees C in the absence of ATP, but it is sensitive to the other treatments including 25 degrees C incubation in the presence of ATP. Once the complex has made two phosphodiester bonds, the properties almost reverse from those of complex 0; complex 2 remains unstable at 25 degrees C in the presence of ATP but is resistant to high levels of Sarkosyl and KCl, to extensive DNase I digestion, and to brief proteolysis. Addition of 10 or more bases to the growing RNA chain results in a complex completely resistant to all of the treatments used. When DNase I-trimmed complex 0 is allowed to initiate RNA synthesis, chains of about 33 bases are obtained. In contrast, DNase-trimmed complex 2 gives only about 23 base transcripts; DNase-treated complex 10 will elongate its nascent chains by about 21 bases as well (to give, on average, 34 base transcripts).
Transcription initiation at the σ54-dependent glnAp2 promoter was studied to follow the state of polymerase as RNA synthesis begins. σ54 polymerase begins transcription in abortive
cycling mode, i.e. after the first bond is made, approximately 75% of the time the short RNA is aborted and synthesis must be restarted. Polymerase
is capable of abortive initiation until it reaches a position beyond +3 and before +7, at which stage polymerase is released
from its promoter contacts and an elongation complex is formed. Initial elongation is accompanied by two transcription bubbles,
one moving with the polymerase and the other remaining at the transcription start site. The σ54-associated polymerase shows
an earlier and more efficient transition out of abortive initiation mode than prior studies of σ70-associated polymerase.
The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals.
Assembly and activity of yeast RNA polymerase II (Pol II) preinitiation complexes (PIC) was investigated with an immobilized promoter assay and extracts made from wild-type cells and from cells containing conditional mutations in components of the Pol II machinery. We describe the following findings: (1) In one step, TFIID and TFIIA assemble at the promoter independently of holoenzyme. In another step, holoenzyme is recruited to the promoter. Mutations in the CTD of Pol II, Srb2, Srb4, and Srb5, and two mutations in TFIIB disrupt recruitment of all holoenzyme components tested without affecting TFIID and TFIIA recruitment. These results indicate that the stepwise assembly pathway is blocked after TFIID/TFIIA binding. (2) Both the Gal4-AH and Gal4-VP16 activators stimulate formation of active PICs by increasing the extent of PIC formation. The Gal4-AH activator stimulated PIC formation by enhancing the binding of TFIID and TFIIA, whereas Gal4-VP16 could enhance the recruitment of TFIID, TFIIA, and holoenzyme. (3) Extracts deficient in TFIIA activity showed reduced assembly of all PIC components. These and other results suggest that TFIIA acts at an early step by enhancing the stable recruitment of TFIID. (4) An extract containing the TFIIB mutant E62G, had no defect in PIC formation, but had a severe defect in transcription. Similarly, mutation of the TATA box reduced PIC formation only two- to fourfold, but severely compromised transcription. These results demonstate an involvement of TFIIB and the TATA box in one or more steps after recruitment of factors to the promoter.
The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation in Saccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization on Drosophila polytene chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo.
Proliferative signals lead to the rapid and transient induction of the c-fos proto-oncogene by targeting the ternary complex assembled on the serum response element (SRE). Transactivation by both components of this complex, serum response factor (SRF) and the ternary complex factor Elk-1, can be potentiated by the coactivator CREB-binding protein (CBP). We report a novel interaction between the bromodomain of CBP, amino acids 1100-1286, and Elk-1. DNA binding and glutathione S-transferase pull-down assays demonstrate that binding requires Elk-1(1-212) but not the C-terminal transactivation domain. Competition and antibody controls show that the bromocomplex involves both SRF and Elk-1 on the c-fos SRE and uniquely Elk-1 on the E74 Ets binding site. Interestingly, methylation interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100-1286) interacts via Elk-1 and does not require specific DNA contacts. Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100-1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters. Repression is relieved moderately or strongly by linking the bromodomain to the N- or C-terminal transactivation domains of CBP, respectively. These results are consistent with a model in which CBP is constitutively bound to the SRE in a higher order complex that would facilitate the rapid transcriptional activation of c-fos by signaling-driven phosphorylation.
The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.
A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (approximately 500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (approximately 2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at approximately 500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 x 10(5) to 6 x 10(5) molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the approximately 2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The approximately 2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.
Studying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole has led to the discovery of a number of proteins involved in the regulation of transcription elongation by RNA polymerase II. We have developed a highly purified elongation control system composed of three purified proteins added back to isolated RNA polymerase II elongation complexes. Two of the proteins, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), act as negative transcription elongation factors by increasing the time the polymerase spent at pause sites. P-TEFb reverses the negative effect of DSIF and NELF through a mechanism dependent on its kinase activity. TFIIF is a general initiation factor that positively affects elongation by decreasing pausing. We show that TFIIF functionally competes with DSIF and NELF, and this competition is dependent on the relative concentrations of TFIIF and NELF.
The C-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit is hyperphosphorylated during transcription. Using an in vivo cross-linking/chromatin immunoprecipitation assay, we found previously that different phosphorylated forms of RNA Pol II predominate at different stages of transcription. At promoters, the Pol II CTD is phosphorylated at Ser 5 by the basal transcription factor TFIIH. However, in coding regions, the CTD is predominantly phosphorylated at Ser 2. Here we show that the elongation-associated phosphorylation of Ser 2 is dependent upon the Ctk1 kinase, a putative yeast homolog of Cdk9/P-TEFb. Furthermore, mutations in the Fcp1 CTD phosphatase lead to increased levels of Ser 2 phosphorylation. Both Ctk1 and Fcp1 cross-link to promoter and coding regions, suggesting that they associate with the elongating polymerase. Both Ctk1 and Fcp1 have been implicated in regulation of transcription elongation. Our results suggest that this regulation may occur by modulating levels of Ser 2 phosphorylation, which in turn, may regulate the association of elongation factors with the polymerase.
The human cofactor complexes ARC (activator-recruited cofactor) and CRSP (cofactor required for Sp1 activation) mediate activator-dependent transcription in vitro. Although these complexes share several common subunits, their structural and functional relationships remain unknown. Here, we report that affinity-purified ARC consists of two distinct multisubunit complexes: a larger complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro transcription with biochemically separated ARC-L and CRSP reveals differential cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the CRSP complex is highly active. Structural determination by electron microscopy (EM) and three-dimensional reconstruction indicate substantial differences in size and shape between ARC-L and CRSP. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.
Activation of gene transcription in mammalian cells requires several classes of coactivators that participate in different steps of the activation cascade. Using conventional and affinity chromatography, we have isolated a human coactivator complex that interacts directly with the C-terminal domain (CTD) of RNA polymerase II (Pol II). The CTD-binding complex is structurally and functionally indistinguishable from our previously isolated CRSP coactivator complex. The closely related, but transcriptionally inactive, ARC-L complex failed to interact with the CTD, indicating a significant biochemical difference between CRSP and ARC-L that may, in part, explain their functional divergence. Electron microscopy and three-dimensional single-particle reconstruction reveals a conformation for CTD-CRSP that is structurally distinct from unliganded CRSP or CRSP bound to SREBP-1a, but highly similar to CRSP bound to the VP16 activator. Together, our findings suggest that the human CRSP coactivator functions, at least in part, by mediating activator-dependent recruitment of RNA Pol II via the CTD.
The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.
The metazoan transcription elongation factor P-TEFb (CDK-9/cyclin T) is essential for HIV transcription, and is recruited by some cellular activators. P-TEFb promotes elongation in vitro by overcoming pausing that requires the SPT-4/SPT-5 complex, but considerable evidence indicates that SPT-4/SPT-5 facilitates elongation in vivo. Here we used RNA interference to investigate P-TEFb functions in vivo, in the Caenorhabditis elegans embryo. We found that P-TEFb is broadly essential for expression of early embryonic genes. P-TEFb is required for phosphorylation of Ser 2 of the RNA Polymerase II C-terminal domain (CTD) repeat, but not for most CTD Ser 5 phosphorylation, supporting the model that P-TEFb phosphorylates CTD Ser 2 during elongation. Remarkably, although heat shock genes are cdk-9-dependent, they can be activated when spt-4 and spt-5 expression is inhibited along with cdk-9. This observation suggests that SPT-4/SPT-5 has an inhibitory function in vivo, and that mutually opposing influences of P-TEFb and SPT-4/SPT-5 may combine to facilitate elongation, or insure fidelity of mRNA production. Other genes are not expressed when cdk-9, spt-4, and spt-5 are inhibited simultaneously, suggesting that these genes require P-TEFb in an additional mechanism, and that they and heat shock genes are regulated through different P-TEFb-dependent elongation pathways.
The adenovirus large E1A protein activates transcription from early viral promoters by a mechanism that requires a forty amino acid zinc finger activation domain in E1A conserved region 3 (CR3). Recent results indicate that activation by a Gal4 DNA-binding domain-E1A-CR3 fusion requires an interaction between the E1A-CR3 zinc finger and the Sur2 subunit of the mammalian Mediator (of transcription) complex. Although several host proteins have been shown to bind stably to E1A proteins in adenovirus-infected and -transformed cells, an in vivo interaction with Mediator complex subunits has not been described previously. Using immunoprecipitation and gel filtration analyses of nuclear extracts prepared from HeLa cells infected with adenovirus 5 or mutants that express either large or small E1A specifically and from adenovirus 5-transformed cells, we report here that large E1A, but not small E1A, binds to Mediator complex in vivo. Only approximately 1 to 10% of large E1A is bound to Mediator complex at 18 h postinfection and in transformed cells, probably explaining why Mediator complex subunits were not identified among cellular E1A-binding proteins described earlier. Surprisingly, even though extracted Mediator can quantitatively bind to an E1A-CR3 affinity column, only on the order of 1% of cellular Mediator complex is bound by E1A in vivo. Much of the large E1A bound to Mediator in 293 cells is in a stable complex that includes RNA polymerase II, leading us to suggest that the interaction of E1A-CR3 with Mediator stabilizes the interaction of Mediator with the polymerase. This stabilization of the interaction between Mediator and RNA polymerase II may contribute to the mechanism of activation by E1A-CR3.
NF-κB induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-κB-responsive
genes are activated rapidly after the cell is stimulated by cytokines and other extracellular signals. However, the mechanism
by which these genes are activated is not entirely understood. Here we report that even though NF-κB interacts directly with
TAFIIs, induction of NF-κB by tumor necrosis factor alpha (TNF-α) does not enhance TFIID recruitment and preinitiation complex
formation on some NF-κB-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-α stimulus.
Using the immediate-early TNF-α-responsive gene A20 as a prototype promoter, we found that the constitutive association of
the general transcription apparatus is mediated by Sp1 and that this is crucial for rapid transcriptional induction by NF-κB.
In vitro transcription assays confirmed that NF-κB plays a postinitiation role since it enhances the transcription reinitiation
rate whereas Sp1 is required for the initiation step. Thus, the consecutive effects of Sp1 and NF-κB on the transcription
process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.
Eukaryotic genomes are organized into condensed, heterogeneous chromatin fibers throughout much of the cell cycle. Here we describe recent studies indicating that even transcriptionally active loci may be encompassed within 80- to 100-nanometer-thick chromonema fibers. These studies suggest that chromatin higher order folding may be a key feature of eukaryotic transcriptional control. We also discuss evidence suggesting that adenosine-5'-triphosphate-dependent chromatin-remodeling enzymes and histone-modifying enzymes may regulate transcription by controlling the extent and dynamics of chromatin higher order folding.
Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.
CBP/p300 recruitment to enhancer-bound complexes is a key determinant in promoter activation by many transcription factors. We present a novel mechanism of activating such complexes and show that pre-assembled Elk-1-p300 complexes become activated following Elk-1 phosphorylation by changes in Elk-1-p300 interactions rather than recruitment. It is known that Elk-1 binds to promoter in the absence of stimuli. However, it is unclear how activation of Elk-1 by mitogen-acivated protein kinase (MAPK)-mediated phosphorylation leads to targeted gene transactivation. We show that Elk-1 can interact with p300 in vitro and in vivo in the absence of a stimulus through the Elk-1 C-terminus and the p300 N-terminus. Phosphorylation on Ser383 and Ser389 of Elk-1 by MAPK enhances this basal binding but, most importantly, Elk-1 exhibits new interactions with p300. These interaction changes render a strong histone acetyltransferase activity in the Elk-1-associated complex that could play a critical role in chromatin remodeling and gene activation. The pre-assembly mechanism may greatly accelerate transcription activation, which is important in regulation of expression of immediate-early response genes, in particular those involved in stress responses.
Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans. Here, we show that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5′ and 3′ ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo.
Keywords
• Pol II pausing
• elongation
• heat shock genes
• Spt5
• Spt6
• P-TEFb
The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation inSaccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization onDrosophila polytene chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo.
Keywords
• Spt
• P-TEFb
• CTD
• elongation
• RNA polymerase
• polytene
The kinetics of formation and dissociation of specific (open) complexes between active Escherichia coli RNA polyrmerase holoenzyme (RNAP) and the λPR promoter have been studied by selective nitrocellulose filter binding assays at two temperatures (25 °C, 37 °C) and over a range of ionic conditions. Competition with a polyanion (heparin) or stabilization of open promoter complexes at PR by incubation with specific combinations of nucleoside triphosphates was employed to obtain selectivity in the filter assay. This study provides a useful example of how information about mechanism may be obtained from the quantitative analysis of the effects of salt concentration and temperature on the rate constants of a protein-DNA interaction.
Rapid, transient induction of the human c-fos proto-oncogene by extracellular signals requires the presence in cis of the serum response element (SRE). Two protein factors that bind to the SRE in vitro are the serum response factor (p67SRF) and polypeptide p62. These polypeptides must interact with one another and the SRE for efficient serum induction of the c-fos gene. Here we use dimethyl sulphate genomic footprinting to establish the in vivo protein contacts on the SRE and flanking sequences. In human A431 cells the patterns of protection and hyper-reactivity that we find are consistent with the presence of p67SRF, p62, and at least one other protein immediately 3' to p67SRF. The protein-DNA contacts we observe within the SRE are present before induction by epidermal growth factor and are unchanged during gene activation and subsequent repression. Our results indicate that a specific DNA-protein architecture may be maintained at the c-fos SRE, regardless of changes in the transcriptional state of the gene. Such established structures could be important generally in rapid transcriptional responses to extracellular signals.
The chain elongation portion of enzymatic RNA synthesis has been studied by employing a ternary complex containing RNA polymerase,
a DNA template, and product RNA. Ternary complexes were isolated by passage through a gel exclusion column and were shown
to carry out RNA chain elongation under conditions that preclude RNA chain initiation or termination. The properties of the
elongation reaction have been studied by employing ternary complexes formed with synthetic DNA templates. A simple ping-pong
kinetic model was derived and was shown to fit the data obtained with alternating copolymer templates. The model is also able
to account for the inhibition observed at high concentrations of nucleoside triphosphates. A general rate equation can be
devised for RNA chain elongation with DNA templates of complex sequence if one assumes that the Michaelis constants for the
substrates are independent of the DNA sequence. This assumption appears to hold for the various synthetic DNA templates that
we have examined but has not been adequately tested for more complex templates.
Using a defined RNA polymerase II (pol II) transcription system, we have investigated the roles of basal factors at discrete stages during the transcription cycle (e.g., initiation, promoter clearance, and transcript elongation). Abortive initiation assays revealed that TATA-binding protein, transcription factors TFIIB and TFIIF, and pol II were necessary and sufficient to form functional initiation complexes on both linear and supercoiled templates. By contrast, TFIIE, TFIIH, and ATP hydrolysis were additionally required during promoter clearance from linear templates, while negative supercoiling obviated the need for these auxiliary factors. Furthermore, TFIIE, TFIIH, and supercoiling were not required during elongation. Our results suggest a role for TFIIH-associated helicase activity or supercoiling during promoter clearance rather than open complex formation. These results establish abortive initiation as a useful assay for studying functional initiation complex formation in defined eukaryotic transcription systems and provide a framework for investigating regulation at different stages of the eukaryotic transcription cycle.
A mediator was isolated from yeast that enabled a response to the activator proteins GAL4-VP16 and GCN4 in a transcription system reconstituted with essentially homogeneous basal factors and RNA polymerase II. The mediator comprised some 20 polypeptides, including the three subunits of TFIIF and other polypeptides cross-reactive with antisera against GAL11, SUG1, SRB2, SRB4, SRB5, and SRB6 proteins. Mediator not only enabled activated transcription but also conferred 8-fold greater activity in basal transcription and 12-fold greater efficiency of phosphorylation of RNA polymerase II by the TFIIH-associated C-terminal repeat domain (CTD) kinase, indicative of mediator-CTD interaction. A holoenzyme form of RNA polymerase II was independently isolated that supported a response to activator proteins with purified basal factors. The holoenzyme proved to consist of mediator associated with core 12-subunit RNA polymerase II.
A plethora of signals induce the c-fos proto-oncogene via phosphorylation of the transcription factor Elk-1 by MAP kinase. We show that the coactivator CBP cooperates with Elk-1 to stimulate c-fos. Elk-1 physically interacts with CBP, which is dependent on the transactivation domain of Elk-1 but is independent of MAP kinase phosphorylation. However, functional cooperation between Elk-1 and CBP requires phosphorylation of Elk-1. Importantly, a carboxy-terminal transactivation domain of CBP itself is phosphorylated by MAP kinase, whereby the transactivation potential of CBP is enhanced. Thus, MAP kinase may not solely activate specific transcription factors but also the coactivator CBP, identifying a novel aspect of MAP kinase function. Thereby MAP kinase stimulation can pleiotropically affect activation of genes regulated by different transcription factors interacting with the same coactivator CBP.
Epigenetic regulation of gene expression through modification of chromatin organization is an important mechanism in the development of eucaryotic organisms. We investigated the developmentally regulated expression of the mouse mBRG1 and mbrm genes, which are homologous to the yeast SWI2 gene. Both proteins are involved in chromatin remodeling as components of the mammalian SWI/SNF complex. The analysis was performed at a time in mouse development when the formation of a functional zygotic nucleus is closely linked to extensive chromatin modifications. Reverse transcription-polymerase chain reaction (RT-PCR) analysis in mature oocytes and through the first cleavage stages showed that both genes were highly expressed as maternal products but that they subsequently exhibited considerable differences in their level of expression when the transition to zygotic transcription occurred. Immunodetection of the two proteins with specific antibodies paralleled the RT-PCR analysis. The mBRG1 protein was present throughout preimplantation development, whereas zygotic mbrm was clearly detectable only when differentiation first occurs at the blastocyst stage. At this stage, mbrm was restricted to the inner cell mass. Cell type-specific expression of mbrm was also observed after in vitro differentiation of embryonic stem cells. These results indicate that the two murine homologues of SWI2 have substantially different roles in chromatin organization during the onset of embryonic development.
Genome-wide expression analysis was used to identify genes whose expression depends on the functions of key components of the transcription initiation machinery in yeast. Components of the RNA polymerase II holoenzyme, the general transcription factor TFIID, and the SAGA chromatin modification complex were found to have roles in expression of distinct sets of genes. The results reveal an unanticipated level of regulation which is superimposed on that due to gene-specific transcription factors, a novel mechanism for coordinate regulation of specific sets of genes when cells encounter limiting nutrients, and evidence that the ultimate targets of signal transduction pathways can be identified within the initiation apparatus.
DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.
Adenovirus E1A proteins prepare the host cell for viral replication, stimulating cell cycling and viral transcription through interactions with critical cellular regulatory proteins such as RB and CBP. Here we show that the E1A zinc-finger domain that is required to activate transcription of viral early genes binds to a host-cell multiprotein complex containing homologues of yeast Srb/Mediator proteins. This occurs through a stable interaction with the human homologue of Caenorhabditis elegans SUR-2, a protein required for many developmental processes in the nematode. This human Srb/Mediator complex stimulates transcription in vitro in response to both the E1A zinc-finger and the herpes simplex virus VP16 activation domains. Interaction with human Sur-2 is also required for transcription to be activated by the activation domain of a transcription factor of the ETS-family in response to activated mitogen-activated protein (MAP) kinase.
In eukaryotes, transcriptional activators have been proposed to function by recruiting the RNA polymerase II (Pol II) machinery, by altering the conformation of this machinery, or by affecting steps after initiation, but the evidence is not definitive. Genomic footprinting of yeast TATA-box elements reveals activator-dependent alterations of chromatin structure and activator-independent protection, but little is known about the association of specific components of the Pol II machinery with promoters in vivo. Here we analyse TATA-box-binding-protein (TBP) occupancy of 30 yeast promoters in vivo. We find that TBP association with promoters is stimulated by activators and inhibited by the Cyc8-Tup1 repressor, and that transcriptional activity correlates strongly with the degree of TBP occupancy. In a small subset of promoters, TBP occupancy is higher than expected when gene activity is low, and the activator-dependent increase is modest. TBP association depends on the Pol II holoenzyme component Srb4, but not on the Kin28 subunit of the transcription factor TFIIH, even though both proteins are generally required for transcription. Thus in yeast cells, TBP association with promoters occurs in concert with the Pol II holoenzyme, activator-dependent recruitment of the Pol II machinery occurs at the vast majority of promoters, and Kin28 acts after the initial recruitment.
Eukaryotic transcriptional activators function, at least in part, by promoting assembly of the preinitiation complex, which comprises RNA polymerase II and its general transcription factors (GTFs). Activator-mediated stimulation of the assembly of the preinitiation complex has been studied in vitro but has been relatively refractory to in vivo analysis. Here we use a DNA-crosslinking/immunoprecipitation assay to study in living cells the first step in the assembly of the preinitiation complex, the interaction between the TATA-box-binding protein (TBP) and its binding site, the TATA box. Analysis of a variety of endogenous yeast genes, and of a series of activators of differing strength, reveals a general correlation between TBP binding and transcriptional activity. Using mutant yeast strains, we show that Mot1 prevents the binding of TBP to inactive promoters and that activator-mediated stimulation of TBP binding requires additional GTFs, including TFIIB and Srb4. Taken together, our results indicate that TBP binding in vivo is stringently controlled, and that the ability of activators to stimulate this step in the assembly of the preinitiation complex is a highly cooperative process involving multiple transcription factors.
Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail. At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators.
A novel multiprotein complex has recently been identified as a coactivator for transcriptional control of protein-encoding genes by RNA polymerase II in higher eukaryotic cells. This complex is evolutionarily related to the Mediator complex from yeast and, on the basis of its structural and functional characteristics, promises to be a key target of diverse regulatory circuits.
Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size. Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily. Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay.
Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interact with transcriptional activator proteins and to play an essential role in transcriptional regulation. Mediator evidently integrates and transduces positive and negative regulatory information from enhancers and operators to promoters. It functions directly through RNA polymerase II, modulating its activity in promoter-dependent transcription. Details of the Mediator mechanism remain obscure. Additional outstanding questions include the patterns of promoter-specificity of the various Mediator subunits, the possible cell-type-specificity of Mediator subunit composition, and the full structures of both free Mediator and RNA polymerase II holoenzyme.
The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5′ end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.
Keywords
• RNA polymerase II
• TFIIH
• capping enzyme
• Kin 28
Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans. Here, we show that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5' and 3' ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo.
The past decade has seen an explosive increase in information about regulation of eukaryotic gene transcription, especially for protein-coding genes. The most striking advances in our knowledge of transcriptional regulation involve the chromatin template, the large complexes recruited by transcriptional activators that regulate chromatin structure and the transcription apparatus, the holoenzyme forms of RNA polymerase II involved in initiation and elongation, and the mechanisms that link mRNA processing with its synthesis. We describe here the major advances in these areas, with particular emphasis on the modular complexes associated with RNA polymerase II that are targeted by activators and other regulators of mRNA biosynthesis.
Sur2 is a metazoan Mediator subunit that interacts with the adenovirus E1A protein and functions in a mitogen-activated protein
kinase pathway required for vulva development in Caenorhabditis elegans. We generated sur2
−/− embryonic stem cells to analyze its function as a mammalian Mediator component. Our results show that Sur2 forms a subcomplex
of the Mediator with two other subunits, TRAP/Med100 and 95. Knock-out of Sur2 prevents activation by E1A-CR3 and the mitogen-activated
protein kinase–regulated ETS transcription factor Elk-1, but not by multiple other transcription factors. These results imply
that specific activation domains stimulate transcription by binding to distinct Mediator subunits. Activation by E1A and Elk-1
requires recruitment of Mediator to a promoter by binding to its Sur2 subunit.
It has been a long-standing challenge to decipher the principles that enable cells to both organize their genomes into compact chromatin and ensure that the genetic information remains accessible to regulatory factors and enzymes within the confines of the nucleus. The discovery of nucleosome remodeling activities that utilize the energy of ATP to render nucleosomal DNA accessible has been a great leap forward. In vitro, these enzymes weaken the tight wrapping of DNA around the histone octamers, thereby facilitating the sliding of histone octamers to neighboring DNA segments, their displacement to unlinked DNA, and the accumulation of patches of accessible DNA on the surface of nucleosomes. It is presumed that the collective action of these enzymes endows chromatin with dynamic properties that govern all nuclear functions dealing with chromatin as a substrate. The diverse set of ATPases that qualify as the molecular motors of the nucleosome remodeling process have a common history and are part of a superfamily. The physiological context of their remodeling action builds on the association with a wide range of other proteins to form distinct complexes for nucleosome remodeling. This review summarizes the recent progress in our understanding of the mechanisms underlying the nucleosome remodeling reaction, the targeting of remodeling machines to selected sites in chromatin, and their integration into complex regulatory schemes.
The biosynthesis of the zinc finger transcription factor Egr-1 is stimulated by many extracellular signaling molecules including hormones, neurotransmitters, growth and differentiation factors, and cytotoxic metabolites. The 5'-flanking region of the Egr-1 gene contains genetic elements that are essential in connecting stimulation of the cells with enhanced transcription of the Egr-1 gene, and subsequently, transcription of Egr-1-responsive genes. Thus, Egr-1 links cellular signaling cascades with changes in the gene expression pattern. Many biological functions have been attributed to Egr-1. Here, we discuss evidence for Egr-1 control of cellular proliferation and programmed cell death.
The SWI/SNF family of complexes utilizes the energy of ATP hydrolysis to remodel chromatin structures, thereby allowing transcription factors to gain access to DNA. Recent studies suggest that these remodelers also participate in other DNA metabolic reactions such as replication and viral integration, and even in control of cell growth and tumor suppression. The SWI/SNF remodelers can be classified into at least two distinct subfamilies: one includes human BAF (also known as hSWI/SNF-A) and yeast SWI/SNF; the other comprises human PBAF (hSWI/SNF-B) and yeast RSC. Although both types of complexes have similar subunit composition and chromatin remodeling activity in vitro, they cannot replace each other during transcription mediated by specific activators. Thus, each remodeler probably works with a specific set of activators during gene activation. The availability of distinct types of remodelers can allow cells to regulate expression of a specific group of genes by modulating the activity of corresponding remodelers.
Chromatin is the physiologically relevant substrate for all genetic processes inside the nuclei of eukaryotic cells. Dynamic changes in the local and global organization of chromatin are emerging as key regulators of genomic function. Indeed, a multitude of signals from outside and inside the cell converges on this gigantic signaling platform. Numerous post-translational modifications of histones, the main protein components of chromatin, have been documented and analyzed in detail. These 'marks' appear to crucially mediate the functional activity of the genome in response to upstream signaling pathways. Different layers of cross-talk between several components of this complex regulatory system are emerging, and these epigenetic circuits are the focus of this review.
Set1, the yeast histone H3-lysine 4 (H3-K4) methylase, is recruited by the Pol II elongation machinery to a highly localized domain at the 5' portion of active mRNA coding regions. Set1 association depends upon the TFIIH-associated kinase that phosphorylates the Pol II C-terminal domain (CTD) and mediates the transition between initiation and elongation, and Set1 interacts with the form of Pol II whose CTD is phosphorylated at serine 5 but not serine 2. The Rtf1 and Paf1 components of the Pol II-associated Paf1 complex are also important for Set1 recruitment. Although the level of dimethylated H3-K4 is fairly uniform throughout the genome, the pattern of trimethylated H3-K4 strongly correlates with Set1 occupancy. Hypermethylated H3-K4 within the mRNA coding region persists for considerable time after transcriptional inactivation and Set1 dissociation from the chromatin, indicating that H3-K4 hypermethylation provides a molecular memory of recent transcriptional activity.