RESUMO
DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3' end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.
Assuntos
RNA Helicases DEAD-box , RNA , Humanos , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Helicases DEAD-box/metabolismo , Processamento Alternativo , Cromatina/genéticaRESUMO
R-loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of two fission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3-transcribed genes and produces long exosome-sensitive 3'-extended transcripts. Importantly, neither of these defects was affected by the removal of R-loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.
Assuntos
DNA Helicases/metabolismo , RNA Helicases/metabolismo , RNA Polimerase III/genética , Schizosaccharomyces/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , RNA de Transferência/química , RNA de Transferência/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Terminação da Transcrição GenéticaRESUMO
Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.
Assuntos
DNA de Cadeia Simples/química , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , Pegada de DNA , DNA Fúngico/química , DNA Fúngico/genética , DNA Recombinante/química , Cloreto de Lítio/farmacologia , Microscopia de Força Atômica , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico/efeitos dos fármacos , Hibridização de Ácido Nucleico , Plasmídeos/genética , RNA Longo não Codificante/química , Proteínas de Schizosaccharomyces pombe/genética , Transcrição GênicaRESUMO
Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differentiation, while in mammals, this mechanism has been supplanted by the testis-determining gene SRY. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic analyses to investigate the precise mechanism through which oestrogen impacts these pathways to activate ß-catenin-a factor essential for ovarian development. We show that oestrogen can activate ß-catenin within 30 min, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to ß-catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.
Assuntos
MAP Quinase Quinase Quinase 1/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Estrogênios/farmacologia , Humanos , MAP Quinase Quinase Quinase 1/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Schizosaccharomyces/fisiologia , Acetiltransferases/metabolismo , Composição de Bases , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The packaging of DNA into chromosomes is a ubiquitous process that enables living organisms to structure and transmit their genome accurately through cell divisions. In the three kingdoms of life, the architecture and dynamics of chromosomes rely upon ring-shaped SMC (Structural Maintenance of Chromosomes) condensin complexes. To understand how condensin rings organize chromosomes, it is essential to decipher how they associate with chromatin filaments. Here, we use recent evidence to discuss the role played by nucleosomes and transcription factors in the loading of condensin at transcribed genes. We propose a model whereby cis-acting features nestled in the promoters of active genes synergistically attract condensin rings and promote their association with DNA.
Assuntos
Adenosina Trifosfatases/genética , Cromatina/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Complexos Multiproteicos/genética , Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Cromatina/química , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Cromossomos/química , Cromossomos/ultraestrutura , DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Genoma/genética , Mitose/genética , Complexos Multiproteicos/química , Nucleossomos/genética , Regiões Promotoras GenéticasRESUMO
Condensin-mediated chromosome condensation is essential for genome stability upon cell division. Genetic studies have indicated that the association of condensin with chromatin is intimately linked to gene transcription, but what transcription-associated feature(s) direct(s) the accumulation of condensin remains unclear. Here we show in fission yeast that condensin becomes strikingly enriched at RNA Pol III-transcribed genes when Swd2.2 and Sen1, two factors involved in the transcription process, are simultaneously deleted. Sen1 is an ATP-dependent helicase whose orthologue in Saccharomyces cerevisiae contributes both to terminate transcription of some RNA Pol II transcripts and to antagonize the formation of DNA:RNA hybrids in the genome. Using two independent mapping techniques, we show that DNA:RNA hybrids form in abundance at Pol III-transcribed genes in fission yeast but we demonstrate that they are unlikely to faciliate the recruitment of condensin. Instead, we show that Sen1 forms a stable and abundant complex with RNA Pol III and that Swd2.2 and Sen1 antagonize both the interaction of RNA Pol III with chromatin and RNA Pol III-dependent transcription. When Swd2.2 and Sen1 are lacking, the increased concentration of RNA Pol III and condensin at Pol III-transcribed genes is accompanied by the accumulation of topoisomerase I and II and by local nucleosome depletion, suggesting that Pol III-transcribed genes suffer topological stress. We provide evidence that this topological stress contributes to recruit and/or stabilize condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1. Our data challenge the idea that a processive RNA polymerase hinders the binding of condensin and suggest that transcription-associated topological stress could in some circumstances facilitate the association of condensin.
Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/genética , Complexos Multiproteicos/genética , RNA Helicases/genética , RNA Polimerase III/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Anáfase/genética , Cromatina/genética , Cromossomos/genética , DNA/genética , Genoma Fúngico , Instabilidade Genômica/genética , Nucleossomos/genética , RNA/genética , RNA Polimerase II/genética , Saccharomyces cerevisiae/genéticaRESUMO
Functional links connecting gene transcription and condensin-mediated chromosome condensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3' end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not affect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1(Dis2) with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologue of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatases PP1 and Ssu72.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Acetilação , Hidrolases Anidrido Ácido/genética , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Complexos Multiproteicos/genética , Fosforilação , Terminação da Transcrição GenéticaRESUMO
The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.
Assuntos
Aciltransferases/genética , Transtorno 46,XY do Desenvolvimento Sexual/genética , Proteínas Hedgehog/metabolismo , Lipoilação/genética , Mutação de Sentido Incorreto , Transdução de Sinais/genética , Aciltransferases/química , Aciltransferases/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Homozigoto , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Linhagem , Homologia de Sequência de Aminoácidos , Testículo/embriologiaRESUMO
The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.
Assuntos
Feto/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Marcadores Genéticos/genética , Ovário/citologia , Ovário/embriologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Fator II de Transcrição COUP/metabolismo , Linhagem da Célula/fisiologia , Feminino , Imunofluorescência , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/metabolismo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Ovário/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.
Assuntos
Adenosina Trifosfatases , Segregação de Cromossomos , Complexos Multiproteicos , Schizosaccharomyces , Proteínas de Ligação a DNA/genética , Cromatina , Cromossomos , DNA , Schizosaccharomyces/genética , RNA Polimerase II/genética , Mitose , Proteínas de Ciclo Celular/genéticaRESUMO
In most mammals, the Y chromosomal Sry gene initiates testis formation within the bipotential gonad, resulting in male development. SRY is a transcription factor and together with SF1 it directly up-regulates the expression of the pivotal sex-determining gene Sox9 via a 1.3-kb cis-regulatory element (TESCO) which contains an evolutionarily conserved region (ECR) of 180 bp. Remarkably, several rodent species appear to determine sex in the absence of Sry and a Y chromosome, including the mole voles Ellobius lutescens and Ellobius tancrei, whereas Ellobius fuscocapillus of the same genus retained Sry. The sex-determining mechanisms in the Sry-negative species remain elusive. We have cloned and sequenced 1.1 kb of E. lutescens TESCO which shares 75% sequence identity with mouse TESCO indicating that testicular Sox9 expression in E. lutescens might still be regulated via TESCO. We have also cloned and sequenced the ECRs of E. tancrei and E. fuscocapillus. While the three Ellobius ECRs are highly similar (94-97% sequence identity), they all display a 14-bp deletion (Δ14) removing a highly conserved SOX/TCF site. Introducing Δ14 into mouse TESCO increased both basal activity and SF1-mediated activation of TESCO in HEK293T cells. We propose a model whereby Δ14 may have triggered up-regulation of Sox9 in XX gonads leading to destabilization of the XY/XX sex-determining mechanism in Ellobius. E. lutescens/E. tancrei and E. fuscocapillus could have independently stabilized their sex determination mechanisms by Sry-independent and Sry-dependent approaches, respectively.
Assuntos
Arvicolinae/genética , Regulação da Expressão Gênica , Fatores de Transcrição SOX9/metabolismo , Processos de Determinação Sexual , Cromossomo Y/metabolismo , Animais , Arvicolinae/metabolismo , Arvicolinae/fisiologia , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Evolução Molecular , Feminino , Variação Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Transcrição SOX9/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Deleção de Sequência , Testículo/citologia , Testículo/metabolismo , Testículo/fisiologia , Cromossomo Y/genéticaRESUMO
The basal lineage of monotremes features an extraordinarily complex sex chromosome system which has provided novel insights into the evolution of mammalian sex chromosomes. Recently, sequence information from autosomes, X chromosomes, and XY-shared pseudoautosomal regions has become available. However, no gene has so far been described on any of the Y chromosome-specific regions. We analyzed sequences derived from Y-specific BAC clones to identify genes with potentially male-specific function. Here, we report the identification and characterization of the mediator complex protein gametologs on platypus Y5 (Crspy). We also identified the X-chromosomal copy which unexpectedly maps to X1 (Crspx). Sequence comparison shows extensive divergence between the X and Y copy, but we found no significant positive selection on either gametolog. Expression analysis shows widespread expression of Crspx. Crspy is expressed exclusively in males with particularly strong expression in testis and kidney. Reporter gene assays to investigate whether Crspx/y can act on the recently discovered mouse Sox9 testis-specific enhancer element did reveal a modest effect together with mouse Sox9 + Sf1, but showed overall no significant upregulation of the reporter gene. This is the first report of a differentiated functional male-specific gene on platypus Y chromosomes, providing new insights into sex chromosome evolution and a candidate gene for male-specific function in monotremes.
Assuntos
Complexo Mediador/genética , Ornitorrinco/genética , Testículo/fisiologia , Cromossomo X/genética , Cromossomo Y/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Cromossomos de Mamíferos/genética , Evolução Molecular , Regulação da Expressão Gênica , Genes Reporter , Genes sry , Células HEK293 , Humanos , Rim/citologia , Rim/metabolismo , Masculino , Camundongos , Filogenia , Mapeamento Físico do Cromossomo , Fatores de Transcrição SOX9/genética , Processos de Determinação Sexual , Testículo/citologia , TransfecçãoRESUMO
The novel long non-coding RNA (lncRNA) Leat1 is extraordinarily conserved in both its location (syntenic with EfnB2, an essential gene in anogenital patterning) and sequence. Here we show that Leat1 is upregulated following the testosterone surge from the developing testis and directly interacts with EfnB2, positively regulating its expression. Leat1 expression is suppressed by estrogen, which in turn suppresses the expression of EfnB2. Moreover, the loss of Leat1 leads to reduced EfnB2, resulting in a severe hypospadias phenotype. The human LEAT1 gene is also co-expressed with EFNB2 in the developing human penis suggesting a conserved function for this gene in urethral closure. Together our data identify Leat1 as a novel molecular regulator of urethral closure and implicate it as a target of endocrine disruption in the etiology of hypospadias.
RESUMO
The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here, we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome remodeler associated with shelterin. We further show that condensin takes part in sister-telomere separation in anaphase, and that this event can be uncoupled from the prior separation of chromosome arms, implying a telomere-specific separation mechanism. Consistent with a cis-acting process, increasing or decreasing condensin occupancy specifically at telomeres modifies accordingly the efficiency of their separation in anaphase. Genetic evidence suggests that condensin promotes sister-telomere separation by counteracting cohesin. Thus, our results reveal a shelterin-based mechanism that enriches condensin at telomeres to drive in cis their separation during mitosis.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Complexo Shelterina , Anáfase , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Fase G1/genética , Fase G2/genética , Proteínas Nucleares/genética , Ligação Proteica , Fase S/genética , Schizosaccharomyces/citologia , Troca de Cromátide Irmã/genética , CoesinasRESUMO
Heterochromatin performs a central role in chromosome segregation and stability by promoting cohesion at centromeres. Establishment of both heterochromatin-mediated silencing and cohesion requires passage through S phase, although the mechanism is unknown. Here we demonstrate that Schizosaccharomyces pombe Hsk1 (CDC7), a conserved Dbf4-dependent protein kinase (DDK) that regulates replication initiation, interacts with and phosphorylates the heterochromatin protein 1 (HP1) equivalent Swi6 (ref. 6). Hsk1 and its regulatory subunit Dfp1 function downstream of Swi6 localization to promote heterochromatin function and cohesion specifically at centromeres. This role for Hsk1-Dfp1 is separable from its replication initiation activity, providing a temporal link between S phase and centromere cohesion that is mediated by heterochromatin.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Centrômero/genética , Segregação de Cromossomos/genética , Heterocromatina/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/genética , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fase S/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaAssuntos
RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Reprodução , Animais , Feminino , Humanos , MasculinoRESUMO
The mechanisms leading to the accumulation of the SMC complexes condensins around specific transcription units remain unclear. Observations made in bacteria suggested that RNA polymerases (RNAPs) constitute an obstacle to SMC translocation, particularly when RNAP and SMC travel in opposite directions. Here we show in fission yeast that gene termini harbour intrinsic condensin-accumulating features whatever the orientation of transcription, which we attribute to the frequent backtracking of RNAP at gene ends. Consistent with this, to relocate backtracked RNAP2 from gene termini to gene bodies was sufficient to cancel the accumulation of condensin at gene ends and to redistribute it evenly within transcription units, indicating that RNAP backtracking may play a key role in positioning condensin. Formalization of this hypothesis in a mathematical model suggests that the inclusion of a sub-population of RNAP with longer dwell-times is essential to fully recapitulate the distribution profiles of condensin around active genes. Taken together, our data strengthen the idea that dense arrays of proteins tightly bound to DNA alter the distribution of condensin on chromosomes.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose/fisiologia , Complexos Multiproteicos/metabolismo , RNA Polimerase II/metabolismo , Adenosina Trifosfatases/genética , Cromossomos/metabolismo , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Mitose/genética , Complexos Multiproteicos/genética , RNA Polimerase II/fisiologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição Gênica/genéticaRESUMO
In mitosis, while the importance of kinetochore (KT)-microtubule (MT) attachment has been known for many years, increasing evidence suggests that telomere dysfunctions also perturb chromosome segregation by contributing to the formation of chromatin bridges at anaphase. Recent evidence suggests that Aurora B kinase ensures proper chromosome segregation during mitosis not only by controlling KT-MT attachment but also by regulating telomere and chromosome arm separation. However, whether and how Aurora B governs telomere separation during meiosis has remained unknown. Here, we show that fission yeast Aurora B localizes at telomeres during meiosis I and promotes telomere separation independently of the meiotic cohesin Rec8. In meiosis II, Aurora B controls KT-MT attachment but appears dispensable for telomere and chromosome arm separation. Likewise, condensin activity is nonessential in meiosis II for telomere and chromosome arm separation. Thus, in meiosis, the requirements for Aurora B are distinct at centromeres and telomeres, illustrating the critical differences in the control of chromosome segregation between mitosis and meiosis II.