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1.
Nat Commun ; 12(1): 5446, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521844

RESUMO

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.


Assuntos
Ciclo Celular/genética , Linhagem da Célula/genética , Células Matadoras Naturais/imunologia , Proteínas com Domínio T/genética , Animais , Sequência de Bases , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Diferenciação Celular , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/imunologia , Epigênese Genética/imunologia , Interleucina-12/farmacologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Baço/citologia , Baço/imunologia , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/imunologia , Transcrição Genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
2.
Mol Cell ; 81(16): 3356-3367.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34297910

RESUMO

RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.


Assuntos
Proteínas de Drosophila/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , RNA Polimerase II/genética , Transcrição Genética , Animais , Drosophila melanogaster/genética , Metiltransferases/genética , Regiões Promotoras Genéticas/genética
3.
Cell Mol Immunol ; 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398809

RESUMO

T cell development proceeds under the influence of a network of transcription factors (TFs). The precise role of Zeb1, a member of this network, remains unclear. Here, we report that Zeb1 expression is induced early during T cell development in CD4-CD8- double-negative (DN) stage 2 (DN2). Zeb1 expression was further increased in the CD4+CD8+ double-positive (DP) stage before decreasing in more mature T cell subsets. We performed an exhaustive characterization of T cells in Cellophane mice that bear Zeb1 hypomorphic mutations. The Zeb1 mutation profoundly affected all thymic subsets, especially DN2 and DP cells. Zeb1 promoted the survival and proliferation of both cell populations in a cell-intrinsic manner. In the periphery of Cellophane mice, the number of conventional T cells was near normal, but invariant NKT cells, NK1.1+ γδ T cells and Ly49+ CD8 T cells were virtually absent. This suggested that Zeb1 regulates the development of unconventional T cell types from DP progenitors. A transcriptomic analysis of WT and Cellophane DP cells revealed that Zeb1 regulated the expression of multiple genes involved in the cell cycle and TCR signaling, which possibly occurred in cooperation with Tcf1 and Heb. Indeed, Cellophane DP cells displayed stronger signaling than WT DP cells upon TCR engagement in terms of the calcium response, phosphorylation events, and expression of early genes. Thus, Zeb1 is a key regulator of the cell cycle and TCR signaling during thymic T cell development. We propose that thymocyte selection is perturbed in Zeb1-mutated mice in a way that does not allow the survival of unconventional T cell subsets.

4.
J Mol Biol ; 432(3): 665-675, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31626801

RESUMO

Chromosomes are folded and organized into topologically associating domains (TADs) which provide a framework for the interaction of enhancers with the promoter of their target gene(s). Structural rearrangements observed during evolution or in disease contexts suggest that changes in genome organization strongly affect gene expression and can have drastic phenotypic effects. In this review, I will discuss how recent genomic engineering experiments reveal a more contrasted picture, suggesting that TADs are important but not always essential for gene expression regulation.


Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica , Expressão Gênica , Rearranjo Gênico , Animais , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Estudos de Associação Genética , Regiões Promotoras Genéticas , Transcrição Genética
5.
Brief Funct Genomics ; 19(2): 92-100, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-31796947

RESUMO

Ever since Thomas Hunt Morgan's discovery of the chromosomal basis of inheritance by using Drosophila melanogaster as a model organism, the fruit fly has remained an essential model system in studies of genome biology, including chromatin organisation. Very much as in vertebrates, in Drosophila, the genome is organised in territories, compartments and topologically associating domains (TADs). However, these domains might be formed through a slightly different mechanism than in vertebrates due to the presence of a large and potentially redundant set of insulator proteins and the minor role of dCTCF in TAD boundary formation. Here, we review the different levels of chromatin organisation in Drosophila and discuss mechanisms and factors that might be involved in TAD formation. The dynamics of TADs and enhancer-promoter interactions in the context of transcription are covered in the light of currently conflicting results. Finally, we illustrate the value of polymer modelling approaches to infer the principles governing the three-dimensional organisation of the Drosophila genome.

6.
Nat Genet ; 51(8): 1272-1282, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308546

RESUMO

Chromatin topology is intricately linked to gene expression, yet its functional requirement remains unclear. Here, we comprehensively assessed the interplay between genome topology and gene expression using highly rearranged chromosomes (balancers) spanning ~75% of the Drosophila genome. Using transheterozyte (balancer/wild-type) embryos, we measured allele-specific changes in topology and gene expression in cis, while minimizing trans effects. Through genome sequencing, we resolved eight large nested inversions, smaller inversions, duplications and thousands of deletions. These extensive rearrangements caused many changes to chromatin topology, disrupting long-range loops, topologically associating domains (TADs) and promoter interactions, yet these are not predictive of changes in expression. Gene expression is generally not altered around inversion breakpoints, indicating that mis-appropriate enhancer-promoter activation is a rare event. Similarly, shuffling or fusing TADs, changing intra-TAD connections and disrupting long-range inter-TAD loops does not alter expression for the majority of genes. Our results suggest that properties other than chromatin topology ensure productive enhancer-promoter interactions.


Assuntos
Cromatina/genética , Cromossomos de Insetos/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Rearranjo Gênico , Genoma de Inseto , Animais , Mapeamento Cromossômico , Feminino , Masculino , Regiões Promotoras Genéticas
7.
EMBO Rep ; 2017 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-28794204

RESUMO

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. Long-range, inter-domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

8.
Genes Dev ; 31(6): 590-602, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28381411

RESUMO

Developmental gene expression is tightly regulated through enhancer elements, which initiate dynamic spatio-temporal expression, and Polycomb response elements (PREs), which maintain stable gene silencing. These two cis-regulatory functions are thought to operate through distinct dedicated elements. By examining the occupancy of the Drosophila pleiohomeotic repressive complex (PhoRC) during embryogenesis, we revealed extensive co-occupancy at developmental enhancers. Using an established in vivo assay for PRE activity, we demonstrated that a subset of characterized developmental enhancers can function as PREs, silencing transcription in a Polycomb-dependent manner. Conversely, some classic Drosophila PREs can function as developmental enhancers in vivo, activating spatio-temporal expression. This study therefore uncovers elements with dual function: activating transcription in some cells (enhancers) while stably maintaining transcriptional silencing in others (PREs). Given that enhancers initiate spatio-temporal gene expression, reuse of the same elements by the Polycomb group (PcG) system may help fine-tune gene expression and ensure the timely maintenance of cell identities.


Assuntos
Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Grupo Polycomb/metabolismo , Elementos de Resposta , Animais , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Desenvolvimento Embrionário/genética
9.
Development ; 143(23): 4533-4542, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27899510

RESUMO

Developmental patterning and tissue formation are regulated through complex gene regulatory networks (GRNs) driven through the action of transcription factors (TFs) converging on enhancer elements. Here, as a point of entry to dissect the poorly defined GRN underlying cardiomyocyte differentiation, we apply an integrated approach to identify active enhancers and TFs involved in Drosophila heart development. The Drosophila heart consists of 104 cardiomyocytes, representing less than 0.5% of all cells in the embryo. By modifying BiTS-ChIP for rare cells, we examined H3K4me3 and H3K27ac chromatin landscapes to identify active promoters and enhancers specifically in cardiomyocytes. These in vivo data were complemented by a machine learning approach and extensive in vivo validation in transgenic embryos, which identified many new heart enhancers and their associated TF motifs. Our results implicate many new TFs in late stages of heart development, including Bagpipe, an Nkx3.2 ortholog, which we show is essential for differentiated heart function.


Assuntos
Drosophila/embriologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Coração/embriologia , Miócitos Cardíacos/citologia , Organogênese/genética , Regiões Promotoras Genéticas/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Cromatina , Drosophila/genética , Redes Reguladoras de Genes/genética , Histonas/metabolismo , Organogênese/fisiologia , Fatores de Transcrição/genética
10.
Methods Mol Biol ; 1478: 263-277, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27730588

RESUMO

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is an invaluable technique to assess transcription factor binding and histone modifications in a genome-wide manner, an essential step towards understanding the mechanisms that govern embryonic development. Here, we provide a detailed protocol for all steps involved in generating a ChIP-seq library, starting from embryo collection, fixation, chromatin preparation, immunoprecipitation, and finally library preparation. The protocol is optimized for Drosophila embryos, but can be easily adapted for any model organism. The resulting library is suitable for sequencing on an Illumina HiSeq or MiSeq platform.


Assuntos
Drosophila melanogaster/genética , Genoma , Histonas/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Transcrição Genética , Animais , Cromatina/química , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina/métodos , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/metabolismo
12.
Bioinformatics ; 31(19): 3085-91, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26034064

RESUMO

MOTIVATION: Circularized Chromosome Conformation Capture (4C) is a powerful technique for studying the spatial interactions of a specific genomic region called the 'viewpoint' with the rest of the genome, both in a single condition or comparing different experimental conditions or cell types. Observed ligation frequencies typically show a strong, regular dependence on genomic distance from the viewpoint, on top of which specific interaction peaks are superimposed. Here, we address the computational task to find these specific peaks and to detect changes between different biological conditions. RESULTS: We model the overall trend of decreasing interaction frequency with genomic distance by fitting a smooth monotonically decreasing function to suitably transformed count data. Based on the fit, z-scores are calculated from the residuals, and high z-scores are interpreted as peaks providing evidence for specific interactions. To compare different conditions, we normalize fragment counts between samples, and call for differential contact frequencies using the statistical method DESEQ2: adapted from RNA-Seq analysis. AVAILABILITY AND IMPLEMENTATION: A full end-to-end analysis pipeline is implemented in the R package FourCSeq available at www.bioconductor.org. CONTACT: felix.klein@embl.de or whuber@embl.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Cromossomos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Conformação de Ácido Nucleico , Estatística como Assunto , Genoma
13.
PLoS One ; 9(7): e102464, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25029256

RESUMO

The BYpass of Ess1 (Bye1) protein is a putative S. cerevisiae transcription factor homologous to the human cancer-associated PHF3/DIDO family of proteins. Bye1 contains a Plant Homeodomain (PHD) and a TFIIS-like domain. The Bye1 PHD finger interacts with tri-methylated lysine 4 of histone H3 (H3K4me3) while the TFIIS-like domain binds to RNA polymerase (Pol) II. Here, we investigated the contribution of these structural features to Bye1 recruitment to chromatin as well as its function in transcriptional regulation. Genome-wide analysis of Bye1 distribution revealed at least two distinct modes of association with actively transcribed genes: within the core of Pol II- and Pol III-transcribed genes concomitant with the presence of the TFIIS transcription factor and, additionally, with promoters of a subset of Pol II-transcribed genes. Specific loss of H3K4me3 abolishes Bye1 association to gene promoters, but doesn't affect its binding within gene bodies. Genetic interactions suggested an essential role of Bye1 in cell fitness under stress conditions compensating the absence of TFIIS. Furthermore, BYE1 deletion resulted in the attenuation of GAL genes expression upon galactose-mediated induction indicating its positive role in transcription regulation. Together, these findings point to a bimodal role of Bye1 in regulation of Pol II transcription. It is recruited via its PHD domain to H3K4 tri-methylated promoters at early steps of transcription. Once Pol II is engaged into elongation, Bye1 binds directly to the transcriptional machinery, modulating its progression along the gene.


Assuntos
Cromatina/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Imunoprecipitação da Cromatina , Histonas/metabolismo , Oligonucleotídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido
14.
Nature ; 512(7512): 96-100, 2014 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-25043061

RESUMO

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the ß-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Loci Gênicos/genética , Genoma de Inseto/genética , Humanos , Iniciação da Transcrição Genética , Ativação Transcricional
15.
Nat Genet ; 44(2): 148-56, 2012 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-22231485

RESUMO

Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. Here, we use a new approach to obtain cell type-specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. We directly assessed the relationship between chromatin modifications and the spatio-temporal activity of enhancers. Rather than having a unique chromatin state, active developmental enhancers show heterogeneous histone modifications and Pol II occupancy. Despite this complexity, combined chromatin signatures and Pol II presence are sufficient to predict enhancer activity de novo. Pol II recruitment is highly predictive of the timing of enhancer activity and seems dependent on the timing and location of transcription factor binding. Chromatin modifications typically demarcate large regulatory regions encompassing multiple enhancers, whereas local changes in nucleosome positioning and Pol II occupancy delineate single active enhancers. This cell type-specific view identifies dynamic enhancer usage, an essential step in deciphering developmental networks.


Assuntos
Drosophila melanogaster/embriologia , Elementos Facilitadores Genéticos , Animais , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo
16.
Methods Mol Biol ; 786: 229-45, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21938630

RESUMO

Accurately assessing the binding of transcription factors to cis-regulatory elements in vivo is an essential step toward understanding the mechanisms that govern embryonic development. Genome-wide transcription factor location analysis has been facilitated by the development of high-density tiling arrays (ChIP-on-chip), and more recently by next-generation sequencing technologies, which are used to sequence the DNA fragments obtained from chromatin immunoprecipitation experiments (ChIP-seq). This chapter provides a detailed protocol of the different steps required to generate a successful ChIP-seq library, starting from embryo collection and fixation to chromatin preparation, immunoprecipitation, and finally library preparation. The protocol is optimized for Drosophila embryos, but can be adapted to any organism. The obtained library is suitable for sequencing on an Illumina GAIIx platform.


Assuntos
Imunoprecipitação da Cromatina/métodos , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fatores de Transcrição/metabolismo , Animais , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos
17.
Nucleic Acids Res ; 40(1): 270-83, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21911356

RESUMO

RNA polymerase (Pol) III synthesizes the tRNAs, the 5S ribosomal RNA and a small number of untranslated RNAs. In vitro, it also transcribes short interspersed nuclear elements (SINEs). We investigated the distribution of Pol III and its associated transcription factors on the genome of mouse embryonic stem cells using a highly specific tandem ChIP-Seq method. Only a subset of the annotated class III genes was bound and thus transcribed. A few hundred SINEs were associated with the Pol III transcription machinery. We observed that Pol III and its transcription factors were present at 30 unannotated sites on the mouse genome, only one of which was conserved in human. An RNA was associated with >80% of these regions. More than 2200 regions bound by TFIIIC transcription factor were devoid of Pol III. These sites were associated with cohesins and often located close to CTCF-binding sites, suggesting that TFIIIC might cooperate with these factors to organize the chromatin. We also investigated the genome-wide distribution of the ubiquitous TFIIS variant, TCEA1. We found that, as in Saccharomyces cerevisiae, TFIIS is associated with class III genes and also with SINEs suggesting that TFIIS is a Pol III transcription factor in mammals.


Assuntos
Células-Tronco Embrionárias/metabolismo , RNA Polimerase III/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Animais , Sítios de Ligação , Fator 1 de Resposta a Butirato , Linhagem Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , Genoma , Camundongos , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNA , Elementos Nucleotídeos Curtos e Dispersos , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição TFIII/metabolismo
18.
EMBO J ; 27(18): 2411-21, 2008 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-18716630

RESUMO

The yeast URA2 gene, encoding the rate-limiting enzyme of UTP biosynthesis, is transcriptionally activated by UTP shortage. In contrast to other genes of the UTP pathway, this activation is not governed by the Ppr1 activator. Moreover, it is not due to an increased recruitment of RNA polymerase II at the URA2 promoter, but to its much more effective progression beyond the URA2 mRNA start site(s). Regulatory mutants constitutively expressing URA2 resulted from cis-acting deletions upstream of the transcription initiator region, or from amino-acid replacements altering the RNA polymerase II Switch 1 loop domain, such as rpb1-L1397S. These two mutation classes allowed RNA polymerase to progress downstream of the URA2 mRNA start site(s). rpb1-L1397S had similar effects on IMD2 (IMP dehydrogenase) and URA8 (CTP synthase), and thus specifically activated the rate-limiting steps of UTP, GTP and CTP biosynthesis. These data suggest that the Switch 1 loop of RNA polymerase II, located at the downstream end of the transcription bubble, may operate as a specific sensor of the nucleoside triphosphates available for transcription.


Assuntos
Aspartato Carbamoiltransferase/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Regulação da Expressão Gênica , Mutação , Nucleosídeos/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Aspartato Carbamoiltransferase/metabolismo , Sítios de Ligação , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , IMP Desidrogenase/genética , Modelos Biológicos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
19.
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
20.
Genes Dev ; 22(14): 1934-47, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18628399

RESUMO

TFIIS is a transcription elongation factor that stimulates transcript cleavage activity of arrested RNA polymerase II (Pol II). Recent studies revealed that TFIIS has also a role in Pol II transcription initiation. To improve our understanding of TFIIS function in vivo, we performed genome-wide location analysis of this factor. Under normal growth conditions, TFIIS was detected on Pol II-transcribed genes, and TFIIS occupancy was well correlated with that of Pol II, indicating that TFIIS recruitment is not restricted to NTP-depleted cells. Unexpectedly, TFIIS was also detected on almost all Pol III-transcribed genes. TFIIS and Pol III occupancies correlated well genome-wide on this novel class of targets. In vivo, some dst1 mutants were partly defective in tRNA synthesis and showed a reduced Pol III occupancy at the restrictive temperature. In vitro transcription assays suggested that TFIIS may affect Pol III start site selection. These data provide strong in vivo and in vitro evidence in favor of a role of TFIIS as a general Pol III transcription factor.


Assuntos
Regulação Fúngica da Expressão Gênica , Genoma Fúngico , RNA Polimerase III/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Transcrição Genética , Fatores de Elongação da Transcrição/fisiologia , Imunoprecipitação da Cromatina , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Processamento Pós-Transcricional do RNA , Saccharomyces cerevisiae/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo
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