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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.
Nat Commun ; 12(1): 4503, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301927

RESUMO

Promoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.


Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV/genética , HIV-1/genética , Regiões Promotoras Genéticas/genética , Latência Viral/genética , Algoritmos , RNA Polimerases Dirigidas por DNA/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Modelos Genéticos , Processos Estocásticos , Fatores de Tempo , Ativação Viral/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
4.
Nucleic Acids Res ; 49(5): 2488-2508, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33533919

RESUMO

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.


Assuntos
Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Epigênese Genética , Antígeno 2 Relacionado a Fos/metabolismo , Humanos , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/fisiologia , Fator de Transcrição AP-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo
5.
Cell Rep ; 32(7): 108048, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32814051

RESUMO

During thymic development and upon peripheral activation, T cells undergo extensive phenotypic and functional changes coordinated by lineage-specific developmental programs. To characterize the regulatory landscape controlling T cell identity, we perform a wide epigenomic and transcriptional analysis of mouse thymocytes and naive CD4 differentiated T helper cells. Our investigations reveal a dynamic putative enhancer landscape, and we could validate many of the enhancers using the high-throughput CapStarr sequencing (CapStarr-seq) approach. We find that genes using multiple promoters display increased enhancer usage, suggesting that apparent "enhancer redundancy" might relate to isoform selection. Furthermore, we can show that two Runx3 promoters display long-range interactions with specific enhancers. Finally, our analyses suggest a novel function for the PRC2 complex in the control of alternative promoter usage. Altogether, our study has allowed for the mapping of an exhaustive set of active enhancers and provides new insights into their function and that of PRC2 in controlling promoter choice during T cell differentiation.


Assuntos
Proteínas do Grupo Polycomb/genética , Linfócitos T/metabolismo , Animais , Diferenciação Celular , Masculino , Camundongos
6.
PLoS One ; 15(5): e0233191, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453736

RESUMO

The Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-throughput enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus.


Assuntos
Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica/fisiologia , Loci Gênicos/fisiologia , Fator de Transcrição Ikaros/biossíntese , Animais , Linhagem Celular , Epigenômica , Genes Reporter , Fator de Transcrição Ikaros/genética , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
7.
Am J Hum Genet ; 106(3): 356-370, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32109418

RESUMO

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.


Assuntos
Metilação de DNA , Transtornos do Neurodesenvolvimento/genética , Fenótipo , Estudos de Coortes , Heterogeneidade Genética , Humanos , Síndrome
8.
Genet Med ; 22(1): 181-188, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31363182

RESUMO

PURPOSE: Kabuki syndrome (KS) (OMIM 147920 and 300867) is a rare genetic disorder characterized by specific facial features, intellectual disability, and various malformations. Immunopathological manifestations seem prevalent and increase the morbimortality. To assess the frequency and severity of the manifestations, we measured the prevalence of immunopathological manifestations as well as genotype-phenotype correlations in KS individuals from a registry. METHODS: Data were for 177 KS individuals with KDM6A or KMT2D pathogenic variants. Questionnaires to clinicians were used to assess the presence of immunodeficiency and autoimmune diseases both on a clinical and biological basis. RESULTS: Overall, 44.1% (78/177) and 58.2% (46/79) of KS individuals exhibited infection susceptibility and hypogammaglobulinemia, respectively; 13.6% (24/177) had autoimmune disease (AID; 25.6% [11/43] in adults), 5.6% (10/177) with ≥2 AID manifestations. The most frequent AID manifestations were immune thrombocytopenic purpura (7.3% [13/177]) and autoimmune hemolytic anemia (4.0% [7/177]). Among nonhematological manifestations, vitiligo was frequent. Immune thrombocytopenic purpura was frequent with missense versus other types of variants (p = 0.027). CONCLUSION: The high prevalence of immunopathological manifestations in KS demonstrates the importance of systematic screening and efficient preventive management of these treatable and sometimes life-threatening conditions.


Assuntos
Doenças Autoimunes/epidemiologia , Proteínas de Ligação a DNA/genética , Face/anormalidades , Doenças Hematológicas/complicações , Histona Desmetilases/genética , Proteínas de Neoplasias/genética , Doenças da Imunodeficiência Primária/epidemiologia , Doenças Vestibulares/complicações , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/imunologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Doenças Hematológicas/genética , Doenças Hematológicas/imunologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação , Prevalência , Sistema de Registros , Índice de Gravidade de Doença , Doenças Vestibulares/genética , Doenças Vestibulares/imunologia , Adulto Jovem
9.
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
10.
Mol Cell ; 74(3): 555-570.e7, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30956044

RESUMO

L1 retrotransposons are transposable elements and major contributors of genetic variation in humans. Where L1 integrates into the genome can directly impact human evolution and disease. Here, we experimentally induced L1 retrotransposition in cells and mapped integration sites at nucleotide resolution. At local scales, L1 integration is mostly restricted by genome sequence biases and the specificity of the L1 machinery. At regional scales, L1 shows a broad capacity for integration into all chromatin states, in contrast to other known mobile genetic elements. However, integration is influenced by the replication timing of target regions, suggesting a link to host DNA replication. The distribution of new L1 integrations differs from those of preexisting L1 copies, which are significantly reshaped by natural selection. Our findings reveal that the L1 machinery has evolved to efficiently target all genomic regions and underline a predominant role for post-integrative processes on the distribution of endogenous L1 elements.


Assuntos
Elementos de DNA Transponíveis/genética , Genoma Humano/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , Mapeamento Cromossômico , Replicação do DNA/genética , Genômica , Células HeLa , Humanos
11.
Nucleic Acids Res ; 47(2): 700-715, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30476274

RESUMO

Mammalian-wide interspersed repeats (MIRs) are retrotransposed elements of mammalian genomes. Here, we report the specific binding of zinc finger protein ZNF768 to the sequence motif GCTGTGTG (N20) CCTCTCTG in the core region of MIRs. ZNF768 binding is preferentially associated with euchromatin and promoter regions of genes. Binding was observed for genes expressed in a cell type-specific manner in human B cell line Raji and osteosarcoma U2OS cells. Mass spectrometric analysis revealed binding of ZNF768 to Elongator components Elp1, Elp2 and Elp3 and other nuclear factors. The N-terminus of ZNF768 contains a heptad repeat array structurally related to the C-terminal domain (CTD) of RNA polymerase II. This array evolved in placental animals but not marsupials and monotreme species, displays species-specific length variations, and possibly fulfills CTD related functions in gene regulation. We propose that the evolution of MIRs and ZNF768 has extended the repertoire of gene regulatory mechanisms in mammals and that ZNF768 binding is associated with cell type-specific gene expression.


Assuntos
Retroelementos , Fatores de Transcrição/metabolismo , Transcrição Genética , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular , DNA/química , DNA/metabolismo , Eucromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Motivos de Nucleotídeos , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/química
12.
Nucleic Acids Res ; 46(7): 3339-3350, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29425303

RESUMO

The transcription factor PLZF (promyelocytic leukemia zinc finger protein) acts as an epigenetic regulator balancing self-renewal and differentiation of hematopoietic cells through binding to various chromatin-modifying factors. First described as a transcriptional repressor, PLZF is also associated with active transcription, although the molecular bases underlying the differences are unknown. Here, we reveal that in a hematopoietic cell line, PLZF is predominantly associated with transcribed genes. Additionally, we identify a new association between PLZF and the histone methyltransferase, EZH2 at the genomic level. We find that co-occupancy of PLZF and EZH2 on chromatin at PLZF target genes is not associated with SUZ12 or trimethylated lysine 27 of histone H3 (H3K27me3) but with the active histone mark H3K4me3 and active transcription. Removal of EZH2 leads to an increase of PLZF binding and increased gene expression. Our results suggest a new role of EZH2 in restricting PLZF positive transcriptional activity independently of its canonical PRC2 activity.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Complexo Repressor Polycomb 2/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Transcrição Genética , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Cromatina/genética , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , Histona Metiltransferases/genética , Histonas/genética , Humanos , Ligação Proteica/genética
13.
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
14.
Nucleic Acids Res ; 45(17): 10229-10241, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973446

RESUMO

Termination of transcription is important for establishing gene punctuation marks. It is also critical for suppressing many of the pervasive transcription events occurring throughout eukaryotic genomes and coupling their RNA products to efficient decay. In human cells, the ARS2 protein has been implicated in such function as its depletion causes transcriptional read-through of selected gene terminators and because it physically interacts with the ribonucleolytic nuclear RNA exosome. Here, we study the role of ARS2 on transcription and RNA metabolism genome wide. We show that ARS2 depletion negatively impacts levels of promoter-proximal RNA polymerase II at protein-coding (pc) genes. Moreover, our results reveal a general role of ARS2 in transcription termination-coupled RNA turnover at short transcription units like snRNA-, replication-dependent histone-, promoter upstream transcript- and enhancer RNA-loci. Depletion of the ARS2 interaction partner ZC3H18 mimics the ARS2 depletion, although to a milder extent, whereas depletion of the exosome core subunit RRP40 only impacts RNA abundance post-transcriptionally. Interestingly, ARS2 is also involved in transcription termination events within first introns of pc genes. Our work therefore establishes ARS2 as a general suppressor of pervasive transcription with the potential to regulate pc gene expression.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/fisiologia , RNA Polimerase II/metabolismo , Terminação da Transcrição Genética , Imunoprecipitação da Cromatina , Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Células HeLa , Humanos , Íntrons , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/fisiologia
15.
Transcription ; 8(3): 179-184, 2017 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-28301306

RESUMO

Pioneer transcription factors are defined by their ability to bind nucleosome-occupied regions. Here, we discuss the properties of nucleosomes bound by pioneers at enhancer regions. We describe how select pioneers bind nucleosome-occupied or -depleted enhancer sites. Importantly, by revisiting and expanding existing data sets, we show differential H2A.Z and p300/CBP association at bound enhancers, highlighting two possible pioneering modes.


Assuntos
Elementos Facilitadores Genéticos/fisiologia , Histonas/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Histonas/genética , Humanos , Nucleossomos/genética , Fatores de Transcrição de p300-CBP/genética
16.
Nat Commun ; 7: 11841, 2016 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-27312418

RESUMO

NFATc1 plays a critical role in double-negative thymocyte survival and differentiation. However, the signals that regulate Nfatc1 expression are incompletely characterized. Here we show a developmental stage-specific differential expression pattern of Nfatc1 driven by the distal (P1) or proximal (P2) promoters in thymocytes. Whereas, preTCR-negative thymocytes exhibit only P2 promoter-derived Nfatc1ß expression, preTCR-positive thymocytes express both Nfatc1ß and P1 promoter-derived Nfatc1α transcripts. Inducing NFATc1α activity from P1 promoter in preTCR-negative thymocytes, in addition to the NFATc1ß from P2 promoter impairs thymocyte development resulting in severe T-cell lymphopenia. In addition, we show that NFATc1 activity suppresses the B-lineage potential of immature thymocytes, and consolidates their differentiation to T cells. Further, in the pTCR-positive DN3 cells, a threshold level of NFATc1 activity is vital in facilitating T-cell differentiation and to prevent Notch3-induced T-acute lymphoblastic leukaemia. Altogether, our results show NFATc1 activity is crucial in determining the T-cell fate of thymocytes.


Assuntos
Linfopenia/imunologia , Fatores de Transcrição NFATC/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Receptor Notch3/imunologia , Linfócitos T/imunologia , Timócitos/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Sobrevivência Celular , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Linfopenia/genética , Linfopenia/patologia , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Receptor Notch3/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/citologia , Timócitos/citologia , Timo/citologia , Timo/imunologia
17.
Bioinformatics ; 32(16): 2528-30, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27153642

RESUMO

UNLABELLED: We describe an R package designed for processing aligned reads from chromatin-oriented high-throughput sequencing experiments. Pasha (preprocessing of aligned sequences from HTS analyses) allows easy manipulation of aligned reads from short-read sequencing technologies (ChIP-seq, FAIRE-seq, MNase-Seq, …) and offers innovative approaches such as ChIP-seq reads elongation, nucleosome midpoint piling strategy for positioning analyses, or the ability to subset paired-end reads by groups of insert size that can contain biologically relevant information. AVAILABILITY AND IMPLEMENTATION: Pasha is a multi-platform R package, available on CRAN repositories under GPL-3 license (https://cran.r-project.org/web/packages/Pasha/). CONTACTS: rfenouil@gmail.com or jean-christophe.andrau@igmm.cnrs.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Software , Nucleossomos
18.
Nucleic Acids Res ; 44(8): 3567-85, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-26673693

RESUMO

Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity.


Assuntos
Diferenciação Celular/genética , Elementos Facilitadores Genéticos/genética , Nucleossomos/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Linfócitos T/citologia , Animais , Sequência de Bases , Sítios de Ligação/genética , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Hematopoese/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleossomos/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , RNA Polimerase II/metabolismo , Análise de Sequência de DNA
19.
Transcription ; 6(5): 91-101, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26566685

RESUMO

Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.


Assuntos
Lisina/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Acetilação , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Espectrometria de Massas , Metilação , Modelos Moleculares , Estrutura Terciária de Proteína , RNA Polimerase II/genética , Iniciação da Transcrição Genética
20.
Genome Res ; 25(12): 1873-85, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26560631

RESUMO

To unveil the still-elusive nature of metazoan replication origins, we identified them genome-wide and at unprecedented high-resolution in mouse ES cells. This allowed initiation sites (IS) and initiation zones (IZ) to be differentiated. We then characterized their genetic signatures and organization and integrated these data with 43 chromatin marks and factors. Our results reveal that replication origins can be grouped into three main classes with distinct organization, chromatin environment, and sequence motifs. Class 1 contains relatively isolated, low-efficiency origins that are poor in epigenetic marks and are enriched in an asymmetric AC repeat at the initiation site. Late origins are mainly found in this class. Class 2 origins are particularly rich in enhancer elements. Class 3 origins are the most efficient and are associated with open chromatin and polycomb protein-enriched regions. The presence of Origin G-rich Repeated elements (OGRE) potentially forming G-quadruplexes (G4) was confirmed at most origins. These coincide with nucleosome-depleted regions located upstream of the initiation sites, which are associated with a labile nucleosome containing H3K64ac. These data demonstrate that specific chromatin landscapes and combinations of specific signatures regulate origin localization. They explain the frequently observed links between DNA replication and transcription. They also emphasize the plasticity of metazoan replication origins and suggest that in multicellular eukaryotes, the combination of distinct genetic features and chromatin configurations act in synergy to define and adapt the origin profile.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Origem de Replicação , Animais , Composição de Bases , Montagem e Desmontagem da Cromatina , Mapeamento Cromossômico , Análise por Conglomerados , Biologia Computacional/métodos , Células-Tronco Embrionárias , Genoma , Genômica , Heterocromatina/genética , Heterocromatina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histonas , Humanos , Camundongos , Nucleossomos/genética , Nucleossomos/metabolismo , Motivos de Nucleotídeos , Complexo de Reconhecimento de Origem , Ativação Transcricional
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