RESUMO
Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.
Assuntos
Complexos Multiproteicos , Mutação , Neoplasias , Proteína SMARCB1 , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/genética , Neoplasias/metabolismo , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteólise , Ubiquitina/metabolismoRESUMO
Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Proteínas F-Box , Histona-Lisina N-Metiltransferase , Histona Desmetilases com o Domínio Jumonji , Proteínas do Grupo Polycomb , Proteína SMARCB1 , Cromatina/genética , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Células Tumorais Cultivadas/metabolismoRESUMO
For cells to initiate and sustain a differentiated state, it is necessary that a 'memory' of this state is transmitted through mitosis to the daughter cells1-3. Mammalian switch/sucrose non-fermentable (SWI/SNF) complexes (also known as Brg1/Brg-associated factors, or BAF) control cell identity by modulating chromatin architecture to regulate gene expression4-7, but whether they participate in cell fate memory is unclear. Here we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but are bound to promoters during mitosis, and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 has a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.
Assuntos
Diferenciação Celular , Proteínas Cromossômicas não Histona , Epigênese Genética , Mitose , Animais , Camundongos , Diferenciação Celular/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Mitose/genética , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Subunidades Proteicas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Divisão Celular/genética , Epigênese Genética/genéticaRESUMO
Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci.
Assuntos
Montagem e Desmontagem da Cromatina , Inativação Gênica , Heterocromatina/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Acetilação , Sítios de Ligação , Ilhas de CpG , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Heterocromatina/química , Heterocromatina/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Modelos Moleculares , Nucleossomos/enzimologia , Nucleossomos/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Fúngico/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In this issue of Molecular Cell, Ishida et al. (2012) and Keller et al. (2012) show distinct outcomes for heterochromatic RNAs that bind different chromodomain proteins; Chp1 tethers transcripts to centromeres, whereas Swi6(HP1)-bound transcripts are evicted from chromatin and destroyed.
RESUMO
Maintenance of the correct level and organisation of nucleosomes is crucial for genome function. Here, we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in the maintenance of nucleosome architecture in fission yeast. Cells lacking abo1(+) experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/genética , Cromatina/metabolismo , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , DNA Intergênico , Inativação Gênica , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Heterocromatina/metabolismo , Metiltransferases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Heterocromatina/genética , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Lisina/metabolismo , Metiltransferases/genética , Mutação , Interferência de RNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Especificidade por Substrato , Telômero/genética , Telômero/metabolismoRESUMO
Vitamin A is an essential nutrient for the protection of children from respiratory tract disease. Supplementation with vitamin A is frequently prescribed in the clinical setting, in part to combat deficiencies among children in developing countries, and in part to treat respiratory infections in clinical trials. This vitamin influences immune responses via multiple, and sometimes seemingly contradictory mechanisms. For example, in separate reports, vitamin A was shown to decrease Th17 T-cell activity by downregulating IL-6, and to promote B cell production of IgA by upregulating IL-6. To explain these apparent contradictions, we evaluated the effects of retinoic acid (RA), a key metabolite of vitamin A, on cell lines of respiratory tract epithelial cells (LETs) and macrophages (MACs). When triggered with LPS or Sendai virus, a mouse respiratory pathogen, these two cell lines experienced opposing influences of RA on IL-6. Both IL-6 protein production and transcript levels were downregulated by RA in LETs, but upregulated in MACs. RA also increased transcript levels of MCP-1, GMCSF, and IL-10 in MACs, but not in LETs. Conversely, when LETs, but not MACs, were exposed to RA, there was an increase in transcripts for RARß, an RA receptor with known inhibitory effects on cell metabolism. Results help explain past discrepancies in the literature by demonstrating that the effects of RA are cell target dependent, and suggest close attention be paid to cell-specific effects in clinical trials involving vitamin A supplements.
Assuntos
Citocinas/biossíntese , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Mucosa Respiratória/metabolismo , Vitamina A/farmacologia , Animais , Linhagem Celular Transformada , Células Epiteliais/citologia , Macrófagos/citologia , Camundongos , Mucosa Respiratória/citologiaRESUMO
In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Sequência de Aminoácidos , Proteínas Argonautas , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Lisina/metabolismo , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Brain tumors are the most common solid tumors in children. Pediatric high-grade glioma (HGG) accounts for â¼8-12 % of these brain tumors and is a devastating disease as 70-90 % of patients die within 2 years of diagnosis. The failure to advance therapy for these children over the last 30 years is largely due to limited knowledge of the molecular basis for these tumors and a lack of disease models. Recently, sequencing of tumor cells revealed that histone H3 is frequently mutated in pediatric HGG, with up to 78 % of diffuse intrinsic pontine gliomas (DIPGs) carrying K27M and 36 % of non-brainstem gliomas carrying either K27M or G34R/V mutations. Although mutations in many chromatin modifiers have been identified in cancer, this was the first demonstration that histone mutations may be drivers of disease. Subsequent studies have identified high-frequency mutation of histone H3 to K36M in chondroblastomas and to G34W/L in giant cell tumors of bone, which are diseases of adolescents and young adults. Interestingly, the G34 mutations, the K36M mutations, and the majority of K27M mutations occur in genes encoding the replacement histone H3.3. Here, we review the peculiar characteristics of histone H3.3 and use this information as a backdrop to highlight current thinking about how the identified mutations may contribute to disease development.
Assuntos
Neoplasias do Tronco Encefálico/genética , Carcinogênese/genética , Glioma/genética , Histonas/genética , Mutação , Sequência de Aminoácidos , Animais , Neoplasias do Tronco Encefálico/diagnóstico , Criança , Glioma/diagnóstico , Humanos , Dados de Sequência Molecular , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.
Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Heterocromatina/genética , Histona Desacetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/enzimologia , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Metilação , Dados de Sequência Molecular , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.
Assuntos
Micrognatismo , Regiões Promotoras Genéticas , Proteínas Repressoras , Tumor Rabdoide , Proteína SMARCB1 , Fatores de Transcrição , Humanos , Proteína SMARCB1/metabolismo , Proteína SMARCB1/genética , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Micrognatismo/genética , Micrognatismo/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Regiões Promotoras Genéticas/genética , Face/anormalidades , Cromatina/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Montagem e Desmontagem da Cromatina , Mutação , Deformidades Congênitas do Pé/genética , Deformidades Congênitas do Pé/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Sistemas CRISPR-Cas , Transcrição Gênica , Animais , Camundongos , Anormalidades Múltiplas , Deformidades Congênitas da Mão , Pescoço/anormalidadesRESUMO
Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of centromeric heterochromatin in fission yeast that integrates our current understanding of the competing forces of transcription, replication, and RNA decay that influence its assembly and propagation.
Assuntos
Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Interferência de RNA , Schizosaccharomyces/metabolismo , Transcrição Gênica , Animais , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Epigênese Genética , Histonas/metabolismo , Mamíferos , Metilação , Complexos Multiproteicos/metabolismo , Estabilidade de RNARESUMO
Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent "priRNAs." The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3(WG)) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi-independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi-independent factors in the assembly of heterochromatin.
Assuntos
Proteínas de Ciclo Celular/genética , Heterocromatina/genética , Metiltransferases/genética , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas Argonautas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/genética , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Lisina/metabolismo , Metilação , Metiltransferases/metabolismo , Mutação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Telomeres play important roles in safeguarding the genome. The specialized repressive chromatin that assembles at telomeres and subtelomeric domains is key to this protective role. However, in many organisms, the repetitive nature of telomeric and subtelomeric sequences has hindered research efforts. The fission yeast S. pombe has provided an important model system for dissection of chromatin biology due to the relative ease of genetic manipulation and strong conservation of important regulatory proteins with higher eukaryotes. Telomeres and the telomere-binding shelterin complex are highly conserved with mammals, as is the assembly of constitutive heterochromatin at subtelomeres. In this review, we seek to summarize recent work detailing the assembly of distinct chromatin structures within subtelomeric domains in fission yeast. These include the heterochromatic SH subtelomeric domains, the telomere-associated sequences (TAS), and ST chromatin domains that assemble highly condensed chromatin clusters called knobs. Specifically, we review new insights into the sequence of subtelomeric domains, the distinct types of chromatin that assemble on these sequences and how histone H3 K36 modifications influence these chromatin structures. We address the interplay between the subdomains of chromatin structure and how subtelomeric chromatin is influenced by both the telomere-bound shelterin complexes and by euchromatic chromatin regulators internal to the subtelomeric domain. Finally, we demonstrate that telomere clustering, which is mediated via the condensed ST chromatin knob domains, does not depend on knob assembly within these domains but on Set2, which mediates H3K36 methylation.
RESUMO
Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.
Assuntos
Histonas/metabolismo , Recombinação Homóloga , Proteínas Mutantes/metabolismo , Schizosaccharomyces/metabolismo , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Histonas/genética , Proteínas Mutantes/genética , Schizosaccharomyces/genéticaRESUMO
The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A(Cnp1) chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A(Cnp1) can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A(Cnp1) chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A(Cnp1) associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A(Cnp1) for assembly into central domain chromatin, resulting in less CENP-A(Cnp1) and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A(Cnp1) influence the extent of DNA at centromeres that is packaged in CENP-A(Cnp1) chromatin and the composition of this chromatin. Thus, CENP-A(Cnp1) chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A(Cnp1) and other core histones.
Assuntos
Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Schizosaccharomyces/metabolismo , Centrômero/metabolismo , Proteína Centromérica A , Cromatina/metabolismo , DNA/química , Primers do DNA/química , Proteínas Fúngicas/química , Genoma Fúngico , Cinetocoros/metabolismo , Modelos Biológicos , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Fuso Acromático/metabolismoRESUMO
In this review, we describe the attributes of histone H3 mutants identified in cancer. H3 mutants were first identified in genes encoding H3.3, in pediatric high-grade glioma, and subsequently in chondrosarcomas and giant cell tumors of bone (GCTB) in adolescents. The most heavily studied are the lysine to methionine mutants K27M and K36M, which perturb the target site for specific lysine methyltransferases and dominantly perturb methylation of corresponding lysines in other histone H3 proteins. We discuss recent progress in defining the consequences of these mutations on chromatin, including a newly emerging view of the central importance of the disruption of H3K36 modification in many distinct K to M histone mutant cancers. We also review new work exploring the role of H3.3 G34 mutants identified in pediatric glioma and GCTB. G34 is not itself post-translationally modified, but G34 mutation impinges on the modification of H3K36. Here, we ask if G34R mutation generates a new site for methylation on the histone tail. Finally, we consider evidence indicating that histone mutations might be more widespread in cancer than previously thought, and if the perceived bias towards mutation of H3.3 is real or reflects the biology of tumors in which the histone mutants were first identified.
RESUMO
A fundamental requirement for life is the ability of cells to divide properly and to pass on to their daughters a full complement of genetic material. The centromere of the chromosome is essential for this process, as it provides the DNA sequences on which the kinetochore (the proteinaceous structure that links centromeric DNA to the spindle microtubules) assembles to allow segregation of the chromosomes during mitosis. It has long been recognized that kinetochore assembly is subject to epigenetic control, and deciphering how centromeres promote faithful chromosome segregation provides a fascinating intellectual challenge. This challenge is made more difficult by the scale and complexity of DNA sequences in metazoan centromeres, thus much research has focused on dissecting centromere function in the single celled eukaryotic yeasts. Interestingly, in spite of similarities in the genome size of budding and fission yeasts, they seem to have adopted some striking differences in their strategy for passing on their chromosomes. Budding yeast have "point" centromeres, where a 125 base sequence is sufficient for mitotic propagation, whereas fission yeast centromeres are more reminiscent of the large repetitive centromeres of metazoans. In addition, the centromeric heterochromatin which coats centromeric domains of fission yeast and metazoan centromeres and is critical for their function, is largely absent from budding yeast centromeres. This review focuses on the assembly and maintenance of centromeric chromatin in the fission yeast.