RESUMO
Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , Proteínas de Saccharomyces cerevisiae , DNA Polimerase III/metabolismo , DNA Polimerase III/genética , Histonas/metabolismo , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , DNA Polimerase Dirigida por DNA/metabolismoRESUMO
Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging strands, respectively. Single Dpb3 deletion (dpb3Δ) or Mcm2 mutation (mcm2-3A), which each disrupts one parental histone transfer pathway, leads to the other's predominance. However, the biological impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3Δ mcm2-3A double mutant did not exhibit the asymmetric parental histone patterns caused by a single dpb3Δ or mcm2-3A mutation, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3A and dpb3Δ mcm2-3A mutants, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones during DNA replication is essential for maintaining chromatin structure and that lower homologous recombination activity due to parental histone transfer defects is detrimental to cells.
Assuntos
Replicação do DNA , Histonas , Recombinação Homóloga , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Recombinação Homóloga/genética , Replicação do DNA/genética , Mutação , Cromatina/metabolismo , Cromatina/genética , DNA Polimerase II/metabolismo , DNA Polimerase II/genética , Epigênese Genética , Reparo do DNARESUMO
Background . Human hexokinase 2 ( HK2 ) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2 -driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of DNA and histones in cells, we hypothesized that altering HK2 expression-mediated cellular glycolysis rates could impact the epigenome and, consequently, genome stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 ( HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2 -dependent metabolism on parental nucleosome transfer, a key DNA replication-coupled epigenetic inheritance process, and chromatin stability. Results . By comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ , hxk2Δ , or double-loss of hxk1Δ hxk2Δ to wild-type cells, we demonstrated that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a HK2 inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability. Conclusion . Our findings suggest that in yeast cells lacking HXK2 , alternative HXK s such as HXK1 or glucokinase 1 ( GLK1 ) play a role in supporting glycolysis at a level that adequately maintain epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur independently of Hxk2-mediated glycolysis inhibition. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.
RESUMO
Parental histones, the carriers of posttranslational modifications, are deposited evenly onto the replicating DNA of sister chromatids in a process dependent on the Mcm2 subunit of DNA helicase and the Pole3 subunit of leading-strand DNA polymerase. The biological significance of parental histone propagation remains unclear. Here we show that Mcm2-mutated or Pole3-deleted mouse embryonic stem cells (ESCs) display aberrant histone landscapes and impaired neural differentiation. Mutation of the Mcm2 histone-binding domain causes defects in pre-implantation development and embryonic lethality. ESCs with biased parental histone transfer exhibit increased epigenetic heterogeneity, showing altered histone variant H3.3 and H3K27me3 patterning at genomic sites regulating differentiation genes. Our results indicate that the lagging strand pattern of H3.3 leads to the redistribution of H3K27me3 in Mcm2-2A ESCs. We demonstrate that symmetric parental histone deposition to sister chromatids contributes to cellular differentiation and development.
Assuntos
Histonas , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Histonas/genética , Células-Tronco Embrionárias , Diferenciação Celular/genética , DNA HelicasesRESUMO
Chromatin relaxation is a prerequisite for the DNA repair machinery to access double-strand breaks (DSBs). Local histones around the DSBs then undergo prompt changes in acetylation status, but how the large demands of acetyl-CoA are met is unclear. Here, we report that pyruvate dehydrogenase 1α (PDHE1α) catalyzes pyruvate metabolism to rapidly provide acetyl-CoA in response to DNA damage. We show that PDHE1α is quickly recruited to chromatin in a polyADP-ribosylation-dependent manner, which drives acetyl-CoA generation to support local chromatin acetylation around DSBs. This process increases the formation of relaxed chromatin to facilitate repair-factor loading, genome stability and cancer cell resistance to DNA-damaging treatments in vitro and in vivo. Indeed, we demonstrate that blocking polyADP-ribosylation-based PDHE1α chromatin recruitment attenuates chromatin relaxation and DSB repair efficiency, resulting in genome instability and restored radiosensitivity. These findings support a mechanism in which chromatin-associated PDHE1α locally generates acetyl-CoA to remodel the chromatin environment adjacent to DSBs and promote their repair.
Assuntos
Cromatina , Quebras de DNA de Cadeia Dupla , Acetilcoenzima A/metabolismo , Acetilação , Reparo do DNA , Dano ao DNA , PiruvatosRESUMO
Faithful inheritance of parental histones is essential to maintain epigenetic information and cellular identity during cell division. Parental histones are evenly deposited onto the replicating DNA of sister chromatids in a process dependent on the MCM2 subunit of DNA helicase. However, the impact of aberrant parental histone partition on human disease such as cancer is largely unknown. In this study, we construct a model of impaired histone inheritance by introducing MCM2-2A mutation (defective in parental histone binding) in MCF-7 breast cancer cells. The resulting impaired histone inheritance reprograms the histone modification landscapes of progeny cells, especially the repressive histone mark H3K27me3. Lower H3K27me3 levels derepress the expression of genes associated with development, cell proliferation, and epithelial to mesenchymal transition. These epigenetic changes confer fitness advantages to some newly emerged subclones and consequently promote tumor growth and metastasis after orthotopic implantation. In summary, our results indicate that impaired inheritance of parental histones can drive tumor progression.
Assuntos
Transição Epitelial-Mesenquimal , Histonas , Humanos , Histonas/genética , Histonas/metabolismo , Epigênese Genética , DNA Helicases/metabolismo , Código das HistonasRESUMO
Glycolysis is essential for the classical activation of macrophages (M1), but how glycolytic pathway metabolites engage in this process remains to be elucidated. Glycolysis leads to production of pyruvate, which can be transported into the mitochondria by the mitochondrial pyruvate carrier (MPC) followed by utilization in the tricarboxylic acid cycle. Based on studies that used the MPC inhibitor UK5099, the mitochondrial route has been considered to be of significance for M1 activation. Using genetic approaches, here we show that the MPC is dispensable for metabolic reprogramming and activation of M1 macrophages. In addition, MPC depletion in myeloid cells has no impact on inflammatory responses and macrophage polarization toward the M1 phenotype in a mouse model of endotoxemia. While UK5099 reaches maximal MPC inhibitory capacity at approximately 2-5 µM, higher concentrations are required to inhibit inflammatory cytokine production in M1 and this is independent of MPC expression. Taken together, MPC-mediated metabolism is dispensable for the classical activation of macrophages and UK5099 inhibits inflammatory responses in M1 macrophages due to effects other than MPC inhibition.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos , Camundongos , Animais , Proteínas de Transporte da Membrana Mitocondrial/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Mitocôndrias/metabolismo , Glicólise , Macrófagos/metabolismoRESUMO
Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging DNA strands, respectively. Single Dpb3 deletion ( dpb3Δ ) or Mcm2 mutation ( mcm2-3A ), which each disrupt one parental histone transfer pathway, leads to the other's predominance. However, the impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3Δ / mcm2-3A double mutant did not exhibit the single dpb3Δ and mcm2-3A mutants' asymmetric parental histone patterns, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3A , and dpb3Δ / mcm2-3A mutants relative to the wild-type strain, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones to the leading and lagging strands during DNA replication is essential for maintaining chromatin structure and that high levels of free histones due to parental histone transfer defects are detrimental to cells.
RESUMO
Constitutively activated B cell receptor (BCR) signaling is a primary biological feature of chronic lymphocytic leukemia (CLL). The biological events controlled by BCR signaling in CLL are not fully understood and need investigation. Here, by analysis of the chromatin states and gene expression profiles of CLL B cells from patients before and after Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib treatment, we show that BTKi treatment leads to a decreased expression of APOBEC3 family genes by regulating the activity of their enhancers. BTKi treatment reduces enrichment of enhancer marks (H3K4me1 and H3K27ac) and chromatin accessibility at putative APOBEC3 enhancers. CRISPR-Cas9 directed deletion or inhibition of the putative APOBEC3 enhancers leads to reduced APOBEC3 expression. We further find that transcription factor NFATc1 couples BCR signaling with the APOBEC3 enhancer activity to control APOBEC3 expression. We also find that enhancer-regulated APOBEC3 expression contributes to replication stress in malignant B cells. In total we demonstrate a novel mechanism for BTKi suppression of APOBEC3 expression via direct enhancer regulation in an NFATc1-dependent manner, implicating BCR signaling as a potential regulator of leukemic genomic instability.
Assuntos
Desaminases APOBEC , Leucemia Linfocítica Crônica de Células B , Receptores de Antígenos de Linfócitos B , Desaminases APOBEC/biossíntese , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Cromatina , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Chromatin structure contains critical epigenetic information in various forms, such as histone post-translational modifications (PTMs). The deposition of certain histone PTMs can remodel the chromatin structure, resulting in gene expression alteration. The epigenetic information carried by histone PTMs could be inherited by daughter cells to maintain the gene expression status. Recently, studies revealed that several conserved replisome proteins regulate the recycling of parental histones carrying epigenetic information in Saccharomyces cerevisiae. Hence, the proper recycling and deposition of parental histones onto newly synthesized DNA strands is presumed to be essential for epigenetic inheritance. Here, we first reviewed the fundamental mechanisms of epigenetic modification establishment and maintenance discovered within fungal models. Next, we discussed the functions of parental histone chaperones and the potential impacts of the parental histone recycling process on heterochromatin-mediated transcriptional silencing inheritance. Subsequently, we summarized novel synthetic biology approaches developed to analyze individual epigenetic components during epigenetic inheritance in fungal and mammalian systems. These newly emerged research paradigms enable us to dissect epigenetic systems in a bottom-up manner. Furthermore, we highlighted the approaches developed in this emerging field and discussed the potential applications of these engineered regulators to building synthetic epigenetic systems.
Assuntos
Cromatina , Histonas , Animais , Cromatina/genética , Epigênese Genética/genética , Epigenômica , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Mamíferos/genética , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.
Assuntos
Histonas , Proteoma , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Biotina/química , Biotina/metabolismo , Biotinilação , Código das Histonas , Histonas/metabolismo , Nucleossomos , Proteoma/metabolismo , Proteína Estafilocócica A/metabolismo , Estreptavidina/metabolismoRESUMO
In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Replicação do DNA/genética , DNA Fúngico/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMO
Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates.
Assuntos
Autofagia , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Tacrolimo/metabolismo , Quinases da Família src/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Piperidinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidoresRESUMO
The recent discovery of the cancer-associated E76K mutation in histone H2B (H2BE76-to-K) in several types of cancers revealed a new class of oncohistone. H2BE76K weakens the stability of histone octamers, alters gene expression, and promotes colony formation. However, the mechanism linking the H2BE76K mutation to cancer development remains largely unknown. In this study, we knock in the H2BE76K mutation in MDA-MB-231 breast cancer cells using CRISPR/Cas9 and show that the E76K mutant histone H2B preferentially localizes to genic regions. Interestingly, genes upregulated in the H2BE76K mutant cells are enriched for the E76K mutant H2B and are involved in cell adhesion and proliferation pathways. We focused on one H2BE76K target gene, ADAM19 (a disintegrin and metalloproteinase-domain-containing protein 19), a gene highly expressed in various human cancers including breast invasive carcinoma, and demonstrate that H2BE76K directly promotes ADAM19 transcription by facilitating efficient transcription along the gene body. ADAM19 depletion reduced the colony formation ability of the H2BE76K mutant cells, whereas wild-type MDA-MB-231 cells overexpressing ADAM19 mimics the colony formation phenotype of the H2BE76K mutant cells. Collectively, our data demonstrate the mechanism by which H2BE76K deregulates the expression of genes that control oncogenic properties through a combined effect of its specific genomic localization and nucleosome destabilization effect.
Assuntos
Proteínas ADAM/genética , Neoplasias da Mama/genética , Histonas/genética , Proteínas ADAM/metabolismo , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Histonas/metabolismo , Humanos , Mutação/genética , Nucleossomos , Oncogenes/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.
Assuntos
Regiões 3' não Traduzidas/genética , Antígenos de Neoplasias/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/patologia , RNA Mensageiro/metabolismo , Ubiquitina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Antígenos de Neoplasias/genética , Apoptose , Biomarcadores Tumorais , Carcinogênese , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Poliadenilação , Splicing de RNA , RNA Mensageiro/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Establishment and subsequent maintenance of distinct chromatin domains during embryonic stem cell (ESC) differentiation are crucial for lineage specification and cell fate determination. Here we show that the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which is recruited to DNA replication forks through its interaction with proliferating cell nuclear antigen (PCNA) for nucleosome assembly, participates in the establishment of H3K27me3-mediated silencing during differentiation. Deletion of CAF-1 p150 subunit impairs the silencing of many genes including Oct4, Sox2 and Nanog as well as the establishment of H3K27me3 at these gene promoters during ESC differentiation. Mutations of PCNA residues involved in recruiting CAF-1 to the chromatin also result in defects in differentiation in vitro and impair early embryonic development as p150 deletion. Together, these results reveal that the CAF-1-PCNA nucleosome assembly pathway plays an important role in the establishment of H3K27me3-mediated silencing during cell fate determination.
Assuntos
Diferenciação Celular/genética , Fator 1 de Modelagem da Cromatina/fisiologia , Células-Tronco Embrionárias/fisiologia , Heterocromatina/metabolismo , Células-Tronco Pluripotentes/fisiologia , Animais , Células Cultivadas , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Replicação do DNA/genética , Inativação Gênica/fisiologia , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Cellular senescence defines an irreversible cell growth arrest state linked to loss of tissue function and aging in mammals. This transition from proliferation to senescence is typically characterized by increased expression of the cell-cycle inhibitor p16INK4a and formation of senescence-associated heterochromatin foci (SAHF). SAHF formation depends on HIRA-mediated nucleosome assembly of histone H3.3, which is regulated by the serine/threonine protein kinase Pak2. However, it is unknown if Pak2 contributes to cellular senescence. Here, we show that depletion of Pak2 delayed oncogene-induced senescence in IMR90 human fibroblasts and oxidative stress-induced senescence of mouse embryonic fibroblasts (MEFs), whereas overexpression of Pak2 accelerated senescence of IMR90 cells. Importantly, depletion of Pak2 in BubR1 progeroid mice attenuated the onset of aging-associated phenotypes and extended life span. Pak2 is required for expression of genes involved in cellular senescence and regulated the deposition of newly synthesized H3.3 onto chromatin in senescent cells. Together, our results demonstrate that Pak2 is an important regulator of cellular senescence and organismal aging, in part through the regulation of gene expression and H3.3 nucleosome assembly.
Assuntos
Envelhecimento , Senescência Celular , Quinases Ativadas por p21/fisiologia , Envelhecimento/metabolismo , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Histonas/metabolismo , Longevidade , Camundongos Knockout , Estresse Oxidativo , Reação em Cadeia da Polimerase em Tempo Real , Quinases Ativadas por p21/metabolismoRESUMO
Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Polα axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging-strand DNA at replication forks. Mutating the conserved histone-binding domain of the Mcm2 subunit of the CMG (Cdc45-MCM-GINS) DNA helicase, which translocates along the leading-strand template, results in a marked enrichment of parental (H3-H4)2 on leading strand, due to the impairment of the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Polα primase mutants that disrupt the connection of the CMG helicase to Polα that resides on lagging-strand template. Our results support a model whereby parental (H3-H4)2 complexes displaced from nucleosomes by DNA unwinding at replication forks are transferred by the CMG-Ctf4-Polα complex to lagging-strand DNA for nucleosome assembly at the original location.
Assuntos
DNA Polimerase III/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Epigênese Genética , Histonas/genética , Complexos Multiproteicos/genética , Nucleossomos/genética , Ligação Proteica , Saccharomyces cerevisiae/genéticaRESUMO
How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging-strand preference increases markedly in budding yeast cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, owing to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading versus lagging strands and that Dbp3-Dpb4 plays an important role in this poorly understood process.
Assuntos
Replicação do DNA , Epigênese Genética , Histonas/metabolismo , Saccharomycetales/metabolismo , DNA/genética , DNA/metabolismo , DNA Polimerase II/genética , Deleção de Genes , Heterocromatina/química , Heterocromatina/metabolismo , Nucleossomos/metabolismo , Multimerização Proteica , Saccharomycetales/genéticaRESUMO
Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.