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1.
Methods Mol Biol ; 2351: 93-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382185

RESUMO

MNase-Seq is a genome-wide procedure that allows mapping of DNA associated to nucleosomes following micrococcal nuclease digestion. It is a rapid and robust technology useful for the analysis of chromatin properties genome-wide at the resolution of mono-nucleosomes. Here, we describe how to produce high-resolution nucleosome maps of cells grown in suspension or adherent mammalian cells. After only three steps: nuclei or cell preparation, native MNase digestion and DNA purification, libraries for high-throughput sequencing can be prepared. Genome-wide nucleosome maps allow analyzing chromatin opening at promoters or enhancers, nucleosome displacement, or labile nucleosome occupancy depending on the digestion condition used. As presented, MNase-Seq is a versatile tool for investigating chromatin dynamics, regulation, and to define open chromatin regions of regulatory elements in mammalian genomes.


Assuntos
Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Animais , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Biologia Computacional/métodos , Biblioteca Gênica
2.
Front Artif Intell ; 3: 559927, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33733209

RESUMO

Addressing the heterogeneity of both the outcome of a disease and the treatment response to an intervention is a mandatory pathway for regulatory approval of medicines. In randomized clinical trials (RCTs), confirmatory subgroup analyses focus on the assessment of drugs in predefined subgroups, while exploratory ones allow a posteriori the identification of subsets of patients who respond differently. Within the latter area, subgroup discovery (SD) data mining approach is widely used-particularly in precision medicine-to evaluate treatment effect across different groups of patients from various data sources (be it from clinical trials or real-world data). However, both the limited consideration by standard SD algorithms of recommended criteria to define credible subgroups and the lack of statistical power of the findings after correcting for multiple testing hinder the generation of hypothesis and their acceptance by healthcare authorities and practitioners. In this paper, we present the Q-Finder algorithm that aims to generate statistically credible subgroups to answer clinical questions, such as finding drivers of natural disease progression or treatment response. It combines an exhaustive search with a cascade of filters based on metrics assessing key credibility criteria, including relative risk reduction assessment, adjustment on confounding factors, individual feature's contribution to the subgroup's effect, interaction tests for assessing between-subgroup treatment effect interactions and tests adjustment (multiple testing). This allows Q-Finder to directly target and assess subgroups on recommended credibility criteria. The top-k credible subgroups are then selected, while accounting for subgroups' diversity and, possibly, clinical relevance. Those subgroups are tested on independent data to assess their consistency across databases, while preserving statistical power by limiting the number of tests. To illustrate this algorithm, we applied it on the database of the International Diabetes Management Practice Study (IDMPS) to better understand the drivers of improved glycemic control and rate of episodes of hypoglycemia in type 2 diabetics patients. We compared Q-Finder with state-of-the-art approaches from both Subgroup Identification and Knowledge Discovery in Databases literature. The results demonstrate its ability to identify and support a short list of highly credible and diverse data-driven subgroups for both prognostic and predictive tasks.

3.
Proc Natl Acad Sci U S A ; 116(51): 25839-25849, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776254

RESUMO

Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 ß and γ isoforms (HP1ß/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K-AKT-mTOR axis and switch to glycolysis. While differentiation of naive TRIM28-/- T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28-/- regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1ß/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.


Assuntos
Diferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética/fisiologia , Linfócitos T/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Autoimunidade/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Plasticidade Celular/fisiologia , Reprogramação Celular/genética , Colo/patologia , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transcriptoma , Proteína 28 com Motivo Tripartido/genética
4.
Diabetes Obes Metab ; 21(8): 1925-1934, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31050099

RESUMO

AIMS: To investigate the effects of sodium-glucose co-transporter-2 (SGLT2) inhibitors vs. dipeptidyl peptidase-4 (DPP-4) inhibitors on renal function preservation (RFP) using real-world data of patients with type 2 diabetes in Japan, and to identify which subgroups of patients obtained greater RFP benefits with SGLT2 inhibitors vs. DPP-4 inhibitors. METHODS: We retrospectively analysed claims data recorded in the Medical Data Vision database in Japan of patients with type 2 diabetes (aged ≥18 years) prescribed any SGLT2 inhibitor or any DPP-4 inhibitor between May 2014 and September 2016 (identification period), in whom estimated glomerular filtration rate (eGFR) was measured at least twice (baseline, up to 6 months before the index date; follow-up, 9 to 15 months after the index date) with continuous treatment until the follow-up eGFR. The endpoint was the percentage of patients with RFP, defined as no change or an increase in eGFR from baseline to follow-up. A proprietary supervised learning algorithm (Q-Finder; Quinten, Paris, France) was used to identify the profiles of patients with an additional RFP benefit of SGLT2 inhibitors vs. DPP-4 inhibitors. RESULTS: Data were available for 990 patients prescribed SGLT2 inhibitors and 4257 prescribed DPP-4 inhibitors. The proportion of patients with RFP was significantly greater in the SGLT2 inhibitor group (odds ratio 1.27; P = 0.01). The Q-Finder algorithm identified four clinically relevant subgroups showing superior RFP with SGLT2 inhibitors (P < 0.1): no hyperlipidaemia and eGFR ≥79 mL/min/1.73 m2 ; eGFR ≥79 mL/min/1.73 m2 and diabetes duration ≤1.2 years; eGFR ≥75 mL/min/1.73 m2 and use of antithrombotic agents; and haemoglobin ≤13.4 g/dL and LDL cholesterol ≥95.1 mg/dL. In each profile, glycaemic control was similar in the two groups. CONCLUSION: SGLT2 inhibitors were associated with more favourable RFP vs. DPP-4 inhibitors in patients with certain profiles in real-world settings in Japan.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Hipoglicemiantes/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Adulto , Algoritmos , Bases de Dados Factuais , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Japão , Rim/efeitos dos fármacos , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Aprendizado de Máquina Supervisionado , Resultado do Tratamento
5.
Blood Adv ; 2(20): 2691-2703, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30337297

RESUMO

Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds DNA at CArG (CC[A/T]6GG) domains in association with myocardin-family proteins (eg, myocardin-related transcription factor A [MRTFA]) or the ternary complex factor family of E26 transformation-specific (ETS) proteins. In primary hematopoietic cells, knockout of either SRF or MRTFA decreases megakaryocyte (Mk) maturation causing thrombocytopenia. The human erythroleukemia (HEL) cell line mimics the effects of MRTFA on Mk maturation, and MRTFA overexpression (MRTFAOE) in HEL cells enhances megakaryopoiesis. To identify the mechanisms underlying these effects, we performed integrated analyses of anti-SRF chromatin immunoprecipitation (ChIP) and RNA-sequencing data from noninduced and phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])-induced HEL cells, with and without MRTFAOE We found that 11% of genes were upregulated with TPA induction, which was enhanced by MRTFAOE, resulting in an upregulation of 25% of genes. MRTFAOE increased binding of SRF to genomic sites and enhanced TPA-induced expression of SRF target genes. The TPA-induced genes are predicted to be regulated by SRF and ETS factors, whereas those upregulated by TPA plus MRTFAOE lack ETS binding motifs, and MRTFAOE skews SRF binding to genomic regions with CArG sites in regions relatively lacking in ETS binding motifs. Finally, ChIP-polymerase chain reaction using HEL cells and primary human CD34+ cell-derived subpopulations confirms that both SRF and MRTFA have increased binding during megakaryopoiesis at upregulated target genes (eg, CORO1A). We show for the first time that MRTFA increases both the genomic association and activity of SRF and upregulates genes that enhance primary human megakaryopoiesis.


Assuntos
Megacariócitos/metabolismo , Fator de Resposta Sérica/genética , Transativadores/genética , Humanos , Transativadores/metabolismo
6.
Mol Cell ; 69(1): 48-61.e6, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29304333

RESUMO

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3' direction several hundred kilobases downstream of genes. The YFFF mutant shows reduced Pol II at promoter-proximal pause sites, a loss of interaction with the Mediator and Integrator complexes, and impaired recruitment of these complexes to chromatin. Consistent with these observations, Pol II loading at enhancers and maturation of snRNAs are altered in the YFFF context genome-wide. We conclude that tyrosine1 residues of the CTD control termination of transcription by Pol II.


Assuntos
RNA Polimerase II/genética , RNA Mensageiro/biossíntese , Terminação da Transcrição Genética/fisiologia , Transcrição Genética/fisiologia , Tirosina/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Humanos , Mutação/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/genética
7.
Mol Cell ; 65(6): 1081-1095.e5, 2017 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-28286024

RESUMO

We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Histonas/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição TCF/metabolismo , Transcrição Genética , Animais , Linhagem Celular , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ativação Enzimática , Camundongos , Camundongos Knockout , Mutação , Fosforilação , Interferência de RNA , Fator de Resposta Sérica/genética , Transdução de Sinais , Fatores de Transcrição TCF/genética , Acetato de Tetradecanoilforbol/farmacologia , Sítio de Iniciação de Transcrição , Transcrição Genética/efeitos dos fármacos , Transfecção , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo
8.
Mol Cell ; 64(6): 1048-1061, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27867007

RESUMO

The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression.


Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica , Fator de Resposta Sérica/genética , Fatores de Complexo Ternário/genética , Animais , Sequência de Bases , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Perfilação da Expressão Gênica , Teste de Complementação Genética , Humanos , Camundongos , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Fatores de Complexo Ternário/antagonistas & inibidores , Fatores de Complexo Ternário/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo
9.
EMBO J ; 34(11): 1554-71, 2015 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-25896507

RESUMO

Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF-dependent transcriptional program that supports survival of epithelial cells.


Assuntos
Apoptose/fisiologia , Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Resposta Sérica/metabolismo , Transcrição Genética/fisiologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Epiteliais/citologia , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Glândulas Mamárias Humanas/citologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Resposta Sérica/genética
10.
Blood ; 125(8): 1244-55, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25573994

RESUMO

Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells (HSCs) during development. Serum response factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the myocardin-related transcription factor (MRTF) family of G-actin-regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact but does not become established in fetal bone marrow. Srf-null HSC progenitor cells (HSC/Ps) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarize in response to SDF-1 and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. Srf-null HSC/Ps exhibit substantial deficits in cytoskeletal gene expression. MRTF-SRF signaling is thus critical for expression of genes required for the response to chemokine signaling during hematopoietic development.


Assuntos
Medula Óssea/embriologia , Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Fator de Resposta Sérica/fisiologia , Nicho de Células-Tronco , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Medula Óssea/crescimento & desenvolvimento , Movimento Celular/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Nicho de Células-Tronco/genética
11.
Genes Dev ; 28(9): 943-58, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24732378

RESUMO

The transcription factor SRF (serum response factor) recruits two families of coactivators, the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors), to couple gene transcription to growth factor signaling. Here we investigated the role of the SRF network in the immediate transcriptional response of fibroblasts to serum stimulation. SRF recruited its cofactors in a gene-specific manner, and virtually all MRTF binding was directed by SRF. Much of SRF DNA binding was serum-inducible, reflecting a requirement for MRTF-SRF complex formation in nucleosome displacement. We identified 960 serum-responsive SRF target genes, which were mostly MRTF-controlled, as assessed by MRTF chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) and/or sensitivity to MRTF-linked signals. MRTF activation facilitates RNA polymerase II (Pol II) recruitment or promoter escape according to gene context. MRTF targets encode regulators of the cytoskeleton, transcription, and cell growth, underpinning the role of SRF in cytoskeletal dynamics and mechanosensing. Finally, we show that specific activation of either MRTFs or TCFs can reset the circadian clock.


Assuntos
Actinas/metabolismo , Fibroblastos/fisiologia , Soro/metabolismo , Transdução de Sinais , Transcrição Genética/genética , Animais , Proteínas CLOCK/genética , Relógios Circadianos/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fator de Resposta Sérica/metabolismo
12.
Cell ; 152(3): 492-503, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23374345

RESUMO

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.


Assuntos
Actinas/metabolismo , Ritmo Circadiano , Regulação da Expressão Gênica , Técnicas Genéticas , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Masculino , Camundongos , Proteínas Circadianas Period/metabolismo , Ratos , Fatores de Transcrição/metabolismo
13.
Mol Cell ; 31(3): 337-46, 2008 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-18691966

RESUMO

In vitro, without Mediator, the association of general transcription factors (GTF) and RNA polymerase II (Pol II) in preinitiation complexes (PIC) occurs in an orderly fashion. In this work, we explore the in vivo function of Mediator in GTF recruitment to PIC. A direct interaction between Med11 Mediator head subunit and Rad3 TFIIH subunit was identified. We explored the significance of this interaction and those of Med11 with head module subunits Med17 and Med22 and found that impairing these interactions could differentially affect the recruitment of TFIIH, TFIIE, and Pol II in the PIC. A med11 mutation that altered promoter occupancy by the TFIIK kinase module of TFIIH genome-wide also reduced Pol II CTD serine 5 phosphorylation. We conclude that the Mediator head module plays a critical role in TFIIH and TFIIE recruitment to the PIC. We identify steps in PIC formation that suggest a branched assembly pathway.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição Genética , Centrômero/metabolismo , Imunoprecipitação da Cromatina , DNA Helicases/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Complexo Mediador , Modelos Biológicos , Mutação/genética , Fosforilação , Fosfotransferases/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição TFII/metabolismo
14.
Proc Natl Acad Sci U S A ; 104(41): 16062-7, 2007 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-17901206

RESUMO

The transcription initiation and elongation steps of protein-coding genes usually rely on unrelated protein complexes. However, the TFIIS elongation factor is implicated in both processes. We found that, in the absence of the Med31 Mediator subunit, yeast cells required the TFIIS polymerase II (Pol II)-binding domain but not its RNA cleavage stimulatory activity that is associated with its elongation function. We also found that the TFIIS Pol II-interacting domain was needed for the full recruitment of Pol II to several promoters in the absence of Med31. This work demonstrated that, in addition to its thoroughly characterized role in transcription elongation, TFIIS is implicated through its Pol II-binding domain in the formation or stabilization of the transcription initiation complex in vivo.


Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Cisteína Sintase , Genes Fúngicos , Teste de Complementação Genética , Complexo Mediador , Modelos Moleculares , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutagênese , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Deleção de Sequência , Transcrição Genética , Fatores de Elongação da Transcrição/química
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