RESUMO
Mammalian SWI/SNF chromatin remodeling complexes move and evict nucleosomes at gene promoters and enhancers to modulate DNA access. Although SWI/SNF subunits are commonly mutated in disease, therapeutic options are limited by our inability to predict SWI/SNF gene targets and conflicting studies on functional significance. Here, we leverage a fast-acting inhibitor of SWI/SNF remodeling to elucidate direct targets and effects of SWI/SNF. Blocking SWI/SNF activity causes a rapid and global loss of chromatin accessibility and transcription. Whereas repression persists at most enhancers, we uncover a compensatory role for the EP400/TIP60 remodeler, which reestablishes accessibility at most promoters during prolonged loss of SWI/SNF. Indeed, we observe synthetic lethality between EP400 and SWI/SNF in cancer cell lines and human cancer patient data. Our data define a set of molecular genomic features that accurately predict gene sensitivity to SWI/SNF inhibition in diverse cancer cell lines, thereby improving the therapeutic potential of SWI/SNF inhibitors.
Assuntos
Proteínas Nucleares , Fatores de Transcrição , Animais , Humanos , Cromatina , Montagem e Desmontagem da Cromatina , Proteínas Nucleares/metabolismo , Nucleossomos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , CamundongosRESUMO
Gene regulation arises out of dynamic competition between nucleosomes, transcription factors, and other chromatin proteins for the opportunity to bind genomic DNA. The timescales of nucleosome assembly and binding of factors to DNA determine the outcomes of this competition at any given locus. Here, we review how these properties of chromatin proteins and the interplay between the dynamics of different factors are critical for gene regulation. We discuss how molecular structures of large chromatin-associated complexes, kinetic measurements, and high resolution mapping of protein-DNA complexes in vivo set the boundary conditions for chromatin dynamics, leading to models of how the steady state behaviors of regulatory elements arise.
Assuntos
Cromatina , Nucleossomos , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Nucleossomos/genética , Fatores de Transcrição/genéticaRESUMO
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.
Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Polimerase II/genética , UbiquitinaçãoRESUMO
Transcription and translation are two main pillars of gene expression. Due to the different timings, spots of action, and mechanisms of regulation, these processes are mainly regarded as distinct and generally uncoupled, despite serving a common purpose. Here, we sought for a possible connection between transcription and translation. Employing an unbiased screen of multiple human promoters, we identified a positive effect of TATA box on translation and a general coupling between mRNA expression and translational efficiency. Using a CRISPR-Cas9-mediated approach, genome-wide analyses, and in vitro experiments, we show that the rate of transcription regulates the efficiency of translation. Furthermore, we demonstrate that m6A modification of mRNAs is co-transcriptional and depends upon the dynamics of the transcribing RNAPII. Suboptimal transcription rates lead to elevated m6A content, which may result in reduced translation. This study uncovers a general and widespread link between transcription and translation that is governed by epigenetic modification of mRNAs.
Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Transcrição Gênica , Adenosina/metabolismo , Humanos , Metilação , Iniciação Traducional da Cadeia Peptídica , RNA Polimerase II/metabolismo , TATA BoxRESUMO
The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana/virologia , RNA Polimerase II/metabolismo , Células A549 , Animais , Imunoprecipitação da Cromatina , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/metabolismo , Humanos , Espectrometria de Massas , Camundongos , Mutação , Doenças Neurodegenerativas/virologia , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Transcrição GênicaRESUMO
DNA replication produces a global disorganization of chromatin structure that takes hours to be restored. However, how these chromatin rearrangements affect the regulation of gene expression and the maintenance of cell identity is not clear. Here, we use ChOR-seq and ChrRNA-seq experiments to analyze RNA polymerase II (RNAPII) activity and nascent RNA synthesis during the first hours after chromatin replication in human cells. We observe that transcription elongation is rapidly reactivated in nascent chromatin but that RNAPII abundance and distribution are altered, producing heterogeneous changes in RNA synthesis. Moreover, this first wave of transcription results in RNAPII blockages behind the replication fork, leading to changes in alternative splicing. Altogether, our results deepen our understanding of how transcriptional programs are regulated during cell division and uncover molecular mechanisms that explain why chromatin replication is an important source of gene expression variability.
Assuntos
Processamento Alternativo , Cromatina , Humanos , Cromatina/genética , Transcrição Gênica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA/metabolismo , Splicing de RNA , Replicação do DNARESUMO
RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.
Assuntos
Linhagem da Célula , DNA Helicases , Elementos Facilitadores Genéticos , Proteínas Nucleares , Fatores de Transcrição , Animais , Humanos , Linhagem da Célula/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The positive transcription elongation factor b (P-TEFb) is composed of cyclins T1 or T2 and cyclin-dependent kinase 9 that regulate the elongation phase of transcription by RNA polymerase II. By antagonizing negative elongation factors and phosphorylating the C-terminal domain of RNA polymerase II, P-TEFb facilitates the elongation and co-transcriptional processing of nascent transcripts. This step is critical for the expression of most eukaryotic genes. In growing cells, P-TEFb is regulated negatively by its reversible associations with HEXIM1/2 in the 7SK snRNP and positively by a number of transcription factors, as well as the super elongation complex. In resting cells, P-TEFb falls apart, and cyclin T1 is degraded by the proteasome. This complex regulation of P-TEFb has evolved for the precise temporal and spatial regulation of gene expression in the organism. Its dysregulation contributes to inflammatory and neoplastic conditions.
Assuntos
Fator B de Elongação Transcricional Positiva , RNA Polimerase II , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células HeLa , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Ciclina T/genética , Ciclina T/metabolismo , Transcrição Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.
Assuntos
Replicação do DNA , RNA Polimerase II , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , DNA Helicases/genética , DNA Helicases/metabolismo , Instabilidade GenômicaRESUMO
Accumulation of fat above the waist is an important risk factor in developing obesity-related comorbidities independently of BMI or total fat mass. Deciphering the gene regulatory programs of the adipose tissue precursor cells within upper body or abdominal (ABD) and lower body or gluteofemoral (GF) depots is important to understand their differential capacity for lipid accumulation, maturation, and disease risk. Previous studies identified the HOX transcript antisense intergenic RNA (HOTAIR) as a GF-specific lncRNA; however, its role in adipose tissue biology is still unclear. Using three different approaches (silencing of HOTAIR in GF human adipose-derived stem cells [GF hASCs], overexpression of HOTAIR in ABD hASCs, and ChIRP-seq) to localize HOTAIR binding in GF hASC chromatin, we found that HOTAIR binds and modulates expression, both positively and negatively, of genes involved in adipose tissue-specific pathways, including adipogenesis. We further demonstrate a direct interaction between HOTAIR and genes with high RNAPII binding in their gene bodies, especially at their 3' ends or transcription end sites. Computational analysis suggests HOTAIR binds preferentially to the 3' ends of genes containing predicted strong RNA-RNA interactions with HOTAIR. Together, these results reveal a unique function for HOTAIR in hASC depot-specific regulation of gene expression.
Assuntos
RNA Longo não Codificante , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Expressão Gênica , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células-Tronco/metabolismoRESUMO
As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.
Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Neoplasias Colorretais/enzimologia , DNA Topoisomerases Tipo I/metabolismo , Fase G1 , Mitose , RNA Polimerase II/metabolismo , Transcrição Gênica , Proliferação de Células/efeitos dos fármacos , Sequenciamento de Cromatina por Imunoprecipitação , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA Topoisomerases Tipo I/genética , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Inibidores de MTOR/farmacologia , Mitose/efeitos dos fármacos , RNA Polimerase II/genéticaRESUMO
The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elongação da Transcrição Genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Linhagem Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase II/genética , Transcrição Gênica/genética , Animais , Sistemas de Liberação de Medicamentos , Humanos , Fosfoproteínas Fosfatases/genéticaRESUMO
Cells have evolved a robust and highly regulated DNA damage response to preserve their genomic integrity. Although increasing evidence highlights the relevance of RNA regulation, our understanding of its impact on a fully efficient DNA damage response remains limited. Here, through a targeted CRISPR-knockout screen, we identify RNA-binding proteins and modifiers that participate in the p53 response. Among the top hits, we find the m6A reader YTHDC1 as a master regulator of p53 expression. YTHDC1 binds to the transcription start sites of TP53 and other genes involved in the DNA damage response, promoting their transcriptional elongation. YTHDC1 deficiency also causes the retention of introns and therefore aberrant protein production of key DNA damage factors. While YTHDC1-mediated intron retention requires m6A, TP53 transcriptional pause-release is promoted by YTHDC1 independently of m6A. Depletion of YTHDC1 causes genomic instability and aberrant cancer cell proliferation mediated by genes regulated by YTHDC1. Our results uncover YTHDC1 as an orchestrator of the DNA damage response through distinct mechanisms of co-transcriptional mRNA regulation.
Assuntos
Dano ao DNA , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Adenosina/metabolismo , Adenosina/análogos & derivados , Instabilidade Genômica , Proliferação de Células , Regulação da Expressão Gênica , Proteínas do Tecido NervosoRESUMO
The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting NOT4 or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of RSP5, where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.
Assuntos
RNA Polimerase II/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Mutantes/metabolismo , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The RNA polymerase II (RNAPII) C-terminal domain kinase, CDK12, regulates genome stability, expression of DNA repair genes, and cancer cell resistance to chemotherapy and immunotherapy. In addition to its role in mRNA biosynthesis of DNA repair genes, we show here that CDK12 phosphorylates the mRNA 5' cap-binding repressor, 4E-BP1, to promote translation of mTORC1-dependent mRNAs. In particular, we found that phosphorylation of 4E-BP1 by mTORC1 (T37 and T46) facilitates subsequent CDK12 phosphorylation at two Ser-Pro sites (S65 and T70) that control the exchange of 4E-BP1 with eIF4G at the 5' cap of CHK1 and other target mRNAs. RNA immunoprecipitation coupled with deep sequencing (RIP-seq) revealed that CDK12 regulates release of 4E-BP1, and binding of eIF4G, to many mTORC1 target mRNAs, including those needed for MYC transformation. Genome-wide ribosome profiling (Ribo-seq) further identified specific CDK12 "translation-only" target mRNAs, including many mTORC1 target mRNAs as well as many subunits of mitotic and centromere/centrosome complexes. Accordingly, confocal imaging analyses revealed severe chromosome misalignment, bridging, and segregation defects in cells deprived of CDK12 or CCNK. We conclude that the nuclear RNAPII-CTD kinase CDK12 cooperates with mTORC1, and controls a specialized translation network that is essential for mitotic chromosome stability.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Instabilidade Genômica/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Mitose/genética , Fosforilação/genética , Ligação Proteica/genéticaRESUMO
DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , UbiquitinaçãoRESUMO
The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.
RESUMO
Eukaryotic RNA polymerase II (RNAPII) is responsible for the transcription of the protein-coding genes in the cell. Enormous progress has been made in discovering the protein activities that are required for transcription to occur, but the effects of post-translational modifications (PTMs) on RNAPII transcriptional regulation are much less understood. Most of our understanding relates to the cyclin-dependent kinases (CDKs), which appear to act relatively early in transcription. However, it is becoming apparent that other PTMs play a crucial role in the transcriptional cycle, and it is doubtful that any sort of complete understanding of this regulation is attainable without understanding the spectra of PTMs that occur on the transcriptional machinery. Among these is O-GlcNAcylation. Recent experiments have shown that the O-GlcNAc PTM likely has a prominent role in transcription. This review will cover the role of the O-GlcNAcylation in RNAPII transcription during initiation, pausing, and elongation, which will hopefully be of interest to both O-GlcNAc and RNAPII transcription researchers.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II , Transcrição Gênica , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
The current understanding of how specific distributions of histone post-translational modifications (PTMs) are achieved throughout the chromatin remains incomplete. This review focuses on the role of RNA polymerase II (RNAPII) in establishing H2BK120/K123 ubiquitination and H3K4/K36 methylation distribution. The rate of RNAPII transcription is mainly a function of the RNAPII elongation and recruitment rates. Two major mechanisms link RNAPII's transcription rate to the distribution of PTMs. First, the phosphorylation patterns of Ser2P/Ser5P in the C-terminal domain of RNAPII change as a function of time, since the start of elongation, linking them to the elongation rate. Ser2P/Ser5P recruits specific histone PTM enzymes/activators to the nucleosome. Second, multiple rounds of binding and catalysis by the enzymes are required to establish higher methylations (H3K4/36me3). Thus, methylation states are determined by the transcription rate. In summary, the first mechanism determines the location of methylations in the gene, while the second mechanism determines the methylation state.