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Transcriptional and Developmental Functions of the H3.3 Histone Variant in Drosophila
Abstract
Changes in chromatin composition accompany cellular differentiation in eukaryotes. Although bulk chromatin is duplicated during DNA replication, replication-independent (RI) nucleosome replacement occurs in transcriptionally active chromatin and during specific developmental transitions where the genome is repackaged. In most animals, replacement uses the conserved H3.3 histone variant, but the functions of this variant have not been defined. Using mutations for the two H3.3 genes in Drosophila, we identify widespread transcriptional defects in H3.3-deficient animals. We show that mutant animals compensate for the lack of H3.3 in two ways: they upregulate the expression of the major histone H3 genes, and they maintain chromatin structure by using H3 protein for RI nucleosome replacement at active genes. Rescue experiments show that increased expression of H3 is sufficient to relieve transcriptional defects. In contrast, H3.3 is essential for male fertility, and germline cells specifically require the histone variant. Defects without H3.3 first occur around meiosis, resulting in a failure to condense, segregate, and reorganize chromatin. Rescue experiments with mutated transgenes demonstrate that H3.3-specific residues involved in RI nucleosome assembly-but not major histone modification sites-are required for male fertility. Our results imply that the H3.3 variant plays an essential role in chromatin transitions in the male germline.
... In Drosophila, where H3.3 makes up approximately 25% of the total H3 protein in the cell, single H3.3 knockouts where phenotypically normal, but double knockouts were infertile and had reduced viability [41]. Examination of the testes of H3.3-double-deficient flies showed defects in chromosome condensation and segregation during meiosis, with lagging chromosomes frequently observed in Anaphase I and chromosome bridges in Anaphase II, suggesting a crucial role for H3.3 in chromosome organization and condensation during gamete production [41]. ...
... In Drosophila, where H3.3 makes up approximately 25% of the total H3 protein in the cell, single H3.3 knockouts where phenotypically normal, but double knockouts were infertile and had reduced viability [41]. Examination of the testes of H3.3-double-deficient flies showed defects in chromosome condensation and segregation during meiosis, with lagging chromosomes frequently observed in Anaphase I and chromosome bridges in Anaphase II, suggesting a crucial role for H3.3 in chromosome organization and condensation during gamete production [41]. These defects can be rescued by ectopic expression of the canonical histone H3.2 [41], suggesting the total level of histone H3 could be more important than the specific variant being expressed in some contexts. ...
... Examination of the testes of H3.3-double-deficient flies showed defects in chromosome condensation and segregation during meiosis, with lagging chromosomes frequently observed in Anaphase I and chromosome bridges in Anaphase II, suggesting a crucial role for H3.3 in chromosome organization and condensation during gamete production [41]. These defects can be rescued by ectopic expression of the canonical histone H3.2 [41], suggesting the total level of histone H3 could be more important than the specific variant being expressed in some contexts. The infertility and chromosome segregation defects observed in H3.3 deficient Drosophila are similar to defects seen in H3.3 knockout mice and suggest an evolutionarily conserved role for H3.3 in fertility and, more specifically, chromosome segregation during gamete production. ...
Histone variant H3.3 plays novel roles in development as compared to canonical H3 proteins and is the most commonly mutated histone protein of any kind in human disease. Here we discuss how gene targeting studies of the two H3.3-coding genes H3f3a and H3f3b have provided important insights into H3.3 functions including in gametes as well as brain and lung development. Knockouts have also provided insights into the important roles of H3.3 in maintaining genomic stability and chromatin organization, processes that are also affected when H3.3 is mutated in human diseases such as pediatric tumors and neurodevelopmental syndromes. Overall, H3.3 is a unique histone linking development and disease via epigenomic machinery.
... The essentiality of H3.3 appears to be species dependent. For example, RD H3 can compensate for the loss of H3.3 in somatic tissues during Drosophila melanogaster development [25]. Similarly, H3.3 is not essential in Caenorhabditis elegans [26]. ...
... Both organisms, however, contain CAF1 and HIRA-like chaperones. In certain organisms, such as Drosophila, the deposition of H3.3 can occur via both RD and RI pathways [25]. Evolutionary studies have suggested that H3.3 is the ancestral form of H3.1/2 [4]. ...
... In mammalian cells, H3.3 is deposited in distinct chromatin regions including telomeric heterochromatin, gene bodies, enhancers, and promoters [5]. H3.3 deposition at highly expressed genes appears to be conserved in plants, Drosophila and mammals [5,25,62]. Tetrahymena H3.3 exhibited enrichment over promoters, gene bodies, and transcription termination regions of highly transcribed genes. In Drosophila and human cells, enrichment of H3.3 in the gene body and after the TES has been found to be correlated with transcriptional activity [5,25]. ...
Background:
Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts.
Results:
We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes.
Conclusion:
Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.
... Accordingly, functional redundancies between H3.2 and H3.3 have been described. One study showed that H3.2 can compensate for loss of H3.3 (31). Another example is highlighted by the H3K9 residue (14) as the combination of H3.2 K9R and H3.3 K9R mutations resulted in more severe developmental and transcriptional defects than did either mutation alone. ...
... Thus, the viability of H3.3B K36R animals was unaffected by deletion of H3.3A. Given that animals lacking both H3.3A and H3.3B (H3.3∆) had previously been shown to complete development (31,38), and those lacking only H3.3A are fully viable and fertile (31), this result was expected. However, unlike H3.3 K9R or H3.3 K27R mutants (14, 39), H3.3 K36R males are fertile. ...
... Thus, the viability of H3.3B K36R animals was unaffected by deletion of H3.3A. Given that animals lacking both H3.3A and H3.3B (H3.3∆) had previously been shown to complete development (31,38), and those lacking only H3.3A are fully viable and fertile (31), this result was expected. However, unlike H3.3 K9R or H3.3 K27R mutants (14, 39), H3.3 K36R males are fertile. ...
Polycomb complexes regulate cell type–specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent ( H3.2 K36R ) or replication-independent ( H3.3 K36R ) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined ( H3.3 K36R H3.2 K36R ) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2 K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3 K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.
... In the His4r Δ42 mutant we generated the entire coding region is deleted making it an amorphic (genetic null) allele. Similarly to carriers of loss of function mutations of histone H3 variants His3.3A or His3.3B 28,29 homozygous His4r Δ42 mutants proved to be viable without any obvious morphological defects. Furthermore, in contrast to the sterility caused by complete loss of H3.3 in H3.3B; H3.3A double mutants 28,29 both genders are fertile in the absence of His4r. ...
... Similarly to carriers of loss of function mutations of histone H3 variants His3.3A or His3.3B 28,29 homozygous His4r Δ42 mutants proved to be viable without any obvious morphological defects. Furthermore, in contrast to the sterility caused by complete loss of H3.3 in H3.3B; H3.3A double mutants 28,29 both genders are fertile in the absence of His4r. We observed mild (~ 32%) sublethality of His4r mutant animals that occurred in part before pupariation and in part during metamorphosis. ...
... In transcriptomic analysis we found that only a small subset of genes showed significantly altered transcriptional activity in His4r Δ42 male flies and this set was not enriched for genes specific for any biological process. The general trend of gene expression changes in the absence of His4r (67 genes up-regulated and 34 genes down-regulated) was similar to what was observed by others in the absence of H3.3 (288 up-and 99 down-regulated genes) 28 i.e. in accordance with their role in chromatin formation up-regulation of transcriptional activity was more common in the absence of either His4r or H3.3. ...
His4r is the only known variant of histone H4 in Drosophila. It is encoded by the His4r single-copy gene that is located outside of the histone gene cluster and expressed in a different pattern than H4, although the encoded polypeptides are identical. We generated a null mutant (His4rΔ42) which is homozygous viable and fertile without any apparent morphological defects. Heterozygous His4rΔ42 is a mild suppressor of position-effect variegation, suggesting that His4r has a role in the formation or maintenance of condensed chromatin. Under standard conditions loss of His4r has a modest effect on gene expression. Upon heat-stress the induction of the Heat shock protein (HSP) genes Hsp27 and Hsp68 is stronger in His4rΔ42 mutants with concordantly increased survival rate. Analysis of chromatin accessibility after heat shock at a Hsp27 regulatory region showed less condensed chromatin in the absence of His4r while there was no difference at the gene body. Interestingly, preconditioning before heat shock led to increased chromatin accessibility, HSP gene transcription and survival rate in control flies while it did not cause notable changes in His4rΔ42. Thus, our results suggest that His4r might play a role in fine tuning chromatin structure at inducible gene promoters upon environmental stress conditions.
... Individual homozygous disruption of either one of the H3.3 genes in Drosophila (H3.3A and H3.3B) has little phenotypic effect on the overall organism. In contrast, combined disruption of both genes results sterility in both sexes and slightly reduced viability, but with no obvious morphological defects in adult flies (Hodl and Basler, 2009;Sakai et al., 2009 is also essential for fertility. Specifically, during fertilization it helps in decondensing paternal chromatin by assembling H3.3-containing nucleosomes in the male pronucleus (Bonnefoy et al., 2007;Loppin et al., 2005). ...
... Specifically, during fertilization it helps in decondensing paternal chromatin by assembling H3.3-containing nucleosomes in the male pronucleus (Bonnefoy et al., 2007;Loppin et al., 2005). In Drosophila, both Hira and H3.3 are required for fertility and for transcriptional regulation of specific genes, but not for viability in development and adult animals (Bonnefoy et al., 2007;Hodl and Basler, 2009;Nakayama et al., 2007;Sakai et al., 2009). Furthermore, histone H3 variants can replace each other in both replication-dependent and replication-independent chromatin assembly (Sakai et al., 2009). ...
... In Drosophila, both Hira and H3.3 are required for fertility and for transcriptional regulation of specific genes, but not for viability in development and adult animals (Bonnefoy et al., 2007;Hodl and Basler, 2009;Nakayama et al., 2007;Sakai et al., 2009). Furthermore, histone H3 variants can replace each other in both replication-dependent and replication-independent chromatin assembly (Sakai et al., 2009). ...
Numerous mutations in histone H3 lysine 4 (H3K4) and H3 lysine 36 (H3K36) modifying enzymes have been reported in human disease, yet the role of the H3K4 and H3K36 residues in mammals remain unclear due to the clustered arrays of many histone genes. Replication-dependent canonical H3 (H3.1/H3.2) exists as multiple gene copies and supplies nucleosomes for packaging of newly synthesized DNA during replication. The histone variant H3.3 differs from canonical H3 by only 4 to 5 amino acids, which allow nucleosome assembly independent of DNA replication throughout the cell cycle and in post-mitotic cells. In this study, I set out to investigate the role of the K4 and K36 residues in the histone H3.3 variant, which is enriched at active regions of the mammalian genome and encoded by two isolated genes; therefore amenable to functional analysis. Using CRISPR-Cas9, I mutated the K4 or K36 residue of endogenous H3.3 to unmodifable alanine (A) in mouse embryonic stem cells (ESCs) and revealed that the K4A mutation, but not K36A, resulted in widespread gene expression changes and impairment of neuronal differentiation into glutamatergic neurons. Furthermore, K4A resulted in significant H3.3 protein depletion at transcription start sites and active enhancers of ESCs - without effects at other sites. Genomic regions depleted of H3.3K4A showed concerted alterations of histone modifications (decreased K27 acetylation and increased K4 methylation) regardless of gene expression changes. In differentiated neurons, the K4A mutation impacted protein stability and resulted in widespread proteasomal degradation of the mutant histone. Thus, H3.3K4 is required for site-specific nucleosome maintenance at regulatory regions, histone stability and cellular differentiation of ESCs. H3.3K36 is not required for H3.3 deposition and turnover inside coding regions, and the K36A mutation affected gene expression at later stages of neurodevelopment. Furthermore, the K36A mutation globally depleted H3K36 di-metylation levels in ESCs, which resulted in a spread of the repressive mark H3K27me3, suggesting that H3K36 di-methylation is required to restrict the activity of PRC2. This study demonstrates a direct link between a specific histone residue (H3K4) and histone maintenance at promoters and enhancers, and that H3.3 provides a platform for analyzing the role of histone residues in mammals.
... Single knockouts of the two H3.3 coding genes (H3.3A and H3.3B) in Drosophila did not strongly affect development and the double H3.3A/H3.3B knockout in the fly was compatible with life but led to infertility (Hodl & Basler, 2009;Sakai, Schwartz, Goldstein, & Ahmad, 2009). Single mouse knockout studies have also found that H3f3a and H3f3b have roles in mammalian fertility and more specifically in gamete and early embryonic states including specifying appropriate chromatin states (Bush et al., 2013;Jang et al., 2015;Tang et al., 2015;Tang, Binos, Ong, Wong, & Mann, 2014;Yuen et al., 2014). ...
... Transient and variable phenotypes with homozygous loss of one of the two H3.3-coding genes could be due to variable compensation by the remaining H3.3-coding gene or other compensatory mechanisms in cells and organisms such as changes in H3.3 chaperone function. In fully H3.3 deficient, double knockout flies, canonical H3 was also reported to have elevated expression and begin functioning in a partially replication-independent manner compensating for loss of H3.3 (Sakai et al., 2009), perhaps explaining the mechanism by which some double knockouts could survive. It is unknown whether loss of H3.3 induces H3.1 or H3.2 to function in a more H3.3-like ...
Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others have produced generally similar phenotypes to what we found in our H3f3b nulls, but the relative impacts of the loss of either H3f3a or H3f3b have varied depending on the approach and genetic background. Here we used a knockout‐first approach to target the H3f3a gene for inactivation in C57BL6 mice. Homozygous H3f3a targeting produced a lethal phenotype at or before birth. E13.5 null embryos had some potential morphological differences from WT littermates including smaller size and reduced head size. An E18.5 null embryo was smaller than its control littermates with several potential defects including small head and brain size as well as small lungs, which would be consistent with a late gestation lethal phenotype. Despite a reduction in H3.3 and total H3 protein levels, the only histone H3 post‐translational modification in the small panel assessed that was significantly altered was the unique H3.3 mark phospho‐Serine31, which was consistently increased in null neurospheres. H3f3a null neurospheres also exhibited consistent gene expression changes including in protocadherins. Overall, our findings are consistent with the model that there are differential, cell‐type‐specific contributions of H3f3a and H3f3b to H3.3 functions in epigenetic and developmental processes.
... Thus, the viability of H3.3B K36R animals was unaffected by deletion of H3.3A. Given that animals lacking both H3.3A and H3.3B (H3.3∆) had previously been shown to complete development (31,38), and those lacking only H3.3A are fully viable and fertile (31), this result was unsurprising. However, unlike H3.3 K9R or H3.3 K27R mutants (14,39), H3.3 K36R males are fertile. ...
... Thus, the viability of H3.3B K36R animals was unaffected by deletion of H3.3A. Given that animals lacking both H3.3A and H3.3B (H3.3∆) had previously been shown to complete development (31,38), and those lacking only H3.3A are fully viable and fertile (31), this result was unsurprising. However, unlike H3.3 K9R or H3.3 K27R mutants (14,39), H3.3 K36R males are fertile. ...
Polycomb complexes regulate cell-type specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2 K36R ) or -independent (H3.3 K36R ) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3 K36R H3.2 K36R ) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2 K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3 K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive PREs (Polycomb Response Elements) located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.
Short summary
Histone H3.2 and H3.3 K36 residues ensure Hox gene silencing and enable development by different, but synergistic mechanisms.
... Although H3.1 plays an essential role in the chromatin assembly of doubled genome during DNA replication, the function of H3.3 is complex and remains undetermined. In both animals and plants, the lack of H3.3 causes defects in development (Hodl and Basler, 2009;Sakai et al., 2009;Szenker et al., 2012;Jang et al., 2015;Wollmann et al., 2017), demonstrating its important role in multicellular eukaryotes. ...
... On the genome, H3.3 is associated with actively transcribed genes and gene regulatory elements (Wirbelauer et al., 2005;Goldberg et al., 2010;Szenker et al., 2011;Stroud et al., 2012;Wollmann et al., 2012). However, H3.3 is nonessential for most of the transcriptional events in Drosophila (Drosophila melanogaster; Hodl and Basler, 2009) and it is largely interchangeable with the replicative H3 (Sakai et al., 2009). In Arabidopsis (Arabidopsis thaliana), only a small number of H3.3-enriched genes show transcriptional defects when H3.3 levels are reduced, indicating that H3.3 may not be directly required for transcription (Wollmann et al., 2017). ...
The histone H3 family in animals and plants includes replicative H3 and non-replicative H3.3 variants. H3.3 preferentially associates with active transcription, yet its function in development and transcription regulation remains elusive. The floral transition in Arabidopsis (Arabidopsis thaliana) involves complex chromatin regulation at a central flowering repressor FLOWERING LOCUS C (FLC). Here we show that H3.3 upregulates FLC expression and promotes active histone modifications Histone H3 lysine 4 trimethylation (H3K4me3) and Histone H3 lysine 36 trimethylation (H3K36me3) at the FLC locus. The FLC activator FRIGIDA (FRI) directly mediates H3.3 enrichment at FLC, leading to chromatin conformation changes and further induction of active histone modifications at FLC. Moreover, the antagonistic H3.3 and H2A.Z act in concert to activate FLC expression, likely by forming unstable nucleosomes ideal for transcription processing. We also show that H3.3 knockdown leads to H3K4me3 reduction at a subset of particularly short genes, suggesting the general role of H3.3 in promoting H3K4me3. The finding that H3.3 stably accumulates at FLC in the absence of H3K36me3 indicates that the H3.3 deposition may serve as a prerequisite for active histone modifications. Our results reveal the important function of H3.3 in mediating the active chromatin state for flowering repression.
... In addition, in macrophages, H3.3S31ph over gene bodies interacts with SETD2 to promote H3K36me3 in genes stimulated by bacterial lipopolysaccharides, suggesting a role in regulating rapid response genes (2). However, in Caenorhabditis, H3.3 is not required for viability (21), and in Drosophila H3.3 knockout mutants, an overexpressed H3.2 (H3A31) construct was able to rescue viability and nearly all transcription defects (115), implying minimally that the requirement for H3.3S31 differs in different organisms. In both flies and mice, H3.3 is required 6. 16 Talbert for male fertility (115,158). ...
... However, in Caenorhabditis, H3.3 is not required for viability (21), and in Drosophila H3.3 knockout mutants, an overexpressed H3.2 (H3A31) construct was able to rescue viability and nearly all transcription defects (115), implying minimally that the requirement for H3.3S31 differs in different organisms. In both flies and mice, H3.3 is required 6. 16 Talbert for male fertility (115,158). In H3.3-reduced male mice, apoptosis of spermatogonia and spermatocytes occurs and the transition to protamines is incomplete (158), implying a defect at or prior to meiosis. ...
Nucleosomes wrap DNA and impede access for the machinery of transcription. The core histones that constitute nucleosomes are subject to a diversity of posttranslational modifications, or marks, that impact the transcription of genes. Their functions have sometimes been difficult to infer because the enzymes that write and read them are complex, multifunctional proteins. Here, we examine the evidence for the functions of marks and argue that the major marks perform a fairly small number of roles in either promoting transcription or preventing it. Acetylations and phosphorylations on the histone core disrupt histone-DNA contacts and/or destabilize nucleosomes to promote transcription. Ubiquitylations stimulate methylations that provide a scaffold for either the formation of silencing complexes or resistance to those complexes, and carry a memory of the transcriptional state. Tail phosphorylations deconstruct silencing complexes in particular contexts. We speculate that these fairly simple roles form the basis of transcriptional regulation by histone marks.
Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 22 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... CHD1 is indeed involved in the loading of H3.3 in the fly brain, where it seems to contribute to the regulation of genes that control the homeostasis of hunger and satiety signals [51]. On the other hand, as a demonstration of the wide range of tissues and functions in which H3.3 histone is probably involved, it was also found to be essential for the chromatin transitions that accompany Drosophila male germline maturation [52]. ...
All the cells of an organism contain the same genome. However, each cell expresses only a minor fraction of its potential and, in particular, the genes encoding the proteins necessary for basal metabolism and the proteins responsible for its specific phenotype. The ability to use only the right and necessary genes involved in specific functions depends on the structural organization of the nuclear chromatin, which in turn depends on the epigenetic history of each cell, which is stored in the form of a collection of DNA and protein modifications. Among these modifications, DNA methylation and many kinds of post-translational modifications of histones play a key role in organizing the complex indexing of usable genes. In addition, non-canonical histone proteins (also known as histone variants), the synthesis of which is not directly linked with DNA replication, are used to mark specific regions of the genome. Here, we will discuss the role of the H3.3 histone variant, with particular attention to its loading into chromatin in the mammalian nervous system, both in physiological and pathological conditions. Indeed, chromatin modifications that mark cell memory seem to be of special importance for the cells involved in the complex processes of learning and memory.
... Another example is the histone H3.3, which is required for reprogramming events during development across species and is associated with genes that are actively transcribing in both animals and plants [93]. Histone H3.3 is encoded by two genes in Drosophila (H3.3A and H3.3B) and human (H3f3a and H3f3b), and the knock-out mutants of one of either genes show decreased viability, partial lethality or infertility of the survivors [69,94,95]. Knock-down studies in mice also showed that the complete depletion of H3.3 caused early embryonic lethality, suggesting the significance of H3.3 in animal development [96]. ...
Histones are subjected to extensive covalent modifications that affect inter-nucleosomal interactions as well as alter chromatin structure and DNA accessibility. Through switching the corresponding histone modifications, the level of transcription and diverse downstream biological processes can be regulated. Although animal systems are widely used in studying histone modifications, the signalling processes that occur outside the nucleus prior to histone modifications have not been well understood due to the limitations including non viable mutants, partial lethality, and infertility of survivors. Here, we review the benefits of using Arabidopsis thaliana as the model organism to study histone modifications and their upstream regulations. Similarities among histones and key histone modifiers such as the Polycomb group (PcG) and Trithorax group (TrxG) in Drosophila, Human, and Arabidopsis are examined. Furthermore, prolonged cold-induced vernalization system has been well-studied and revealed the relationship between the controllable environment input (duration of vernalization), its chromatin modifications of FLOWERING LOCUS C (FLC), following gene expression, and the corresponding phenotypes. Such evidence suggests that research on Arabidopsis can bring insights into incomplete signalling pathways outside of the histone box, which can be achieved through viable reverse genetic screenings based on the phenotypes instead of direct monitoring of histone modifications among individual mutants. The potential upstream regulators in Arabidopsis can provide cues or directions for animal research based on the similarities between them.
... In contrast, PTMs associated with more repressive chromatin, such as H3K27me2/3 and H3K9me2/3, occur preferentially on H3 (23)(24)(25). Furthermore, recent studies in several organisms have suggested a conserved role for H3.3 during gametogenesis and early embryonic development in mice (27)(28)(29)(30), Drosophila (31)(32)(33), and Xenopus laevis (34). In C. elegans, removal of H3.3 is not lethal but reduces fertility and viability in response to stress (35). ...
During metazoan development, the marked change in developmental potential from the parental germline to the embryo raises an important question regarding how the next life cycle is reset. As the basic unit of chromatin, histones are essential for regulating chromatin structure and function and, accordingly, transcription. However, the genome-wide dynamics of the canonical, replication-coupled (RC) histones during gametogenesis and embryogenesis remain unknown. In this study, we use CRISPR-Cas9-mediated gene editing in Caenorhabditis elegans to investigate the expression pattern and role of individual RC histone H3 genes and compare them to the histone variant, H3.3. We report a tightly regulated epigenome landscape change from the germline to embryos that are regulated through differential expression of distinct histone gene clusters. Together, this study reveals that a change from a H3.3- to H3-enriched epigenome during embryogenesis restricts developmental plasticity and uncovers distinct roles for individual H3 genes in regulating germline chromatin.
... Perhaps surprisingly, H3.3-mediated reduction of enhancer acetylation is not correlated with global reduction of transcription in mouse embryonic stem cells (ESCs) [25,33]. However, a number of studies in mammalian cell lines suggest that H3.3 plays a role in de novo transcription [34,35] in response to extracellular stimuli [25,[36][37][38][39][40], and H3.3 knockout in animal models results in embryonic lethality or sterility [41][42][43]. Together, these observations suggest that H3.3 may be functionally important to initiate new transcription programs. Tafessu et al. ...
Background
The histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood.
Results
We perform genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and find evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 results in reduced chromatin accessibility and transcription factor (TF) binding at promoters of expressed genes in ESCs. Likewise, enrichment of the transcriptional coactivator p300 and downstream histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the acetyl lysine reader BRD4. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TF motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate.
Conclusions
H3.3 facilitates DNA accessibility, transcription factor binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it does promote de novo transcription factor binding which may contribute to the dysregulation of cellular differentiation in the absence of H3.3.
... In contrast, PTMs associated with more repressive chromatin, such as H3K27me2/3 and H3K9me2/3, occur preferentially on H3 (17)(18)(19). Furthermore, recent studies in several organisms have suggested a conserved role for H3.3 during gametogenesis and early embryonic development in mice (21)(22)(23)(24), Drosophila (25)(26)(27), and X. Laevis (28). In C. elegans, removal of H3.3 is not lethal, but reduces fertility and viability in response to stress (29). ...
During metazoan development, the dramatic potency change from germline to embryos raises an important question regarding how the new life cycle is reset. Here, we report a tightly regulated epigenome landscape change from the parental germline to embryos in C. elegans . The epigenome is enriched with histone H3 in early-stage germ cells but switches to a histone variant H3.3-enriched epigenome in the mature egg. This H3.3-dominant epigenome persists in early-stage embryos until gastrulation, when the epigenome becomes H3 abundant again. We further demonstrate that this developmentally programmed H3 → H3.3 → H3 epigenome landscape change is regulated through differential expression of distinct histone gene clusters and is required for both germline integrity and early embryonic cellular plasticity. Together, this study reveals that a bimodal expression of H3 versus H3.3 is important for epigenetic reprogramming during gametogenesis and embryonic plasticity.
One Sentence Summary
Developmentally programmed epigenome resets cellular plasticity at the parental-to-zygote transition in C. elegans .
... The histone variant H3.3 is essential for development in both animals and plants (Sakai et al., 2009;Szenker et al., 2012;Jang et al., 2015;Wollmann et al., 2017;Zhao et al., 2021Zhao et al., , 2022. In Arabidopsis, loss of H3.3 particularly affects the expression of responsive genes, suggesting that H3.3 is also required for plant responsiveness (Wollmann et al., 2017). ...
Plants can sense temperature changes and adjust their development and morphology accordingly in a process called thermomorphogenesis. This phenotypic plasticity implies complex mechanisms regulating gene expression reprogramming in response to environmental alteration. Histone variants often associate with specific chromatin states; yet, how their deposition/eviction modulates transcriptional changes induced by environmental cues remains elusive.
In Arabidopsis thaliana , temperature elevation‐induced transcriptional activation at thermo‐responsive genes entails the chromatin eviction of a histone variant H2A.Z by INO80, which is recruited to these loci via interacting with a key thermomorphogenesis regulator PIF4.
Here, we show that both INO80 and the deposition chaperones of another histone variant H3.3 associate with ELF7, a critical component of the transcription elongator PAF1 complex. H3.3 promotes thermomorphogenesis and the high temperature‐enhanced RNA Pol II transcription at PIF4 targets, and it is broadly required for the H2A.Z removal‐induced gene activation. Reciprocally, INO80 and ELF7 regulate H3.3 deposition, and are necessary for the high temperature‐induced H3.3 enrichment at PIF4 targets.
Our findings demonstrate close coordination between H2A.Z eviction and H3.3 deposition in gene activation induced by high temperature, and pinpoint the importance of histone variants dynamics in transcriptional regulation.
... Interestingly, Drosophila embryos can develop to adulthood in the absence of H3.3. However, Drosophila mutants of H3.3A and H3.3B have reduced viability and both males and females are unfertile (Hödl and Basler 2009;Sakai et al. 2009). ...
Nuclear structure influences genome architecture, which contributes to determine patterns of gene expression. Global changes in chromatin dynamics are essential during development and differentiation, and are one of the hallmarks of ageing. This chapter describes the molecular dynamics of chromatin structure that occur during development and ageing. In the first part, we introduce general information about the nuclear lamina, the chromatin structure, and the 3D organization of the genome. Next, we detail the molecular hallmarks found during development and ageing, including the role of DNA and histone modifications, 3D genome dynamics, and changes in the nuclear lamina. Within the chapter we discuss the implications that genome structure has on the mechanisms that drive development and ageing, and the physiological consequences when these mechanisms fail.
... Second, H3.3 enriched at gene bodies and regulatory elements such as promoters and enhancers (Ahmad and Henikoff, 2002;Mito et al., 2007), suggesting an association with active transcription. Although, absence of H3.3 in Drosophila led to transcriptional defects, the latter could be compensated by increased expression of variant H3.1 (Sakai et al., 2009). This suggests that transcription was affected by lack of histone replacement rather than by the variant of H3. ...
Dynamic regulation of the epigenome underlies cellular plasticity and allows cells to respond to developmental and differentiation programs. Epigenomic effectors can mediate changes such as modification of histone tails, nucleosome positioning or the incorporation of specific histone variants, altering chromatin structure and thus gene expression. Yet, the molecular mechanisms underpinning these processes remain largely unknown. A major challenge in the field is to establish direct functional connections between upstream chromatin factors, which generally have broad impact on chromatin, and the controlled expression of specific cellular effectors. My PhD project was precisely based on the discovery of a specific functional link between a series of epigenomic effectors and the highly regulated terminal effector of zygote formation in Drosophila melanogaster: the maternal thioredoxin Deadhead (Dhd). Dhd is critical to ensure paternal chromatin remodeling at fertilization, and thus zygote formation. First, an shRNA-based genetic screen in the female germline led me to identify (i) the H3K4me3 demethylase Lid, (ii) the members of the deacetylase complex Sin3A and Rpd3, (iii) the Snr1 subunit of the chromatin remodeler Swi/Snf and (iv) the chromatin factor Mod(mdg4), as essential for the expression of dhd. For further analyses I focused on Lid, Sin3A, Snr1 and Mod(mdg4). Transcriptomic analyses showed that dhd is among the most highly expressed genes in ovaries and this expression is completely abolished when I deplete Lid, Sin3A, Snr1 or Mod(mdg4) specifically in the female germline. Remarkably, there is a paucity of misregulated genes in knockdown ovaries. This suggested that these broadly conserved, ubiquitous complexes are mostly dedicated to regulation of dhd in this tissue. This paradigmatic case presented the opportunity to dissect the mechanisms at play in the epigenomic control of the establishment of specific transcriptional programs, as is the one set during oogenesis. Next, using Cut&Run chromatin profiling with a dedicated data analysis strategy, I found that dhd is embedded in a heterochromatic H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3A, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, I showed that these effectors activate dhd 11 independently of H3K27me3/H3K9me3 and that these marks are not required to repress dhd in adult tissues. Altogether, my work uncovered multiple unusual genomic and epigenomic characteristics at the dhd locus, but did not identify a single feature that was truly defining ovarian hyperactivation. The dramatic regulation of dhd may rely not on an individual trait but rather on a unique combination of such rare features. Through the example of dhd, my work demonstrates the puzzling process that is gene activation in the right place, at the right time and in the right amount. It also illustrates the difficulty to establish general rules on chromatin-based regulatory systems, which are very often context-dependent.
... Instead, absolute requirement for H3.1 or H3.3 may depend on organisms and developmental stages. In Drosophila, H3.1 can replace H3.3 in somatic tissues and restore transcriptional defects of h3.3 mutants [39], while in Arabidopsis, complete loss of H3.3 causes lethality [40]. Despite the specific enrichment of H3.3 in the 3' end of transcriptionally active genes [10], reduction of H3. [42], suggesting a more general role for H3.3 or its deposition machinery in favoring small chromatin loops at gene bodies and therefore in the 3D organization of the genome. ...
The organization of DNA with histone proteins into chromatin is fundamental for the regulation of gene expression. Incorporation of different histone variants into the nucleosome together with post-translational modifications of these histone variants allows modulating chromatin accessibility and contributes to the establishment of functional chromatin states either permissive or repressive for transcription. This review highlights emerging mechanisms required to deposit or evict histone variants in a timely and locus-specific manner. This review further discusses how assembly of specific histone variants permits to reinforce transmission of chromatin states during replication, to maintain heterochromatin organization and stability and to reprogram existing epigenetic information.
... H3f3b and H3f3a both encode the highly conserved protein, histone H3.3. Lack of histone H3.3 induces dysfunction of heterochromatin structures at pericentric regions and telomeres, resulting in mitotic and meiotic defects [38][39][40] . It is likely that H3f3b (H3.3) regulates LINE1 elements, as H3.3 deposited in heterochromatic genomic regions over LINE1 elements represses LINE1 19,20,41 . ...
We identified two NEXMIF variants in two unrelated individuals with non-autoimmune diabetes and autistic traits, and investigated the expression of Nexmif in mouse and human pancreas and its function in pancreatic beta cells in vitro and in vivo. In insulin-secreting INS-1E cells, Nexmif expression increased strongly in response to oxidative stress. CRISPR Cas9-generated Nexmif knockout mice exhibited a reduced number of proliferating beta cells in pancreatic islets. RNA sequencing of pancreatic islets showed that the downregulated genes in Nexmif mutant islets are involved in stress response and the deposition of epigenetic marks. They include H3f3b, encoding histone H3.3, which is associated with the regulation of beta-cell proliferation and maintains genomic integrity by silencing transposable elements, particularly LINE1 elements. LINE1 activity has been associated with autism and neurodevelopmental disorders in which patients share characteristics with NEXMIF patients, and can cause genomic instability and genetic variation through retrotransposition. Nexmif knockout mice exhibited various other phenotypes. Mortality and phenotypic abnormalities increased in each generation in both Nexmif mutant and non-mutant littermates. In Nexmif mutant mice, LINE1 element expression was upregulated in the pancreas, brain, and testis, possibly inducing genomic instability in Nexmif mutant mice and causing phenotypic variability in their progeny.
... Inactivation of H3.3 has been performed in several organisms and enabled a better understanding of its roles in development. In Drosophila, loss of H3.3 does not impair embryonic and postnatal development but leads to fertility defects (Hodl and Basler 2009;Sakai et al. 2009), which can be rescued by ectopic expression of H3.2 (Hodl and Basler, 2012). ...
Brain tumors are the second most frequent pediatric cancer after leukemia and the absence of efficient treatment leads to a poor prognosis with a survival rate of less than two years. Histone variant H3.3 mutations have been described as drivers of pediatric high-grade glioma, but the underlying mechanisms remain unknown. The main goal of this thesis is to dissect the mechanistic aspects of H3.3 mutation functions and the molecular mechanisms through which these mutations contribute to oncogenesis. Using a new mouse embryonic stem cell model, we showed that recently integrated endogenous retroviruses (ERV) are enriched in H3.3 and become overexpressed under mutant H3.3. The direct link between H3.3 and ERV regulation could be clinically confirmed on patient samples. In addition, a novel role for H3.3 in neural differentiation has been highlighted through the regulation of recently integrated retrotransposons, with H3.3 mutants leading to their deregulation and failure of differentiation. ERV deregulation under H3.3 K27M and G34R represents a novel mechanism potentially implicated in pediatric high-grade glioma development and progression.
... H3.3 is also involved in spermatogenesis. A very strong phenotype, including severe infertility and reduced viability were observed in Drosophila loss of functions mutants, where both H3.3A and H3.3B were deleted by P element transposition (18). Inactivation of both H3f3a and H3f3b genes in mice led to early embryonic lethality and double H3f3a KO/WT /H3f3b KO mutant males are totally infertile, while double H3f3a KO/WT /H3f3b KO females are fertile, revealing a specific role of H3.3 in spermatogenesis (16). ...
The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions.
... In C. elegans, the H3.3 301 requirement only stands out upon heat shock to ensure an appropriate transcriptional activation 302 and the timely firing of replication origins [78,79]. In flies, H3.3 is mainly critical for male 303 germline development [80,81], while in Xenopus a specific requirement during gastrulation 304 could be revealed [56]. Finally, mice lacking H3. ...
Histone variant H3.3 is incorporated into chromatin throughout the cell cycle and even in non-cycling cells. This histone variant marks actively transcribed chromatin regions with high nucleosome turnover, as well as silent pericentric and telomeric repetitive regions. In the past few years, significant progress has been made in our understanding of mechanisms involved in the transcription-coupled deposition of H3.3. Here we review how, during transcription, new H3.3 deposition intermingles with the fate of the old H3.3 variant and its recycling. First, we describe pathways enabling the incorporation of newly synthesized vs old H3.3 histones in the context of transcription. We then review the current knowledge concerning differences between these two H3.3 populations, focusing on their PTMs composition. Finally, we discuss the implications of H3.3 recycling for the maintenance of the transcriptional state and underline the emerging importance of H3.3 as a potent epigenetic regulator for both maintaining and switching a transcriptional state.
... Several studies in flies, worms and plants have demonstrated a role for H3.3 in controlling gene expression, DNA repair, cell differentiation and embryonic development (24)(25)(26)(27)(28)(29)(30)(31). In mouse, knockout of H3.3-encoding genes leads to infertility and embryo lethality (32)(33)(34)(35)(36), providing support for the importance of H3.3 in development and survival. ...
Histone H3.3 is an H3 variant which differs from the canonical H3.1/2 at four residues, including a serine residue at position 31 which is evolutionarily conserved. The H3.3 S31 residue is phosphorylated (H3.3 S31Ph) at heterochromatin regions including telomeres and pericentric repeats. However, the role of H3.3 S31Ph in these regions remains unknown. In this study, we find that H3.3 S31Ph regulates heterochromatin accessibility at telomeres during replication through regulation of H3K9/K36 histone demethylase KDM4B. In mouse embryonic stem (ES) cells, substitution of S31 with an alanine residue (H3.3 A31 -phosphorylation null mutant) results in increased KDM4B activity that removes H3K9me3 from telomeres. In contrast, substitution with a glutamic acid (H3.3 E31, mimics S31 phosphorylation) inhibits KDM4B, leading to increased H3K9me3 and DNA damage at telomeres. H3.3 E31 expression also increases damage at other heterochromatin regions including the pericentric heterochromatin and Y chromosome-specific satellite DNA repeats. We propose that H3.3 S31Ph regulation of KDM4B is required to control heterochromatin accessibility of repetitive DNA and preserve chromatin integrity.
... The replacement of histones by the oocyte-specific histone variants is critically important for transcriptional regulation and oocyte reprogramming (Nashun et al., 2015). Histone variant H3.3 is required for female fertility (Hödl and Basler, 2009;Sakai et al., 2009). Similar to spermatogenesis, continuous H3.3-H4 turnover and the expression of HIRA are critical for the transcriptional regulation of genes for developmental progression. ...
Dynamicity and flexibility of the chromatin landscape are critical for most of the DNA-dependent processes to occur. This higher-order packaging of the eukaryotic genome into the chromatin is mediated by histones and associated non-histone proteins that determine the states of chromatin. Histone chaperones- “the guardian of genome stability and epigenetic information” controls the chromatin accessibility by escorting the nucleosomal and non-nucleosomal histones as well as their variants. This distinct group of molecules is involved in all facets of histone metabolism. The selectivity and specificity of histone chaperones to the histones determine the maintenance of the chromatin in an open or closed state. This review highlights the functional implication of the network of histone chaperones in shaping the chromatin function in the development of an organism. Seminal studies have reported embryonic lethality at different stages of embryogenesis upon perturbation of some of the chaperones, suggesting their essentiality in development. We hereby epitomize facts and functions that emphasize the relevance of histone chaperones in orchestrating different embryonic developmental stages starting from gametogenesis to organogenesis in multicellular organisms.
... This notion supports the assumption that these histone variants play role in/during transcription activation. H3.3 has already been described to be involved in transcription activation 8 . H4r seems to behave similarly upon heat induction. ...
Histone variants are different from their canonical counterparts in structure and are encoded by solitary genes with unique regulation to fulfill tissue or differentiation specific functions. A single H4 variant gene ( His4r or H4r ) that is located outside of the histone cluster and gives rise to a polyA tailed messenger RNA via replication-independent expression is preserved in Drosophila strains despite that its protein product is identical with canonical H4. In order to reveal information on the possible role of this alternative H4 we epitope tagged endogenous H4r and studied its spatial and temporal expression, and revealed its genome-wide localization to chromatin at the nucleosomal level. RNA and immunohistochemistry analysis of H4r expressed under its cognate regulation indicate expression of the gene throughout zygotic and larval development and presence of the protein product is evident already in the pronuclei of fertilized eggs. In the developing nervous system a slight disequibrium in H4r distribution is observable, cholinergic neurons are the most abundant among H4r-expressing cells. ChIP-seq experiments revealed H4r association with regulatory regions of genes involved in cellular stress response. The data presented here indicate that H4r has a variant histone function.
... In Arabidopsis thaliana, H3.3 KO is lethal, and H3.3 knockdown impairs male gametogenesis (Wollmann et al. 2017). In Drosophila Melanogaster, males and females depleted for H3.3 are viable but sterile (Hödl and Basler 2009;Sakai et al. 2009). ...
The packaging of DNA into nucleosomes represents a challenge for transcription. Nucleosome disruption and histone eviction enables RNA Polymerase II progression through DNA, a process that compromises chromatin integrity and the maintenance of epigenetic information. Here, we used the imaging SNAP-tag system to distinguish new and old histones and monitor chromatin re-assembly coupled to transcription incells. First, we uncovered a loss of both old variants H3.1 and H3.3 that depends on transcriptional activity, with a major effect on H3.3. Focusing on transcriptionally active domains, we revealed a local enrichment in H3.3 with dynamics involving both new H3.3 incorporation and old H3.3 retention. Mechanistically, we demonstrate that the HIRA chaperone is critical to handle both new and old H3.3, and showed that this implicates different pathways. The de novo H3.3 deposition depends strictly on HIRA trimerization as well as its partner UBN1 while ASF1 interaction with HIRA can be bypassed. In contrast, the recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows an absolute dependency on ASF1-HIRA interaction. Therefore, we propose a model where HIRA can coordinate these distinct pathways for old H3.3 recycling and new H3.3 deposition during transcription to finetune chromatin states.
... Or, l'expression du transcrit H3.3Bse restreint au stade méiotique(Bramlage, Kosciessa, et Doenecke 1997). Les drosophiles mâles déficientes en H3.3 sont infertiles(Sakai et al. 2009). Des mutations germinales et somatiques dans des composants de la voie d'incorporation du génome H3.3 ou dans des gènes codant pour H3.3 ont été associées à des maladies congénitales humaines et à des cancers. ...
L’ADN s’organise via les histones, les principaux acteurs dans la compaction du matériel génétique. L’évolution technologique a favorisé la découverte de nombreux variants protéique. Toutefois, l’annotation de ces derniers s’est faite de manière non conventionnelle, compliquant leur identification par spectrométrie de masse. Ainsi, j’ai développé une banque de données exhaustive, intitulée MS_HistoneDB, dédiée à la détection des variants d’histone par spectrométrie de masse. MS_HistoneDB permet l’utilisation d’échantillons murin et humain. En outre, l’utilisation de tests immunologiques permet difficilement de discriminer au niveau protéique des variants quasi-similaire. Ainsi, j’ai développé une méthode d’analyse de spectrométrie de masse ciblée pour détecter et quantifier les variants d’histones en un essai multiplexe. Cette méthodologie a été appliqué dans l’investigation de la chromatine au cours de la spermatogenèse, dans des modèles murins, physiologique ou pathologique mimant l’infertilité masculine.Un autre aspect de mon travail s’est intéressé aux protéines liant les formes modifiées des histones. Ainsi, j’ai étudié les « readers »de la famille BET (Bromodomaine et Extra-terminal domain). Ces protéines sont recrutées sur la chromatine via leurs bromodomaines, module spécifique reconnaissant les histones acétylées. Leur domaine extra terminal joue le rôle de plateforme de recrutement de régulateurs transcriptionnels. Ces protéines sont conservées chez la levure Saccharomyces cerevisiae,et également chez les pathogènes fongiques responsables d’infections invasives, où l’unique membre est appelé Bdf1. Ainsi, j’ai étudié le domaine extra-terminale de Bdf1 et démontré qu’il est essentiel à la survie des levures. Puis, j’ai exploré les mécanismes moléculaires impliqués. Enfin, des inhibiteurs sélectifs sont en cours de développement dans des espèces de levure pathogène. L’ensemble de ces travaux ouvrent la voie au développement d’une nouvelle classe thérapeutique d’antifongiques.
... Although Caenorhabditis H3.3 is not required for viability (Delaney et al., 2018), in H3.3-deficient Drosophila males, chromosomes fail to condense properly for meiosis and undergo segregation defects (Sakai et al., 2009). In mice with H3.3 knockout mutations, failure to maintain heterochromatin leads to mitotic abnormalities and embryonic lethality (Jang et al., 2015). ...
Eukaryotic nucleosomes organize chromatin by wrapping 147 bp of DNA around a histone core particle comprising two molecules each of histone H2A, H2B, H3 and H4. The DNA entering and exiting the particle may be bound by the linker histone H1. Whereas deposition of bulk histones is confined to S-phase, paralogs of the common histones, known as histone variants, are available to carry out functions throughout the cell cycle and accumulate in post-mitotic cells. Histone variants confer different structural properties on nucleosomes by wrapping more or less DNA or by altering nucleosome stability. They carry out specialized functions in DNA repair, chromosome segregation and regulation of transcription initiation, or perform tissue-specific roles. In this Cell Science at a Glance article and the accompanying poster, we briefly examine new insights into histone origins and discuss variants from each of the histone families, focusing on how structural differences may alter their functions.
... H3.3 is associated with early replication domains and is recruited to sites of DNA repair in human cells (Adam et al. 2013;Clément et al. 2018). Loss of H3.3 results in lethality or sterility in most organisms (Hödl and Basler 2009;Sakai et al. 2009;Jang et al. 2015;Tang et al. 2015;Wollmann et al. 2017). The C. elegans genome contains five genes encoding H3.3 homologs, and we recently showed that knockout of all five genes resulted in viable worms with a reduced number of viable offspring at higher temperatures (Delaney et al. 2018). ...
Histone H3.3 is a replication-independent variant of histone H3 with important roles in development, differentiation, and fertility. Here, we show that loss of H3.3 results in replication defects in Caenorhabditis elegans embryos at elevated temperatures. To characterize these defects, we adapt methods to determine replication timing, map replication origins, and examine replication fork progression. Our analysis of the spatiotemporal regulation of DNA replication shows that despite the very rapid embryonic cell cycle, the genome is replicated from early and late firing origins and is partitioned into domains of early and late replication. We find that under temperature stress conditions, additional replication origins become activated. Moreover, loss of H3.3 results in altered replication fork progression around origins, which is particularly evident at stress-activated origins. These replication defects are accompanied by replication checkpoint activation, a delayed cell cycle, and increased lethality in checkpoint-compromised embryos. Our comprehensive analysis of DNA replication in C. elegans reveals the genomic location of replication origins and the dynamics of their firing, and uncovers a role of H3.3 in the regulation of replication origins under stress conditions.
... When both H3f3a and H3f3b are knocked out in mice, it causes embryonic lethality at embryonic day 6.5, and reduced expression leads to sterility, growth retardation, and increased neonatal lethality (10). Disrupting either His3.3A or His3.3B in Drosophila is tolerated; however, disrupting both leads to sterility and increased lethality (11). While knockout models have been studied in mice and Drosophila, germline missense mutations have not. ...
Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.
... Another scenario would be that excess H3.1 protein during M phase may directly compete with H3.3 variant for deposition onto genomic loci with higher H3.3 turnover. This appears to be possible since the overexpression of canonical H3.1 increased replication-independent H3.1 assembly outside of S phase in flies (Sakai et al., 2009). Moreover, a study showed that altering the ratio of H3.1 to H3.3 by increasing H3.1 protein resulted in replacement of H3.3 with H3.1 in regulatory regions of skeletal muscle cells (Harada et al., 2015). ...
Replication-dependent canonical histone messenger RNAs (mRNAs) do not terminate with a poly(A) tail at the 3’ end. We previously demonstrated that exposure to arsenic, an environmental carcinogen, induces polyadenylation of canonical histone H3.1 mRNA, causing transformation of human cells in vitro. Here we report that polyadenylation of H3.1 mRNA increases H3.1 protein, resulting in displacement of histone variant H3.3 at active promoters, enhancers, and insulator regions, leading to transcriptional deregulation, G2/M cell cycle arrest, chromosome aneuploidy and aberrations. In support of these observations, knocking down the expression of H3.3 induced cell transformation, whereas ectopic expression of H3.3 attenuated arsenic-induced cell transformation. Notably, arsenic exposure also resulted in displacement of H3.3 from active promoters, enhancers, and insulator regions. These data suggest that H3.3 displacement might be central to carcinogenesis caused by polyadenylation of H3.1 mRNA upon arsenic exposure. Our findings illustrate the importance of proper histone stoichiometry in maintaining genome integrity.
... Paradoxically, however, in humanized S. cerevisiae strains where all histones are exchanged for human orthologs, replacement with hH3.1 more readily produced colonies than with the hH3.3 variant 31 . In metazoans such as Drosophila melanogaster, the replicative variant can compensate for the loss of H3.3 during development in somatic tissues, although the adults are sterile [32][33][34][35][36] . As sterility could simply reflect a shortage of maternal H3.3 to replace protamine from sperm chromatin after fertilization, the most parsimonious hypothesis suggests that the nature of the variant itself might not be critical. ...
Vertebrates exhibit specific requirements for replicative H3 and non-replicative H3.3 variants during development. To disentangle whether this involves distinct modes of deposition or unique functions once incorporated into chromatin, we combined studies in Xenopus early development with chromatin assays. Here we investigate the extent to which H3.3 mutated at residues that differ from H3.2 rescue developmental defects caused by H3.3 depletion. Regardless of the deposition pathway, only variants at residue 31—a serine that can become phosphorylated—failed to rescue endogenous H3.3 depletion. Although an alanine substitution fails to rescue H3.3 depletion, a phospho-mimic aspartate residue at position 31 rescues H3.3 function. To explore mechanisms involving H3.3 S31 phosphorylation, we identified factors attracted or repulsed by the presence of aspartate at position 31, along with modifications on neighboring residues. We propose that serine 31-phosphorylated H3.3 acts as a signaling module that stimulates the acetylation of K27, providing a chromatin state permissive to the embryonic development program. Replicative and non-replicative H3 variants have different cell cycle regulation and incorporation modes. Here, the authors identify H3.3 S31 deposition as key at Xenopus gastrulation, independently of the pathways; and characterize H3.3 S31 phosphorylation importance for the underlying mechanisms.
Dosage compensation in Drosophila involves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions of MSL3 (male-specific lethal 3) with histone marks; in particular, Set2-dependent H3 lysine-36 trimethylation (H3K36me3). However, Set2 might affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Further, it is difficult to parse male-specific effects from those that are simply X-specific. To discriminate among these possibilities, we employed genomic approaches in H3K36 residue and Set2 writer mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global, male-specific reduction of X-genes in Set2/H3K36 mutants, we observe heterogeneous effects. Interestingly, we identified cohorts of genes whose expression was significantly altered following loss of H3K36 or Set2, but changes were in opposite directions, suggesting that H3K36me states have reciprocal functions. In contrast to H4K16R controls, analysis of combined H3.2K36R/H3.3K36R mutants neither showed consistent reduction in X-gene expression, nor any correlation with MSL3 binding. Examination of other developmental stages/tissues revealed additional layers of context-dependence. Our studies implicate BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 directly recruits the MSL complex. We propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.
Histones are the core components of the eukaryote chromosome, and have been implicated in transcriptional gene regulation. There are three major isoforms of histone H3 in Arabidopsis. Studies have shown that the H3.3 variant is pivotal in modulating nucleosome structure and gene transcription. However, the function of H3.3 during development remains to be further investigated in plants. In this study, we disrupted all three H3.3 genes in Arabidopsis. Two triple mutants, h3.3cr-4 and h3.3cr-5, were created by the CRISPR/Cas9 system. The mutant plants displayed smaller rosettes and decreased fertility. The stunted growth of h3.3cr-4 may result from reduced expression of cell cycle regulators. The shorter stamen filaments, but not the fertile ability of the gametophytes, resulted in reduced fertility of h3.3cr-4. The transcriptome analysis suggested that the reduced filament elongation of h3.3cr-4 was probably caused by the ectopic expression of several JASMONATE-ZIM DOMAIN (JAZ) genes, which are the key repressors of the signaling pathway of the phytohormone jasmonic acid (JA). These observations suggest that the histone variant H3.3 promotes plant growth, including rosette growth and filament elongation.
Aging is a multifactorial process that disturbs homeostasis, increases disease susceptibility, and ultimately results in death. Although the definitive set of molecular mechanisms responsible for aging remain to be discovered, epigenetic change over time is proving to be a promising piece of the puzzle. Several posttranslational histone modifications (PTMs) have been linked to the maintenance of longevity. Here, we focus on lysine-36 of the replication-independent histone protein, H3.3 (H3.3K36). To interrogate the role of this residue in Drosophila developmental gene regulation, we generated a lysine to arginine mutant that blocks the activity of its cognate modifying enzymes. We found that an H3.3BK36R mutation causes a significant reduction in adult lifespan, accompanied by dysregulation of the genomic and transcriptomic architecture. Transgenic co-expression of wild-type H3.3B completely rescues the longevity defect. Because H3.3 is known to accumulate in non-dividing tissues, we carried out transcriptome profiling of young vs aged adult fly heads. The data show that loss of H3.3K36 results in age-dependent misexpression of NF-κB and other innate immune target genes, as well as defects in silencing of heterochromatin. We propose H3.3K36 maintains the postmitotic epigenomic landscape, supporting longevity by regulating both pericentric and telomeric retrotransposons and by suppressing aberrant immune signaling.
Reprogramming of chromatin structure and changes of gene expression are critical for plant male gamete development and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3-1 is sterile due to male sterility. Transcriptome analysis of young inflorescence have identified 2348 genes downregulated in h3.3-1 mutant among which 1087 target genes are directly bound by H3.3, especially at their 3ʹ ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation, and pollen tube growth. And the function of H3.3 in male gamete development is dependent on the ASF1A/1B-HIRA-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development.
The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is also reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.
Hunger is an ancient drive, yet the molecular nature of pressures of this sort and how they modulate physiology are unknown. We find that hunger modulates aging in Drosophila. Limitation of branched-chain amino acids (BCAAs) or activation of hunger-promoting neurons induced a hunger state that extended life span despite increased feeding. Alteration of the neuronal histone acetylome was associated with BCAA limitation, and preventing these alterations abrogated the effect of BCAA limitation to increase feeding and extend life span. Hunger acutely increased feeding through usage of the histone variant H3.3, whereas prolonged hunger seemed to decrease a hunger set point, resulting in beneficial consequences for aging. Demonstration of the sufficiency of hunger to extend life span reveals that motivational states alone can be deterministic drivers of aging.
Adult pluripotent stem cell (aPSC) populations underlie whole-body regeneration in many distantly-related animal lineages, but how the underlying cellular and molecular mechanisms compare across species is unknown. Here, we apply single-cell RNA sequencing to profile transcriptional cell states of the acoel worm Hofstenia miamia during postembryonic development and regeneration. We identify cell types shared across stages and their associated gene expression dynamics during regeneration. Functional studies confirm that the aPSCs, also known as neoblasts, are the source of differentiated cells and reveal transcription factors needed for differentiation. Subclustering of neoblasts recovers transcriptionally distinct subpopulations, the majority of which are likely specialized to differentiated lineages. One neoblast subset, showing enriched expression of the histone variant H3.3, appears to lack specialization. Altogether, the cell states identified in this study facilitate comparisons to other species and enable future studies of stem cell fate potentials.
The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is also reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.
The term epigenetics dates back to the 1940s, when Conrad Waddington introduced it to refer to gene expression changes that occur during development and do not involve alterations in the DNA sequence. Subsequently the definition expanded beyond development, and the field became one of the most rapidly developing ones in life sciences. Advances in epigenetics transformed our understanding of cellular and molecular events that occur during development, homeostasis, and disease and helped explain processes that have long fascinated and puzzled scientists, such as the link between inflammation and disease, the intricacies of memory formation and maintenance, and the connection between the social environment/social adversity and chronic disease risk. Epigenetically mediated gene expression changes were described in a broad group of medical conditions, including cancer and neurodegenerative, metabolic, autoimmune, psychiatric, and cardiovascular diseases and, of these, the most advanced understanding of their contribution, so far, has occurred for cancer. Changes in DNA methylation, histone posttranslational modifications, and microRNA alterations, described in a broad group of human cancers, were implicated in all stages of carcinogenesis, including initiation, progression, invasion, and metastasis. The discovery of epigenetic biomarkers facilitated novel strategies for the early detection of disease, helped better monitor progression, therapeutic response, and prognosis, and revolutionized personalized medicine. Moreover, the reversible nature of epigenetic marks opened the possibility to therapeutically reverse aberrant gene expression patterns and catalyzed the emergence of epigenetic drugs. Besides their promise as monotherapies, epigenetic drugs show considerable interest thanks to the possibility to combine them with other cancer therapeutic modalities, such as chemotherapy, hormone therapy, and radiation therapy.KeywordsCancerDNA methylationEpigeneticsGene expressionHistone post-translational modificationsRNA interference
Histones serve many purposes in eukaryotic cells in the regulation of diverse genomic processes, including transcription, replication, DNA repair, and chromatin organization. As such, experimental systems to assess histone function are fundamental resources toward elucidating the regulation of activities occurring on chromatin. One set of important tools for investigating histone function are histone replacement systems, in which endogenous histone expression can be partially or completely replaced with a mutant histone. Histone replacement systems allow systematic screens of histone regulatory functions and the direct assessment of functions for histone residues. In this review, we describe existing histone replacement systems in model organisms, the benefits and limitations of these systems, and opportunities for future research with histone replacement strategies.
The nuclear environment changes dramatically over the course of early development. Histones are core chromatin components that play critical roles in regulating gene expression and nuclear architecture. Additionally, the embryos of many species, including Drosophila, Zebrafish, and Xenopus use the availability of maternally deposited histones to time critical early embryonic events including cell cycle slowing and zygotic genome activation. Here, we review recent insights into how histones control early development. We first discuss the regulation of chromatin functions through interaction of histones and transcription factors, incorporation of variant histones, and histone post-translational modifications. We also highlight emerging roles for histones as developmental regulators independent of chromatin association.
The histone chaperone DAXX targets H3.3 to pericentric and telomeric heterochromatin in a replication independent manner. The mechanism that ensures appropriate recruitement of DAXX to heterochromatic region is largely unknown. Using proteomic and buochemical approaches, we show that in addition to the well-known ATRX/H3.3 complex, DAXX forms an alternative complex with CAF-1, ADNP, HP1 and trim28. DAXX physically interact with the C-terminus of CAF1-p150 subunit through its N-terminal domain. The DAXX SIM domain was found to be essential for targeting DAXX to PML-NBs, for the recruitment of CAF-1 to heterochromatin. Our genomic date further revealed that DAXX targets CAF-1 to X-chromosome and repetitive elements. Inactivation of DAXX results in major depletion of CAF-1 from X-chromosome SINEs and LINEs. Our data point to a novel function of DAXX and CAF-1 in targeting H3.3 to X-chromosome.
The role of DNA is to store an individual's genetic information so that it can be used during development and can be copied accurately during the divisions of the cell. DNA has to be packed within a small nucleus in an organized manner to be accessible for a variety of vital cell processes (transcription, DNA replication, repair, mitosis and meiosis). This is achieved by DNA associating with different proteins to form chromatin.
Different levels of compaction are involved, from the nucleosome fibre to higher order chromatin structures. All the chromatin components involved at different levels of DNA compaction are highly dynamic, allowing the required accessibility.
The understanding of chromatin components, their dynamics and interactions in plants would allow us to provide plant breeders with new tools to help fulfil the increased demand for food that will be required in future decades of increased global population and climate change.
The genome of eukaryotic cells is packaged into chromatin, which establishment and maintenance require mechanisms of assembly and remodelling. This thesis work was dedicated to the characterization of two factors of chromatin assembly machinery. The first factor studied in this work was HIRIP3, a mammalian homologue of yeast H2A.Z chaperone Chz1. We aimed to test whether HIRIP3 is a histone chaperone by itself. At first, we established HIRIP3 interaction with histones in vivo. After then, we studied the structural specificity of this interaction in vitro. We have characterized HIRIP3 as a novel H2A histone chaperone that utilizes the CHZ motif for its function. The second part of this work was focused on SRCAP chromatin remodelling complex. We aimed to decipher its interaction network and to describe its sub-complexes. We have reconstituted YL1, SRCAP, TIP49A, TIP49B and H2A.Z/H2B core complex using baculovirus expression system. Our protocol allowed us to purify core complex suitable for future structural studies by cryo-electron microscopy.
Nucleosome dynamics and properties are central to all forms of genomic activities. Among the core histones, H3 variants play a pivotal role in modulating nucleosome structure and function. Here, we focus on the impact of H3 variants on various facets of development. The deposition of replicative H3 variant following DNA replication is essential for the transmission of the epigenomic information encoded in posttranscriptional modifications. Through this process, replicative H3 maintains cell fate while, in contrast, the replacement H3.3 variant opposes cell differentiation during early embryogenesis. In later steps of development, H3.3 and specialized H3 variants are emerging as new, important regulators of terminal cell differentiation, including neurons and gametes. The specific pathways that regulate the dynamics of the deposition of H3.3 are paramount during reprogramming events that drive zygotic activation and the initiation of a new cycle of development.
Expected final online publication date for the Annual Review of Genetics, Volume 54 is November 23, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer. Histone variants differ from canonical histones in their genomic localization, regulation and function. Incorporation of histone variants endows specific genomic regions with unique features to fine-tune gene expression, contributing to animal development and disease pathogenesis.
Maternally inherited RNA and proteins control much of embryonic development. The effect of such maternal information beyond embryonic development is largely unclear. Here, we report that maternal contribution of histone H3.3 assembly complexes can prevent the expression of late-onset anatomical, physiologic, and behavioral abnormalities of C. elegans. We show that mutants lacking hira-1, an evolutionarily conserved H3.3-deposition factor, have severe pleiotropic defects that manifest predominantly at adulthood. These late-onset defects can be maternally rescued, and maternally derived HIRA-1 protein can be detected in hira-1(−/−) progeny. Mitochondrial stress likely contributes to the late-onset defects, given that hira-1 mutants display mitochondrial stress, and the induction of mitochondrial stress results in at least some of the hira-1 late-onset abnormalities. A screen for mutants that mimic the hira-1 mutant phenotype identified PQN-80—a HIRA complex component, known as UBN1 in humans—and XNP-1—a second H3.3 chaperone, known as ATRX in humans. pqn-80 and xnp-1 abnormalities are also maternally rescued. Furthermore, mutants lacking histone H3.3 have a late-onset defect similar to a defect of hira-1, pqn-80, and xnp-1 mutants. These data demonstrate that H3.3 assembly complexes provide non-DNA-based heritable information that can markedly influence adult phenotype. We speculate that similar maternal effects might explain the missing heritability of late-onset human diseases, such as Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes.
Transcription of the four yeast histone gene pairs (HTA1-HTB1, HTA2-HTB2, HHT1-HHF1, and HHT2-HHF2) is repressed during G1, G2, and M. For all except HTA2-HTB2, this repression requires several trans-acting factors, including the products of the HIR genes, HIR1, HIR2, and HIR3. ASF1 is a highly conserved protein that has been implicated in transcriptional silencing and chromatin assembly. In this analysis, we show that HIR1 interacts with ASF1 in a two-hybrid analysis. Further, asf1 mutants, like hir mutants, are defective in repression of histone gene transcription during the cell cycle and in cells arrested in early S phase in response to hydroxyurea. asf1 and hir1 mutations also show very similar synergistic interactions with mutations in cac2, a subunit of the yeast chromatin assembly factor CAF-I. The results suggest that ASF1 and HIR1 function in the same pathway to create a repressive chromatin structure in the histone genes during the cell cycle.
Histones are the fundamental components of the nucleosome. Physiologically relevant variation is introduced into this structure through chromatin remodeling, addition of covalent modifications, or replacement with specialized histone variants. The histone H3 family contains an evolutionary conserved variant, H3.3, which differs in sequence in only five amino acids from the canonical H3, H3.1, and was shown to play a role in the transcriptional activation of genes. Histone H3.3 contains a serine (S) to alanine (A) replacement at amino acid position 31 (S31). Here, we demonstrate by both MS and biochemical methods that this serine is phosphorylated (S31P) during mitosis in mammalian cells. In contrast to H3 S10 and H3 S28, which first become phosphorylated in prophase, H3.3 S31 phosphorylation is observed only in late prometaphase and metaphase and is absent in anaphase. Additionally, H3.3 S31P forms a speckled staining pattern on the metaphase plate, whereas H3 S10 and H3 S28 phosphorylation localizes to the outer regions of condensed DNA. Furthermore, in contrast to phosphorylated general H3, H3.3 S31P is localized in distinct chromosomal regions immediately adjacent to centromeres. These findings argue for a unique function for the phosphorylated isoform of H3.3 that is distinct from its suspected role in gene activation.
• mitosis
• cell cycle
• subtype modification
In Tetrahymena, HHT1 and HHT2 genes encode the same major histone H3; HHT3 and HHT4 encode similar minor H3 variants (H3s), H3.3 and H3.4. Green fluorescent protein (GFP)-tagged H3 is deposited onto chromatin
through a DNA replication-coupled (RC) pathway. GFP-tagged H3.3 and H3.4 can be deposited both by a transcription-associated,
replication-independent (RI) pathway and also weakly by an RC pathway. Although both types of H3s can be deposited by the
RC pathway, DNA repair synthesis associated with meiotic recombination utilizes H3 specifically. The regions distinguishing
H3 and H3.3 for their deposition pathways were identified. RC major H3 is not essential. Cells can grow without major H3 if
the minor H3s are expressed at high levels. Surprisingly, cells lacking RI H3s are also viable and maintain normal nucleosome
density at a highly transcribed region. The RC H3 is not detectably deposited by the RI pathway, even when there are no RI
H3s available, indicating that transcription-associated RI H3 deposition is not essential for transcription. Minor H3s are
also required to produce viable sexual progeny and play an unexpected role in the germ line micronuclei late in conjugation
that is unrelated to transcription.
Histone mRNA metabolism is tightly coupled to cell cycle progression and to rates of DNA synthesis. The recent identification of several novel proteins involved in histone gene transcription and pre-mRNA processing has shed light on the variety of mechanisms cells employ to achieve this coupling.
Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem–loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3′ end. Stem–loop–binding protein (SLBP) binds to the histone mRNA 3′ end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations indSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3′ ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3′ end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle.
Keywords
• Drosophila
• cell cycle
• SLBP
• histone
• replication
• RNA processing
Drosophila melanogaster is a widely used model organism for genetic dissection of developmental processes. To exploit its full potential for studying the genetic basis of male fertility, we performed a large-scale screen for male-sterile (ms) mutations. From a collection of 12,326 strains carrying ethyl-methanesulfonatetreated, homozygous viable second or third chromosomes, 2216 ms lines were identified, constituting the largest collection of ms mutations described to date for any organism. Over 2000 lines were cytologically characterized and, of these, 81% failed during spermatogenesis while 19% manifested postspermatogenic processes. Of the phenotypic categories used to classify the mutants, the largest groups were those that showed visible defects in meiotic chromosome segregation or cytokinesis and those that failed in sperm individualization. We also identified 62 fertile or subfertile lines that showed high levels of chromosome loss due to abnormal mitotic or meiotic chromosome transmission in the male germ line or due to paternal chromosome loss in the early embryo. We argue that the majority of autosomal genes that function in male fertility in Drosophila are represented by one or more alleles in the ms collection. Given the conservation of molecular mechanisms underlying important cellular processes, analysis of these mutations should provide insight into the genetic networks that control male fertility in Drosophila and other organisms, including humans.
Di- and trimethylation of histone H3 lysine 4 (H3K4me2 and H3K4me3) are hallmarks of chromatin at active genes. The major fraction of K4-methylated histone H3 is the variant H3 (termed H3.3 in Drosophila), which replaces canonical H3 (H3.2) in transcribed genes. Here, we genetically address the in vivo significance of such K4 methylation by replacing wild-type H3.3 with a mutant form (H3.3K4A) that cannot be methylated. We monitored the transcription that occurs in response to multiple well-described signaling pathways. Surprisingly, the transcriptional outputs of these pathways remain intact in H3.3K4A mutant cells. Even the complete absence of both H3.3 genes does not noticeably affect viability or function of cells: double mutant animals are viable but sterile. Fertility can be rescued by K4-containing versions of H3.3, but not with mutant H3.3 (H3.3K4A) or with canonical H3.2. Together, these data suggest that in Drosophila, presence of H3.3K4me in the chromatin of active genes is dispensable for successful transcription in most cells and only plays an important role in reproductive tissues.
We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.
We demonstrate that in Drosophila melanogaster the histone H3.3 replacement variant is encoded by two genes, H3.3A and H3.3B. We have isolated cDNA clones for H3.3A and cDNA and genomic clones for H3.3B. The genes encode exactly the same protein but are widely divergent in their untranslated regions (UTR). Both genes are expressed in embryos and adults; they are expressed in the gonads as well as in somatic tissues of the flies. However, only one of them, H3.3A, shows strong testes expression. The 3' UTR of the H3.3A gene is relatively short (approximately 250 nucleotides (nt)). H3.3B transcripts can be processed at several polyadenylation sites, the longest with a 3' UTR of more than 1500 nt. The 3' processing sites, preferentially used in the gonads and somatic tissues, are different. We have also isolated the Drosophila hydei homologues of the two H3.3 genes. They are quite similar to the D. melanogaster genes in their expression patterns. However, in contrast to their vertebrate counterparts, which are highly conserved in their noncoding regions, the Drosophila genes display only limited sequence similarity in these regions.
Heterochromatin is a cytologically visible form of condensed chromatin capable of repressing genes in eukaryotic cells. For the yeast Saccharomyces cerevisiae, despite the absence of observable heterochromatin, there is genetic and chromatin structure data which indicate that there are heterochromatin-like repressive structures. Genes experience position effects at the silent mating loci and the telomeres, resulting in a repressed state that is inherited in an epigenetic manner. The histone H4 amino terminus is required for repression at these loci. Additional studies have indicated that the histone H3 N terminus is not important for silent mating locus repression, but redundancy of repressive elements at the silent mating loci may be responsible for masking its role. Here we report that histone H3 is required for full repression at yeast telomeres and at partially disabled silent mating loci, and that the acetylatable lysine residues of H3 play an important role in silencing.
Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem-loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3' end. Stem-loop-binding protein (SLBP) binds to the histone mRNA 3' end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3' ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3' end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle.
Two very similar H3 histones-differing at only four amino acid positions-are produced in Drosophila cells. Here we describe a mechanism of chromatin regulation whereby the variant H3.3 is deposited at particular loci, including active rDNA arrays. While the major H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition. In contrast to replication-coupled (RC) deposition, RI deposition does not require the N-terminal tail. H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast. RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification. Inheritance of newly deposited nucleosomes may then mark sites as active loci.
Histone mRNA metabolism is tightly coupled to cell cycle progression and to rates of DNA synthesis. The recent identification of several novel proteins involved in histone gene transcription and pre-mRNA processing has shed light on the variety of mechanisms cells employ to achieve this coupling.
Chromatin states can be distinguished by differential covalent modifications of histones or by utilization of histone variants. Chromatin associated with transcriptionally active loci becomes enriched for histones with particular lysine modifications and accumulates the H3.3 histone variant, the substrate for replication-independent nucleosome assembly. However, studies of modifications at particular loci have not distinguished between histone variants, so the relationship among modifications, histone variants, and nucleosome assembly pathways is unclear. To address this uncertainty, we have quantified the relative abundance of H3 and H3.3 and their lysine modifications. Using a Drosophila cell line system in which H3.3 has been shown to specifically package active loci, we found that H3.3 accounts for approximately 25% of total histone 3 in bulk chromatin, enough to package essentially all actively transcribed genes. MS and antibody characterization of separated histone 3 fractions revealed that H3.3 is relatively enriched in modifications associated with transcriptional activity and deficient in dimethyl lysine-9, which is abundant in heterochromatin. To explain enrichment on alternative variants, we propose that histone modifications are tied to the alternative nucleosome assembly pathways that use primarily H3 at replication forks and H3.3 at actively transcribed genes in a replication-independent manner.
Drosophila melanogaster is a widely used model organism for genetic dissection of developmental processes. To exploit its full potential for studying the genetic basis of male fertility, we performed a large-scale screen for male-sterile (ms) mutations. From a collection of 12,326 strains carrying ethyl-methanesulfonate-treated, homozygous viable second or third chromosomes, 2216 ms lines were identified, constituting the largest collection of ms mutations described to date for any organism. Over 2000 lines were cytologically characterized and, of these, 81% failed during spermatogenesis while 19% manifested postspermatogenic processes. Of the phenotypic categories used to classify the mutants, the largest groups were those that showed visible defects in meiotic chromosome segregation or cytokinesis and those that failed in sperm individualization. We also identified 62 fertile or subfertile lines that showed high levels of chromosome loss due to abnormal mitotic or meiotic chromosome transmission in the male germ line or due to paternal chromosome loss in the early embryo. We argue that the majority of autosomal genes that function in male fertility in Drosophila are represented by one or more alleles in the ms collection. Given the conservation of molecular mechanisms underlying important cellular processes, analysis of these mutations should provide insight into the genetic networks that control male fertility in Drosophila and other organisms, including humans.
DNA in eukaryotic cells is packaged into nucleosomes, the structural unit of chromatin. Both DNA and bulk histones are extremely long-lived, because old DNA strands and histones are retained when chromatin duplicates. In contrast, we find that the Drosophila HSP70 genes rapidly lose histone H3 and acquire variant H3.3 histones as they are induced. Histone replacement does not occur at artificial HSP70 promoter arrays, demonstrating that transcription is required for H3.3 deposition. The H3.3 histone is enriched in all active chromatin and throughout large transcription units, implying that deposition occurs during transcription elongation. Strikingly, we observed that the stability of chromatin-bound H3.3 differs between loci: H3.3 turns over at continually active rDNA genes, but becomes stable at induced HSP70 genes that have shut down. We conclude that H3.3 deposition is coupled to transcription, and continues while a gene is active. Repeated histone replacement suggests a mechanism to both maintain the structure of chromatin and access to DNA at active genes.
Chromatin can be differentiated by the deposition of variant histones at centromeres, active genes, and silent loci. Variant histones are assembled into nucleosomes in a replication-independent manner, in contrast to assembly of bulk chromatin that is coupled to replication. Recent in vitro studies have provided the first glimpses of protein machines dedicated to building and replacing alternative nucleosomes. They deposit variant H2A and H3 histones and are targeted to particular functional sites in the genome. Differences between variant and canonical histones can have profound consequences, either for delivery of the histones to sites of assembly or for their function after incorporation into chromatin. Recent studies have also revealed connections between assembly of variant nucleosomes, chromatin remodeling, and histone post-translational modification. Taken together, these findings indicate that chromosome architecture can be highly dynamic at the most fundamental level, with epigenetic consequences.
In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.
Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. We have examined chromatin at Drosophila homeotic gene clusters by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. Our results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements.
The Development of Drosophila
- Mt Fuller
- M Bate
- Martinez
- A Arias
Fuller, MT. Spermatogenesis. In: Bate, M.; Martinez-Arias, A., editors. The Development of Drosophila. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1993. p. 71-147
The Development of Drosophila
- M T Fuller
- Spermatogenesis
Fuller, MT. Spermatogenesis. In: Bate, M.; Martinez-Arias, A., editors. The Development of
Drosophila. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1993. p. 71-147.