Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia.
ABSTRACT: Establishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.Our results demonstrate that histone H3 tri-methylated at lysine 9 (H3K9me3), a hallmark of constitutive heterochromatin, as well as the chromatin remodeling protein ATRX remained associated with pericentric heterochromatin regions in spite of their extensive hypo-methylation. This suggests that in neonatal spermatogonia, chromosomal 5-methyl cytosine patterns are regulated independently of changes in histone methylation, potentially reflecting a crucial mechanism to maintain pericentric heterochromatin silencing. Furthermore, chromatin immunoprecipitation and fluorescence in situ hybridization, revealed that ATRX as well as H3K9me3 associate with Y chromosome-specific DNA sequences and decorate both arms of the Y chromosome, suggesting a possible role in heterochromatinization and the predominant transcriptional quiescence of this chromosome during spermatogenesis.These results are consistent with a role for histone modifications and chromatin remodeling proteins such as ATRX in maintaining transcriptional repression at constitutive heterochromatin domains in the absence of 5-methyl cytosine and provide evidence suggesting that the establishment and/or maintenance of repressive histone and chromatin modifications at pericentric heterochromatin following genome-wide epigenetic reprogramming in the germ line may precede the establishment of chromosomal 5-methyl cytosine patterns as a genomic silencing strategy in neonatal spermatogonia.
Project description:The ?-thalassemia/mental retardation X-linked protein (ATRX) is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein) to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA-containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.
Project description:Histone variant H3.3 is deposited in chromatin at active sites, telomeres, and pericentric heterochromatin by distinct chaperones, but the mechanisms of regulation and coordination of chaperone-mediated H3.3 loading remain largely unknown. We show here that the chromatin-associated oncoprotein DEK regulates differential HIRA- and DAAX/ATRX-dependent distribution of H3.3 on chromosomes in somatic cells and embryonic stem cells. Live cell imaging studies show that nonnucleosomal H3.3 normally destined to PML nuclear bodies is re-routed to chromatin after depletion of DEK. This results in HIRA-dependent widespread chromatin deposition of H3.3 and H3.3 incorporation in the foci of heterochromatin in a process requiring the DAXX/ATRX complex. In embryonic stem cells, loss of DEK leads to displacement of PML bodies and ATRX from telomeres, redistribution of H3.3 from telomeres to chromosome arms and pericentric heterochromatin, induction of a fragile telomere phenotype, and telomere dysfunction. Our results indicate that DEK is required for proper loading of ATRX and H3.3 on telomeres and for telomeric chromatin architecture. We propose that DEK acts as a "gatekeeper" of chromatin, controlling chromatin integrity by restricting broad access to H3.3 by dedicated chaperones. Our results also suggest that telomere stability relies on mechanisms ensuring proper histone supply and routing.
Project description:Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1alpha (HP1alpha)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1alpha-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1alpha with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.
Project description:Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function.
Project description:Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.
Project description:DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.
Project description:BACKGROUND: Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks. RESULTS: To gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin. CONCLUSION: FRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.
Project description:Functionally distinct chromatin domains are delineated by distinct posttranslational modifications of histones, and in some organisms by differences in DNA methylation. Proper establishment and maintenance of chromatin domains is critical but not well understood. We previously demonstrated that heterochromatin in the filamentous fungus Neurospora crassa is marked by cytosine methylation directed by trimethylated Lysine 9 on histone H3 (H3K9me3). H3K9me3 is the product of the DIM-5 Lysine methyltransferase and is recognized by a protein complex containing heterochromatin protein-1 and the DIM-2 DNA methyltransferase. To identify additional components that control the establishment and function of DNA methylation and heterochromatin, we built a strain harboring two selectable reporter genes that are silenced by DNA methylation and employed this strain to select for mutants that are defective in DNA methylation (dim). We report a previously unidentified gene (dim-7) that is essential for H3K9me3 and DNA methylation. DIM-7 homologs are found only in fungi and are highly divergent. We found that DIM-7 interacts with DIM-5 in vivo and demonstrated that a conserved domain near the N terminus of DIM-7 is required for its stability. In addition, we found that DIM-7 is essential for recruitment of DIM-5 to form heterochromatin.
Project description:The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.
Project description:Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins and modifications of histones and DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation (5mC), methylation of histone H3 lysine 9 (H3K9me), and heterochromatin protein 1 (HP1), and is a model to investigate heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3, and H3K4me2 at 100 bp resolution and explored their interplay. HP1, H3K9me3, and 5mC were extensively co-localized and defined 44 heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation. Interestingly, the centromere was found in an approximately 350 kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation in N. crassa. In contrast, we found that localization of H3K9me3 was independent of 5mC or HP1 at virtually all heterochromatin regions. In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the H3K9 methylation machinery. These data show that A:T-rich RIP'd DNA efficiently directs methylation of H3K9, which in turn, directs methylation of associated cytosines.