RESUMO
Histone lysine methyltransferase SUV420H1, which is responsible for site-specific di-/tri-methylation of histone H4 lysine 20 (H4K20), has crucial roles in DNA-templated processes, including DNA replication, DNA damage repair, and chromatin compaction. Its mutations frequently occur in human cancers. Nucleosomes containing the histone variant H2A.Z enhance the catalytic activities of SUV420H1 on H4K20 di-methylation deposition, regulating early replication origins. However, the molecular mechanism by which SUV420H1 specifically recognizes and deposits H4K20 methyl marks on nucleosomes remains poorly understood. Here we report the cryo-electron microscopy structures of SUV420H1 associated with H2A-containing nucleosome core particles (NCPs), and H2A.Z-containing NCPs. We find that SUV420H1 makes extensive site-specific contacts with histone and DNA regions. SUV420H1 C-terminal domain recognizes the H2A-H2B acidic patch of NCPs through its two arginine anchors, thus enabling H4K20 insertion for catalysis specifically. We also identify important residues increasing the catalytic activities of SUV420H1 bound to H2A.Z NCPs. In vitro and in vivo functional analyses reveal that multiple disease-associated mutations at the interfaces are essential for its catalytic activity and chromatin state regulation. Together, our study provides molecular insights into the nucleosome-based recognition and methylation mechanisms of SUV420H1, and a structural basis for understanding SUV420H1-related human disease.
RESUMO
DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1-24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. In vitro and in vivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214-223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome.
Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , Nucleossomos/genética , Metilação , DNA/metabolismo , Replicação do DNARESUMO
The transmission and maintenance of genetic information in eukaryotic cells relies on the faithful duplication of the entire genome. In each round of division, excessive replication origins are licensed, with only a fraction activated to give rise to bi-directional replication forks in the context of chromatin. However, it remains elusive how eukaryotic replication origins are selectively activated. Here we demonstrate that O-GlcNAc transferase (OGT) enhances replication initiation by catalyzing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing reduced phosphorylation of the replicative helicase mini-chromosome maintenance (MCM) complex and compromised DNA unwinding. Our short nascent-strand sequencing results further confirm the importance of H4S47 O-GlcNAcylation in origin activation. We propose that H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the control of replication efficiency by chromatin environment.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Origem de Replicação , Replicação do DNA , Cromatina/metabolismo , Mamíferos/genéticaRESUMO
Histone variants have been implicated in regulating chromatin dynamics and genome functions. Previously, we have shown that histone variant H3.3 actively marks enhancers and cooperates with H2A.Z at promoters to prime the genes into a poised state in mouse embryonic stem cells (mESCs). However, how these two important histone variants collaboratively function in this process still remains elusive. In this study, we found that depletion of different components of HIRA complex, a specific chaperone of H3.3, results in significant decreases of H2A.Z enrichment at genome scale. In addition, CUT&Tag data revealed a genomic colocalization between HIRA complex and SRCAP complex. In vivo and in vitro biochemical assays verified that HIRA complex could interact with SRCAP complex through the Hira subunit. Furthermore, our chromatin accessibility and transcription analyses demonstrated that HIRA complex contributed to preset a defined chromatin feature around TSS region for poising gene transcription. In summary, our results unveiled that while regulating the H3.3 incorporation in the regulatory regions, HIRA complex also collaborates with SRCAP to deposit H2A.Z onto the promoters, which cooperatively determines the transcriptional potential of the poised genes in mESCs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina , CamundongosRESUMO
Spinal ependymomas are the most common spinal cord tumors in adults, but their intratumoral cellular heterogeneity has been less studied, and how spinal microglia are involved in tumor progression is still unknown. Here, our single-cell RNA-sequencing analyses of three spinal ependymoma subtypes dissect the microenvironmental landscape of spinal ependymomas and reveal tumor-associated macrophage (TAM) subsets with distinct functional phenotypes. CCL2+ TAMs are related to the immune response and exhibit a high capacity for apoptosis, while CD44+ TAMs are associated with tumor angiogenesis. By combining these results with those of single-cell ATAC-sequencing data analysis, we reveal that TEAD1 and EGR3 play roles in regulating the functional diversity of TAMs. We further identify diverse characteristics of both malignant cells and TAMs that might underlie the different malignant degrees of each subtype. Finally, assessment of cell-cell interactions reveal that stromal cells act as extracellular factors that mediate TAM diversity. Overall, our results reveal dual functions of TAMs in tumor progression, providing valuable insights for TAM-targeting immunotherapy.
Assuntos
Ependimoma/patologia , Neoplasias da Medula Espinal/patologia , Macrófagos Associados a Tumor/patologia , Apoptose , Comunicação Celular , Ependimoma/genética , Heterogeneidade Genética , Humanos , Neovascularização Patológica/patologia , Fenótipo , Análise de Célula Única , Neoplasias da Medula Espinal/genética , Células Estromais/patologia , Transcriptoma/genética , Microambiente Tumoral , Macrófagos Associados a Tumor/metabolismoRESUMO
Chromatin dynamics play a critical role in cell fate determination and maintenance by regulating the expression of genes essential for development and differentiation. In mouse embryonic stem cells (mESCs), maintenance of pluripotency coincides with a poised chromatin state containing active and repressive histone modifications. However, the structural features of poised chromatin are largely uncharacterized. By adopting mild time-course MNase-seq with computational analysis, the low-compact chromatin in mESCs is featured in two groups: one in more open regions, corresponding to an active state, and the other enriched with bivalent histone modifications, considered the poised state. A parameter called the chromatin opening potential index (COPI) is also devised to quantify the transcription potential based on the dynamic changes of MNase-seq signals at promoter regions. Use of COPI provides effective prediction of gene activation potential and, more importantly, reveals a few developmental factors essential for mouse neural progenitor cell (NPC) differentiation.
Assuntos
Cromatina/genética , Regulação da Expressão Gênica/genética , Células-Tronco Neurais/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Epigênese Genética/genética , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Código das Histonas/genética , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Nucleosome is the basic structural unit of chromatin, and its dynamics plays critical roles in the regulation of genome functions. However, how the nucleosome structure is regulated by histone variants in vivo is still largely uncharacterized. Here, by employing Micrococcal nuclease (MNase) digestion of crosslinked chromatin followed by chromatin immunoprecipitation (ChIP) and paired-end sequencing (MNase-X-ChIP-seq), we mapped unwrapping states of nucleosomes containing histone variant H2A.Z in mouse embryonic stem (ES) cells. We found that H2A.Z nucleosomes are more enriched with unwrapping states compared with canonical nucleosomes. Interestingly, +1 H2A.Z nucleosomes with 30-80 bp DNA is correlated with less active genes compared with +1 H2A.Z nucleosomes with 120-140 bp DNA. We confirmed the unwrapping of H2A.Z nucleosomes under native condition by re-ChIP of H2A.Z and H2A after CTCF CUT&RUN in mouse ES cells. Importantly, we found that depletion of H2A.Z results in decreased unwrapping of H3.3 nucleosomes and increased CTCF binding. Taken together, through MNase-X-ChIP-seq, we showed that histone variant H2A.Z regulates nucleosome unwrapping in vivo and that its function in regulating transcription or CTCF binding is correlated with unwrapping states of H2A.Z nucleosomes.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Montagem e Desmontagem da Cromatina , Camundongos , Nucleossomos/genética , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
DNA replication is a tightly regulated process that ensures the precise duplication of the genome during the cell cycle1. In eukaryotes, the licensing and activation of replication origins are regulated by both DNA sequence and chromatin features2. However, the chromatin-based regulatory mechanisms remain largely uncharacterized. Here we show that, in HeLa cells, nucleosomes containing the histone variant H2A.Z are enriched with histone H4 that is dimethylated on its lysine 20 residue (H4K20me2) and with bound origin-recognition complex (ORC). In vitro studies show that H2A.Z-containing nucleosomes bind directly to the histone lysine methyltransferase enzyme SUV420H1, promoting H4K20me2 deposition, which is in turn required for ORC1 binding. Genome-wide studies show that signals from H4K20me2, ORC1 and nascent DNA strands co-localize with H2A.Z, and that depletion of H2A.Z results in decreased H4K20me2, ORC1 and nascent-strand signals throughout the genome. H2A.Z-regulated replication origins have a higher firing efficiency and early replication timing compared with other origins. Our results suggest that the histone variant H2A.Z epigenetically regulates the licensing and activation of early replication origins and maintains replication timing through the SUV420H1-H4K20me2-ORC1 axis.
Assuntos
Período de Replicação do DNA , Replicação do DNA , Histonas/metabolismo , Origem de Replicação/genética , DNA/metabolismo , Replicação do DNA/genética , Epigênese Genética , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Lisina/metabolismo , Metilação , Nucleossomos/química , Nucleossomos/metabolismo , Complexo de Reconhecimento de Origem/metabolismoRESUMO
Polo-like kinase 1 (Plk1) is a crucial regulator of cell cycle progression; but the mechanism of regulation of Plk1 activity is not well understood. We present evidence that Plk1 activity is controlled by a balanced methylation and phosphorylation switch. The methyltransferase G9a monomethylates Plk1 at Lys209, which antagonizes phosphorylation of T210 to inhibit Plk1 activity. We found that the methyl-deficient Plk1 mutant K209A affects DNA replication, whereas the methyl-mimetic Plk1 mutant K209M prolongs metaphase-to-anaphase duration through the inability of sister chromatids separation. We detected accumulation of Plk1 K209me1 when cells were challenged with DNA damage stresses. Ablation of K209me1 delays the timely removal of RPA2 and RAD51 from DNA damage sites, indicating the critical role of K209me1 in guiding the machinery of DNA damage repair. Thus, our study highlights the importance of a methylation-phosphorylation switch of Plk1 in determining its kinase activity and functioning in DNA damage repair.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Replicação do DNA , Ativação Enzimática , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-LikeRESUMO
Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5, Tgfb1 and Tgfbr2, for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.
Assuntos
Proteínas de Transporte/metabolismo , Histonas/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/fisiologia , Animais , Proteínas de Transporte/genética , Diferenciação Celular/fisiologia , Embrião de Mamíferos , Células Epiteliais/fisiologia , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organogênese/fisiologia , Organoides , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: H3.3 is an ancient and conserved H3 variant and plays essential roles in transcriptional regulation. HIRA complex, which is composed of HIRA, UBN1 or UBN2, and Cabin1, is a H3.3 specific chaperone complex. However, it still remains largely uncharacterized how HIRA complex specifically recognizes and deposits H3.3 to the chromatin, such as promoters and enhancers. RESULTS: In this study, we demonstrate that the UBN1 or UBN2 subunit is mainly responsible for specific recognition and direct binding of H3.3 by the HIRA complex. While the HIRA subunit can enhance the binding affinity of UBN1 toward H3.3, Cabin1 subunit cannot. We also demonstrate that both Ala87 and Gly90 residues of H3.3 are required and sufficient for the specific recognition and binding by UBN1. ChIP-seq studies reveal that two independent HIRA complexes (UBN1-HIRA and UBN2-HIRA) can cooperatively deposit H3.3 to cis-regulatory regions, including active promoters and active enhancers in mouse embryonic stem (mES) cells. Importantly, disruption of histone chaperone activities of UBN1 and UBN2 by FID/AAA mutation results in the defect of H3.3 deposition at promoters of developmental genes involved in neural differentiation, and subsequently causes the failure of activation of these genes during neural differentiation of mES cells. CONCLUSION: Together, our results provide novel insights into the mechanism by which the HIRA complex specifically recognizes and deposits H3.3 at promoters and enhancers of developmental genes, which plays a critical role in neural differentiation of mES cells.
Assuntos
Regulação da Expressão Gênica , Histonas/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/genética , Sequências Reguladoras de Ácido Nucleico , Proteínas Adaptadoras de Transdução de Sinal , Animais , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The hierarchical organization of eukaryotic chromatin plays a central role in gene regulation, by controlling the extent to which the transcription machinery can access DNA. The histone variants H3.3 and H2A.Z have recently been identified as key regulatory players in this process, but the underlying molecular mechanisms by which they permit or restrict gene expression remain unclear. Here, we investigated the regulatory function of H3.3 and H2A.Z on chromatin dynamics and Polycomb-mediated gene silencing. RESULTS: Our ChIP-seq analysis reveals that in mouse embryonic stem (mES) cells, H3K27me3 enrichment correlates strongly with H2A.Z. We further demonstrate that H2A.Z promotes PRC2 activity on H3K27 methylation through facilitating chromatin compaction both in vitro and in mES cells. In contrast, PRC2 activity is counteracted by H3.3 through impairing chromatin compaction. However, a subset of H3.3 may positively regulate PRC2-dependent H3K27 methylation via coordinating depositions of H2A.Z to developmental and signaling genes in mES cells. Using all-trans retinoic acid (tRA)-induced gene as a model, we show that the dynamic deposition of H2A.Z and H3.3 coordinately regulates the PRC2-dependent H3K27 methylation by modulating local chromatin structure at the promoter region during the process of turning genes off. CONCLUSIONS: Our study provides key insights into the mechanism of how histone variants H3.3 and H2A.Z function coordinately to finely tune the PRC2 enzymatic activity during gene silencing, through promoting or impairing chromosome compaction respectively.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Complexo Repressor Polycomb 2/genética , Animais , Linhagem Celular , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas , Complexo Repressor Polycomb 2/metabolismoRESUMO
Genomic DNA of eukaryotic cells is hierarchically packaged into chromatin by histones. The dynamic organization of chromatin fibers plays a critical role in the regulation of gene transcription and other DNA-associated biological processes. Recently, numerous approaches have been developed to map the chromatin organization by characterizing chromatin accessibilities in genome-wide. However, reliable methods to quantitatively map chromatin accessibility are not well-established, especially not on a genome-wide scale. Here, we developed a modified MNase-seq for mouse embryonic fibroblasts, wherein chromatin was partially digested at multiple digestion times using micrococcal nuclease (MNase), allowing quantitative analysis of local yet genome-wide chromatin compaction. Our results provide strong evidence that the chromatin accessibility at promoter regions are positively correlated with gene activity. In conclusion, our assay is an ideal tool for the quantitative study of gene regulation in the perspective of chromatin accessibility.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Mapeamento Cromossômico/métodos , Segregação de Cromossomos/genética , Fibroblastos/fisiologia , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação , Células Cultivadas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Análise de Sequência de DNA/métodosRESUMO
Histones are the main protein components of eukaryotic chromatin. Histone variants and histone modifications modulate chromatin structure, ensuring the precise operation of cellular processes associated with genomic DNA. H3.3, an ancient and conserved H3 variant, differs from its canonical H3 counterpart by only five amino acids, yet it plays essential and specific roles in gene transcription, DNA repair and in maintaining genome integrity. Here, we review the most recent insights into the functions of histone H3.3, and the involvement of its mutant forms in human diseases.
Assuntos
Histonas/genética , Histonas/metabolismo , Neoplasias/genética , Animais , Cromatina/genética , Reparo do DNA/genética , Desenvolvimento Embrionário/genética , Instabilidade Genômica/genética , Código das Histonas/genética , Humanos , Mutação , Transcrição Gênica/genéticaRESUMO
The histone variants H3.3 and H2A.Z have recently emerged as two of the most important features in transcriptional regulation, the molecular mechanism of which still remains poorly understood. In this study, we investigated the regulation of H3.3 and H2A.Z on chromatin dynamics during transcriptional activation. Our in vitro biophysical and biochemical investigation showed that H2A.Z promoted chromatin compaction and repressed transcriptional activity. Surprisingly, with only four to five amino acid differences from the canonical H3, H3.3 greatly impaired higher-ordered chromatin folding and promoted gene activation, although it has no significant effect on the stability of mononucleosomes. We further demonstrated that H3.3 actively marks enhancers and determines the transcriptional potential of retinoid acid (RA)-regulated genes via creating an open chromatin signature that enables the binding of RAR/RXR. Additionally, the H3.3-dependent recruitment of H2A.Z on promoter regions resulted in compaction of chromatin to poise transcription, while RA induction results in the incorporation of H3.3 on promoter regions to activate transcription via counteracting H2A.Z-mediated chromatin compaction. Our results provide key insights into the mechanism of how histone variants H3.3 and H2A.Z function together to regulate gene transcription via the modulation of chromatin dynamics over the enhancer and promoter regions.