RESUMO
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , RNA Polimerase II , Transcrição Gênica , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Regulação da Expressão Gênica , Fosforilação , Ligação Proteica , Resposta ao Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Nucleossomos/genéticaRESUMO
The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.
Assuntos
Doenças Neurodegenerativas , RNA Polimerase II , Animais , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Elongação da Transcrição/genética , Doenças Neurodegenerativas/genética , Transcrição Gênica , Regulação da Expressão Gênica , Envelhecimento/genética , Genes Controladores do DesenvolvimentoRESUMO
It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.
Assuntos
RNA Polimerase II , Fatores de Transcrição , Resposta ao Choque Térmico , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Transcription by RNA polymerase II (RNA Pol II) relies on the elongation factors PAF1 complex (PAF), RTF1, and SPT6. Here, we use rapid factor depletion and multi-omics analysis to investigate how these elongation factors influence RNA Pol II elongation activity in human cells. Whereas depletion of PAF subunits PAF1 and CTR9 has little effect on cellular RNA synthesis, depletion of RTF1 or SPT6 strongly compromises RNA Pol II activity, albeit in fundamentally different ways. RTF1 depletion decreases RNA Pol II velocity, whereas SPT6 depletion impairs RNA Pol II progression through nucleosomes. These results show that distinct elongation factors stimulate either RNA Pol II velocity or RNA Pol II progression through chromatin in vivo. Further analysis provides evidence for two distinct barriers to early elongation: the promoter-proximal pause site and the +1 nucleosome. It emerges that the first barrier enables loading of elongation factors that are required to overcome the second and subsequent barriers to transcription.
Assuntos
RNA Polimerase II/metabolismo , RNA/biossíntese , Fatores de Transcrição/metabolismo , Humanos , Células K562 , Nucleossomos/genética , Nucleossomos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/genéticaRESUMO
SPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.
Assuntos
RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Replicação do DNA , Humanos , Ácidos Indolacéticos/farmacologia , Poliadenilação , Proteólise/efeitos dos fármacos , RNA/biossíntese , RNA Polimerase II/genética , Fatores de Transcrição/genéticaRESUMO
Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica/genética , Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/genética , Mutação , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.
Assuntos
Elementos Facilitadores Genéticos , Protocaderinas , Cromatina , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , RNA , Transcrição GênicaRESUMO
Eukaryotic transcription occurs on chromatin, where RNA polymerase II encounters nucleosomes during elongation. These nucleosomes must unravel for the DNA to enter the active site. However, in most transcribed genes, nucleosomes remain intact due to transcription-coupled chromatin assembly mechanisms. These mechanisms primarily involve the local reassembly of displaced nucleosomes to prevent (epi)genomic instability and the emergence of cryptic transcription. As a fail-safe mechanism, cells can assemble nucleosomes de novo, particularly in highly transcribed genes, but this may result in the loss of epigenetic information. This review examines transcription-coupled chromatin assembly, with an emphasis on studies in yeast and recent structural studies. These studies shed light on how elongation factors and histone chaperones coordinate to enable nucleosome recycling during transcription.
RESUMO
The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
Assuntos
Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinases Ciclina-Dependentes/genética , Chaperonas de Histonas/genética , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.
Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Fatores de Alongamento de Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , RNA Polimerase II/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Transcription elongation requires elaborate coordination between the transcriptional machinery and chromatin regulatory factors to successfully produce RNA while preserving the epigenetic landscape. Recent structural and genomic studies have highlighted that suppressor of Ty 6 (Spt6), a conserved histone chaperone and transcription elongation factor, sits at the crux of the transcription elongation process. Other recent studies have revealed that Spt6 also promotes DNA replication and genome integrity. Here, we review recent studies of Spt6 that have provided new insights into the mechanisms by which Spt6 controls transcription and have revealed the breadth of Spt6 functions in eukaryotic cells.
Assuntos
Histonas , Humanos , Replicação do DNA/genética , Instabilidade Genômica/genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , AnimaisRESUMO
Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.
Assuntos
Chaperonas de Histonas/genética , Chaperonas de Histonas/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Iniciação da Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/fisiologia , Cromatina/fisiologia , Regulação Fúngica da Expressão Gênica/genética , Chaperonas de Histonas/metabolismo , Histonas/fisiologia , Proteínas Nucleares , Nucleossomos , Fatores de Alongamento de Peptídeos/fisiologia , Regiões Promotoras Genéticas/genética , RNA Polimerase II , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Fatores de Transcrição/fisiologia , Sítio de Iniciação de Transcrição/fisiologia , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 also associates with RNA polymerase II (RNAPII) via a tandem Src2 homology domain. However, the significance of Spt6-RNAPII interaction is not well understood. Here, we show that Spt6 recruitment to genes and the nucleosome reassembly functions of Spt6 can still occur in the absence of its association with RNAPII. Surprisingly, we found that Spt6-RNAPII association is required for efficient recruitment of the Ccr4-Not de-adenylation complex to transcribed genes for essential degradation of a range of mRNAs, including mRNAs required for cell-cycle progression. These findings reveal an unexpected control mechanism for mRNA turnover during transcription facilitated by a histone chaperone.
Assuntos
Chaperonas de Histonas/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Chaperonas de Histonas/genética , Histonas/genética , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , RNA Polimerase II/genética , Estabilidade de RNA , RNA Mensageiro/genética , Elementos Reguladores de Transcrição , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genéticaRESUMO
Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.
Assuntos
Proliferação de Células , Senescência Celular , Dano ao DNA , Replicação do DNA , DNA de Neoplasias/biossíntese , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias Uterinas/metabolismo , Animais , Montagem e Desmontagem da Cromatina , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA de Neoplasias/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Histonas/metabolismo , Humanos , Metilação , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estabilidade de RNA , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , Fatores de Transcrição/genética , Transcrição Gênica , Neoplasias Uterinas/genéticaRESUMO
Spt6 coordinates nucleosome dis- and re-assembly, transcriptional elongation, and mRNA processing. Here, we report that depleting Spt6 in embryonic stem cells (ESCs) reduced expression of pluripotency factors, increased expression of cell-lineage-affiliated developmental regulators, and induced cell morphological and biochemical changes indicative of ESC differentiation. Selective downregulation of pluripotency factors upon Spt6 depletion may be mechanistically explained by its enrichment at ESC super-enhancers, where Spt6 controls histone H3K27 acetylation and methylation and super-enhancer RNA transcription. In ESCs, Spt6 interacted with the PRC2 core subunit Suz12 and prevented H3K27me3 accumulation at ESC super-enhancers and associated promoters. Biochemical as well as functional experiments revealed that Spt6 could compete for binding of the PRC2 methyltransferase Ezh2 to Suz12 and reduce PRC2 chromatin engagement. Thus, in addition to serving as a histone chaperone and transcription elongation factor, Spt6 counteracts repression by opposing H3K27me3 deposition at critical genomic regulatory regions.
Assuntos
Regulação para Baixo , Elementos Facilitadores Genéticos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Histonas/genética , Histonas/metabolismo , Camundongos , Complexo Repressor Polycomb 2/genética , Fatores de Transcrição/genéticaRESUMO
Transcriptional elongation by RNA polymerase II (RNAPII) through chromatin is a dynamic and highly regulated step of eukaryotic gene expression. A combination of transcript elongation factors (TEFs) including modulators of RNAPII activity and histone chaperones facilitate efficient transcription on nucleosomal templates. Biochemical and genetic analyses, primarily performed in Arabidopsis, provided insight into the contribution of TEFs to establish gene expression patterns during plant growth and development. In addition to summarising the role of TEFs in plant gene expression, we emphasise in our review recent advances in the field. Thus, mechanisms are presented how aberrant intragenic transcript initiation is suppressed by repressing transcriptional start sites within coding sequences. We also discuss how transcriptional interference of ongoing transcription with neighbouring genes is prevented. Moreover, it appears that plants make no use of promoter-proximal RNAPII pausing in the way mammals do, but there are nucleosome-defined mechanism(s) that determine the efficiency of mRNA synthesis by RNAPII. Accordingly, a still growing number of processes related to plant growth, development and responses to changing environmental conditions prove to be regulated at the level of transcriptional elongation.
RESUMO
Proper differentiation of the epidermis is essential to prevent water loss and to protect the body from the outside environment. Perturbations in this process can lead to a variety of skin diseases that impacts 1 in 5 people. While transcription factors that control epidermal differentiation have been well characterized, other aspects of transcription control such as elongation are poorly understood. Here we show that of the two cyclin-dependent kinases (CDK12 and CDK13), that are known to regulate transcription elongation, only CDK12 is necessary for epidermal differentiation. Depletion of CDK12 led to loss of differentiation gene expression and absence of skin barrier formation in regenerated human epidermis. CDK12 binds to genes that code for differentiation promoting transcription factors (GRHL3, KLF4, and OVOL1) and is necessary for their elongation. CDK12 is necessary for elongation by promoting Ser2 phosphorylation on the C-terminal domain of RNA polymerase II and the stabilization of binding of the elongation factor SPT6 to target genes. Our results suggest that control of transcription elongation by CDK12 plays a prominent role in adult cell fate decisions.
Assuntos
Quinases Ciclina-Dependentes , RNA Polimerase II , Diferenciação Celular/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Humanos , Fosforilação , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.
Assuntos
Chaperonas de Histonas/metabolismo , Metiltransferases/metabolismo , Elementos de Resposta , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Cromatina/genética , Cromatina/metabolismo , Chaperonas de Histonas/genética , Espectrometria de Massas , Metiltransferases/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post-translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP-dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP-A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription-coupled epigenome editing (or surveillance) function of histone chaperones.
Assuntos
Proteínas de Ligação a DNA/genética , Epigênese Genética , Proteínas de Grupo de Alta Mobilidade/genética , Chaperonas de Histonas/genética , Histonas/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Centromérica A , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Humanos , Nucleossomos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismoRESUMO
The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.