RESUMO
Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylation (γH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and γH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , DNA de Cadeia Simples/metabolismo , Histonas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteína BRCA1/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Fosforilação , Proteínas Inibidoras de STAT Ativados/química , Proteínas Inibidoras de STAT Ativados/genética , Estrutura Terciária de Proteína , Proteína de Replicação A/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.
Assuntos
Adutos de DNA , Proteínas de Ligação a DNA , Humanos , Adutos de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diester Fosfórico Hidrolases/genética , DNA Topoisomerases Tipo II/genética , DNA/genética , Instabilidade Genômica , DNA Helicases/genéticaRESUMO
Loss of functional BRCA1 protein leads to defects in DNA double-strand break (DSB) repair by homologous recombination (HR) and renders cells hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibitors used to treat BRCA1/2-deficient cancers. However, upon chronic treatment of BRCA1-mutant cells with PARP inhibitors, resistant clones can arise via several mechanisms, including loss of 53BP1 or its downstream co-factors. Defects in the 53BP1 axis partially restore the ability of a BRCA1-deficient cell to form RAD51 filaments at resected DSBs in a PALB2- and BRCA2-dependent manner, and thereby repair DSBs by HR. Here we show that depleting 53BP1 in BRCA1-null cells restores PALB2 accrual at resected DSBs. Moreover, we demonstrate that PALB2 DSB recruitment in BRCA1/53BP1-deficient cells is mediated by an interaction between PALB2's chromatin associated motif (ChAM) and the nucleosome acidic patch region, which in 53BP1-expressing cells is bound by 53BP1's ubiquitin-directed recruitment (UDR) domain.
Assuntos
Proteína BRCA1/deficiência , Cromatina/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Recombinação Homóloga , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Motivos de Aminoácidos , Proteína BRCA2/deficiência , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/química , Proteína do Grupo de Complementação N da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Humanos , Nucleossomos/metabolismoRESUMO
The Fanconi anemia pathway orchestrates the repair of DNA interstrand cross-links and stalled replication forks. A key step in this pathway is UBE2T and FANCL-dependent monoubiquitylation of the FANCD2-FANCI complex. The Fanconi anemia pathway represents an attractive therapeutic target, because activation of this pathway has been linked to chemotherapy resistance in several cancers. However, to date, very few selective inhibitors of ubiquitin conjugation pathways are known. By using a high-throughput screen-compatible assay, we have identified a small-molecule inhibitor of UBE2T/FANCL-mediated FANCD2 monoubiquitylation that sensitizes cells to the DNA cross-linking agent, carboplatin.
Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/antagonistas & inibidores , Anemia de Fanconi/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Linhagem Celular Tumoral , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Histone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Dano ao DNA , Histonas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/genética , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
The regulation of transcription elongation within the context of chromatin is a topic of great interest. Even though chromatin presents a barrier to transcription by the PolII machinery in vitro, this process is rather efficient in vivo. Importantly, the chromatin structure of the actively transcribed genes is altered as part of this process. A large number of factors implicated in the control of transcript elongation have been identified through genetics, biochemistry and targeted proteomics approaches. However the precise roles and mechanisms of action of these factors remain obscure. A significant advance came about this past year with the elucidation of the roles of FACT and Spt6 in transcription elongation. These factors facilitate PolII passage through chromatin by destabilizing the nucleosome structure as well as reassemble nucleosomes traversed by PolII.
Assuntos
Cromatina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Transcrição Gênica/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Animais , Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Humanos , Proteínas Nucleares/fisiologia , Nucleossomos/fisiologia , RNA Polimerase II/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
Assuntos
Proteína BRCA1/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Reparo do DNA por Junção de Extremidades , Resistencia a Medicamentos Antineoplásicos , Osteossarcoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas/metabolismo , Reparo de DNA por Recombinação , Animais , Proteína BRCA1/deficiência , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células HEK293 , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Camundongos , Complexos Multiproteicos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In eukaryotic cells, genomic DNA is assembled with chromosomal proteins, mainly histones, in a highly compact structure termed chromatin. In this form, DNA is not readily accessible to the cellular machineries, which require DNA as a template. Dynamic changes in chromatin organization play a critical role in regulation of DNA-dependent processes such as transcription, DNA replication, recombination and repair. Chromatin structure is altered in transcriptionally active loci: the basic chromatin unit, the nucleosome, appears to be depleted for one histone H2A/H2B dimer. Previously, reconstitution of RNA polymerase II (PolII)-driven transcription on chromatin templates in a highly purified in vitro system led to identification of FACT (for facilitates chromatin transcription), which was required for productive transcript elongation through nucleosomes. FACT was proposed to promote PolII transcription through nucleosomes by removing either one or both H2A/H2B dimers. Here we present an overview of the earlier studies, which resulted in the initial identification and characterization of FACT, as well as the recent findings that refine the model for the mechanism of FACT function in transcription.
Assuntos
Cromatina/genética , Substâncias Macromoleculares , Transcrição Gênica , Animais , Cromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , RNA Polimerase II , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Mammalian CtIP protein has major roles in DNA double-strand break (DSB) repair. Although it is well established that CtIP promotes DNA-end resection in preparation for homology-dependent DSB repair, the molecular basis for this function has remained unknown. Here we show by biophysical and X-ray crystallographic analyses that the N-terminal domain of human CtIP exists as a stable homotetramer. Tetramerization results from interlocking interactions between the N-terminal extensions of CtIP's coiled-coil region, which lead to a 'dimer-of-dimers' architecture. Through interrogation of the CtIP structure, we identify a point mutation that abolishes tetramerization of the N-terminal domain while preserving dimerization in vitro. Notably, we establish that this mutation abrogates CtIP oligomer assembly in cells, thus leading to strong defects in DNA-end resection and gene conversion. These findings indicate that the CtIP tetramer architecture described here is essential for effective DSB repair by homologous recombination.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerização Proteica/fisiologia , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Endodesoxirribonucleases , HumanosRESUMO
A recent study published in Science reveals the mechanism and biological importance of DNA damage response abrogation in mitotic cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Mitose/fisiologia , Homeostase do Telômero/fisiologia , Telômero/fisiologia , Animais , HumanosRESUMO
The DNA damage response (DDR) is critical for genome stability and the suppression of a wide variety of human malignancies, including neurodevelopmental disorders, immunodeficiency, and cancer. In addition, the efficacy of many chemotherapeutic strategies is dictated by the status of the DDR. Ubiquitin-specific protease 28 (USP28) was reported to govern the stability of multiple factors that are critical for diverse aspects of the DDR. Here, we examined the effects of USP28 depletion on the DDR in cells and in vivo. We found that USP28 is recruited to double-strand breaks in a manner that requires the tandem BRCT domains of the DDR protein 53BP1. However, we observed only minor DDR defects in USP28-depleted cells, and mice lacking USP28 showed normal longevity, immunological development, and radiation responses. Our results thus indicate that USP28 is not a critical factor in double-strand break metabolism and is unlikely to be an attractive target for therapeutic intervention aimed at chemotherapy sensitization.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/citologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA , Instabilidade Genômica , Células HEK293 , Humanos , Switching de Imunoglobulina/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Pontos de Checagem da Fase S do Ciclo Celular/genética , Transdução de Sinais/genética , Timócitos/imunologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina Tiolesterase/genéticaRESUMO
Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (γH2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of γH2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.
Assuntos
Replicação do DNA/genética , Fase G1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fator 1 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Células Cultivadas , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a "primary" DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11-Rad50-Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Mitose/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Reparo do DNA , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Spt-Ada-Gcn5 acetyltransferase (SAGA) is a previously described histone acetyltransferase/transcriptional coactivator complex in yeast. At promoters of certain genes (HIS3 and TRP3), SAGA has an inhibitory function involving a nonproductive TATA-binding protein interaction mediated by the Spt3 and Spt8 subunits. Related to this, Spt8-less SAGA is a major form of the complex under activating conditions for these genes. In the present study, we purify this activation-specific complex, called SALSA (SAGA altered, Spt8 absent). Besides lacking Spt8, SALSA contains Spt7 subunit that is truncated. Examining the role of this subunit, we find that C-terminally truncated SPT7 resulted in derepressed HIS3 transcription. Furthermore, when grown in rich media (repressing conditions), wild-type cells yielded predominantly SAGA, but Spt7 C-terminal truncations resulted primarily in a form of complex similar to SALSA. Thus, SALSA-like structure and activating function can be partially recapitulated in yeast by truncating the C terminus of Spt7. Overall, these results lead to a model that for a subset of promoters SAGA is inhibitory through Spt3, Spt8, and an Spt8-interacting subdomain of Spt7, whereas SALSA is a form of complex for positive transcriptional regulation. These data clarify a mechanism by which a transcriptional regulatory complex can switch between positive and negative modulation.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Dados de Sequência Molecular , Plasmídeos , Testes de Precipitina , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Appreciable advances into the process of transcript elongation by RNA polymerase II (RNAP II) have identified this stage as a dynamic and highly regulated step of the transcription cycle. Here, we discuss the many factors that regulate the elongation stage of transcription. Our discussion includes the classical elongation factors that modulate the activity of RNAP II, and the more recently identified factors that facilitate elongation on chromatin templates. Additionally, we discuss the factors that associate with RNAP II, but do not modulate its catalytic activity. Elongation is highlighted as a central process that coordinates multiple stages in mRNA biogenesis and maturation.
Assuntos
RNA Polimerase II/fisiologia , Precursores de RNA/fisiologia , RNA Mensageiro/metabolismo , Transcrição Gênica/fisiologia , Trifosfato de Adenosina/metabolismo , Cromatina/fisiologia , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Fosforilação , RNA Polimerase II/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/fisiologiaRESUMO
The FACT (facilitates chromatin transcription) complex is required for transcript elongation through nucleosomes by RNA polymerase II (Pol II) in vitro. Here, we show that FACT facilitates Pol II-driven transcription by destabilizing nucleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage. We also demonstrate that FACT possesses intrinsic histone chaperone activity and can deposit core histones onto DNA. Importantly, FACT activity requires both of its constituent subunits and is dependent on the highly acidic C terminus of its larger subunit, Spt16. These findings define the mechanism by which Pol II can transcribe through chromatin without disrupting its epigenetic status.