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1.
Cancers (Basel) ; 14(1)2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35008373

RESUMO

Epithelial-mesenchymal transition (EMT) and its reversal, mesenchymal-epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET, or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We find that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET, and is lower in triple-negative breast cancer cell lines with EMT features. Through an analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs, but also by a diminished enrichment for CTCF binding motifs. Through a loss-of-function analysis, we demonstrate that the suppression of CTCF alters cellular plasticity, strengthening the epithelial phenotype via the upregulation of epithelial markers E-cadherin/CDH1 and downregulation of N-cadherin/CDH2. Conversely, the upregulation of CTCF leads to the upregulation of EMT gene expression and an increase in mesenchymal traits. These findings are indicative of a role of CTCF in regulating epithelial-mesenchymal plasticity and gene expression.

2.
Clin Cancer Res ; 27(11): 3178-3189, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33731366

RESUMO

PURPOSE: Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome. EXPERIMENTAL DESIGN: CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for 1-6 months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA sequencing (RNA-seq) and assay for transpose accessible chromatin sequencing (ATAC-seq). Chromatin accessible regions were used to identify regulatory RNA expression in more than 700 samples from newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass trial (NCT01454297). RESULTS: Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. More than 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with immunoglobulin heavy chain translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma. CONCLUSIONS: These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.


Assuntos
Cromatina/genética , Regulação Neoplásica da Expressão Gênica/genética , Expressão Gênica , Mieloma Múltiplo/genética , RNA/genética , Progressão da Doença , Epigenoma , Estudos de Viabilidade , Humanos , Prognóstico , Análise de Sequência de RNA
3.
Sci Immunol ; 5(51)2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887843

RESUMO

Cell type-specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage-specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage-specific enhancer (R-TEn) and the two B cell-specific elements, R1B and R2B By generating mice lacking either R-TEn or R1B and R2B, we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn, we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules.


Assuntos
Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Proteínas de Ligação a DNA/imunologia , Proteínas de Homeodomínio/imunologia , Linfócitos T/imunologia , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Feminino , Proteínas de Homeodomínio/genética , Masculino , Camundongos
5.
Cell Rep ; 31(1): 107470, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32268089

RESUMO

The transition from the follicular B to the plasma cell stage is associated with large-scale changes in cell morphology. Here, we examine whether plasma cell development is also associated with changes in nuclear architecture. We find that the onset of plasma cell development is concomitant with a decline in remote genomic interactions; a gain in euchromatic character at loci encoding for factors that specify plasma cell fate, including Prdm1 and Atf4; and establishment of de novo inter-chromosomal hubs. We find that, in developing plasma cells and concurrent with transcriptional silencing, the Ebf1 locus repositions from an euchromatic to peri-centromeric heterochromatic environment. Finally, we find that inter-chromosomal hubs are enriched for the deposition of either H3K27Ac or H3K27me3. These data indicate that plasma cell fate is orchestrated by elaborate changes in genome topology and that epigenetic marks, linked with super-enhancers or transcriptionally repressed regions, are enriched at inter-chromosomal hubs.


Assuntos
Histonas/metabolismo , Plasmócitos/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/fisiologia , Cromossomos/genética , Epigênese Genética/genética , Feminino , Genoma/genética , Heterocromatina/genética , Histonas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/citologia , Plasmócitos/patologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
6.
J Bacteriol ; 201(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30782633

RESUMO

Biofilm formation is a complex process that requires a number of transcriptional, proteomic, and physiological changes to enable bacterial survival. The lipid membrane presents a barrier to communication between the machinery within bacteria and the physical and chemical features of their extracellular environment, and yet little is known about how the membrane influences biofilm development. We found that depleting the anionic phospholipid cardiolipin reduces biofilm formation in Escherichia coli cells by as much as 50%. The absence of cardiolipin activates the regulation of colanic acid synthesis (Rcs) envelope stress response, which represses the production of flagella, disrupts initial biofilm attachment, and reduces biofilm growth. We demonstrate that a reduction in the concentration of cardiolipin impairs translocation of proteins across the inner membrane, which we hypothesize activates the Rcs pathway through the outer membrane lipoprotein RcsF. Our study demonstrates a molecular connection between the composition of membrane phospholipids and biofilm formation in E. coli and suggests that altering lipid biosynthesis may be a viable approach for altering biofilm formation and possibly other multicellular phenotypes related to bacterial adaptation and survival.IMPORTANCE There is a growing interest in the role of lipid membrane composition in the physiology and adaptation of bacteria. We demonstrate that a reduction in the anionic phospholipid cardiolipin impairs biofilm formation in Escherichia coli cells. Depleting cardiolipin reduced protein translocation across the inner membrane and activated the Rcs envelope stress response. Consequently, cardiolipin depletion produced cells lacking assembled flagella, which impacted their ability to attach to surfaces and seed the earliest stage in biofilm formation. This study provides empirical evidence for the role of anionic phospholipid homeostasis in protein translocation and its effect on biofilm development and highlights modulation of the membrane composition as a potential method of altering bacterial phenotypes related to adaptation and survival.


Assuntos
Biofilmes/crescimento & desenvolvimento , Cardiolipinas/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Transdução de Sinais , Ácidos Cólicos/metabolismo , Transporte Proteico , Estresse Fisiológico
7.
Blood ; 131(19): 2138-2150, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29519805

RESUMO

Multiple myeloma (MM) is an aggressive cancer that originates from antibody-secreting plasma cells. Although genetically and transcriptionally well characterized, the aberrant gene regulatory networks that underpin this disease remain poorly understood. Here, we mapped regulatory elements, open chromatin, and transcription factor (TF) footprints in primary MM cells. In comparison with normal antibody-secreting cells, MM cells displayed consistent changes in enhancer activity that are connected to superenhancer (SE)-mediated deregulation of TF genes. MM cells also displayed widespread decompaction of heterochromatin that was associated with activation of regulatory elements and in a major subset of patients' deregulation of the cyclic adenosine monophosphate pathway. Finally, building SE-associated TF-based regulatory networks allowed identification of several novel TFs that are central to MM biology. Taken together, these findings significantly add to our understanding of the aberrant gene regulatory network that underpins MM.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Mieloma Múltiplo/genética , Biomarcadores , Linhagem da Célula/genética , Cromatina/metabolismo , Biologia Computacional/métodos , Humanos , Imunofenotipagem , Mieloma Múltiplo/metabolismo , Translocação Genética
8.
Immunity ; 46(5): 818-834.e4, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28514688

RESUMO

Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early T cell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish T cell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated T cell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, T cell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate.


Assuntos
Imunidade Adaptativa , Imunidade Inata , Imunomodulação , Subpopulações de Linfócitos/imunologia , Timócitos/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/imunologia , Análise por Conglomerados , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Imunofenotipagem , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/metabolismo , Células Progenitoras Linfoides/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Timócitos/citologia , Timócitos/metabolismo , Transcriptoma
9.
J Biol Methods ; 3(4): e56, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-31453219

RESUMO

The RNA-guided CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeat-CRISPR-associated 9) system has become a revolutionary technology for targeted genome engineering. The critical step of this technology requires the design of a highly specific and efficient guide RNA (gRNA) that will guide the Cas9 nuclease to the complementary DNA target sequence. CRISPR-Explorer is a new and user-friendly web server for selecting optimal CRISPR sites. It implements the latest scoring schemes of gRNA specificity and efficiency based on published empirical studies. The gRNA design results are generated instantly, thus removing wait times. The user can visualize the high-quality gRNAs with detailed design information through an interactive genome browser. Furthermore, the user can define and specify the parameters for gRNA selection in the Batch Design mode, which recognizes various input formats. CRISPR Explorer is freely accessible at: http://crisprexplorer.org.

10.
Immunity ; 42(2): 239-251, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25692700

RESUMO

T follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1(-/-) GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner.


Assuntos
Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/biossíntese , Fatores de Transcrição Forkhead/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Animais , Ativação Enzimática , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6 , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/imunologia
11.
Curr Opin Genet Dev ; 23(2): 104-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23266214

RESUMO

It is now well established that the mammalian genome is highly organized. Chromosomes are structured as territories that only sporadically intermingle. Chromosome territories themselves are segregated into distinct environments, that is, the transcriptionally inert/repressive (heterochromatic) and permissive (euchromatic) compartments. The transcriptionally permissive compartment is organized into domains (∼0.5-3 Mb) that consist of bundles of loops, are gene-rich and closely associated by activating epigenetic marks. During ontogeny and developmental progression chromatin states are highly dynamic. Recent studies have shown that loci and domains readily switch compartments. Switching nuclear neighborhoods is closely associated with changes in transcriptional activity and extensive chromatin reorganization. Here we discuss the implications of a dynamic genome and how it relates to the control of developmental progression.


Assuntos
Compartimento Celular , Núcleo Celular/genética , Cromossomos/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Montagem e Desmontagem da Cromatina/genética , Genoma , Humanos
12.
Proc Natl Acad Sci U S A ; 109(51): 21028-33, 2012 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-23213261

RESUMO

Recent studies have identified a number of transcriptional regulators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and paired box gene 5 (PAX5), that promote early B-cell development. However, how this ensemble of regulators mechanistically promotes B-cell fate remains poorly understood. Here we demonstrate that B-cell development in FOXO1-deficient mice is arrested in the common lymphoid progenitor (CLP) LY6D(+) cell stage. We demonstrate that this phenotype closely resembles the arrest in B-cell development observed in EBF1-deficient mice. Consistent with these observations, we find that the transcription signatures of FOXO1- and EBF1-deficient LY6D(+) progenitors are strikingly similar, indicating a common set of target genes. Furthermore, we found that depletion of EBF1 expression in LY6D(+) CLPs severely affects FOXO1 mRNA abundance, whereas depletion of FOXO1 activity in LY6D(+) CLPs ablates EBF1 transcript levels. We generated a global regulatory network from EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1- and FOXO1-deficient CLPs. This analysis reveals that EBF1 and FOXO1 act in a positive feedback circuitry to promote and stabilize specification to the B-cell lineage.


Assuntos
Linfócitos B/imunologia , Fatores de Transcrição Forkhead/metabolismo , Transativadores/fisiologia , Animais , Linfócitos B/citologia , Linhagem da Célula , Separação Celular , Elementos Facilitadores Genéticos , Retroalimentação Fisiológica , Citometria de Fluxo , Proteína Forkhead Box O1 , Regulação da Expressão Gênica , Luciferases/metabolismo , Camundongos , Modelos Biológicos , Modelos Genéticos , Transcrição Gênica
13.
Nat Immunol ; 13(12): 1196-204, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23064439

RESUMO

The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage-specific transcription signature.


Assuntos
Linfócitos B/fisiologia , Linhagem da Célula , Núcleo Celular/genética , Linfopoese , Células Precursoras de Linfócitos B/fisiologia , Animais , Linfócitos B/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator de Ligação a CCCTC , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Lâmina Nuclear/metabolismo , Células Precursoras de Linfócitos B/citologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Fatores de Transcrição de p300-CBP/genética
14.
Semin Immunol ; 23(5): 317-25, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21930392

RESUMO

Multiple trajectories have recently been described through which hematopoietic progenitor cells travel prior to becoming lineage-committed effectors. A wide spectrum of transcription factors has recently been identified that modulate developmental progression along such trajectories. Here we describe how distinct families of transcription factors act and are linked together to orchestrate early hematopoiesis.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/imunologia , Fatores de Transcrição/imunologia , Linhagem da Célula , Humanos , Modelos Biológicos
15.
Immunity ; 35(3): 413-25, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21903424

RESUMO

Recent studies have documented genome-wide binding patterns of transcriptional regulators and their associated epigenetic marks in hematopoietic cell lineages. In order to determine how epigenetic marks are established and maintained during developmental progression, we have generated long-term cultures of hematopoietic progenitors by enforcing the expression of the E-protein antagonist Id2. Hematopoietic progenitors that express Id2 are multipotent and readily differentiate upon withdrawal of Id2 expression into committed B lineage cells, thus indicating a causative role for E2A (Tcf3) in promoting the B cell fate. Genome-wide analyses revealed that a substantial fraction of lymphoid and myeloid enhancers are premarked by the poised or active enhancer mark H3K4me1 in multipotent progenitors. Thus, in hematopoietic progenitors, multilineage priming of enhancer elements precedes commitment to the lymphoid or myeloid cell lineages.


Assuntos
Linfócitos B/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem da Célula , Elementos Facilitadores Genéticos , Células-Tronco Hematopoéticas/citologia , Células Mieloides/citologia , Animais , Células Cultivadas , Regulação da Expressão Gênica , Análise Serial de Proteínas
16.
Nat Immunol ; 12(10): 992-1001, 2011 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-21857655

RESUMO

It is established that the transcription factor E2A and its antagonist Id3 modulate the checkpoints consisting of the precursor to the T cell antigen receptor (pre-TCR) and the TCR. Here we demonstrate that Id3 expression was higher beyond the pre-TCR checkpoint, remained high in naive T cells and showed a bimodal pattern in the effector-memory population. We show how E2A promoted T lineage specification and how pre-TCR-mediated signaling affected E2A genome-wide occupancy. Thymi in Id3-deficient mice had aberrant development of effector-memory cells, higher expression of the chemokine receptor CXCR5 and the transcriptional repressor Bcl-6 and, unexpectedly, T cell-B cell conjugates and B cell follicles. Collectively, our data show how E2A acted globally to orchestrate development into the T lineage and that Id3 antagonized E2A activity beyond the pre-TCR checkpoint to enforce the naive fate of T cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas Inibidoras de Diferenciação/fisiologia , Linfócitos T/imunologia , Animais , Memória Imunológica , Imunofenotipagem , Antígenos Comuns de Leucócito/análise , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores CXCR5/análise , Baço/imunologia , Timo/imunologia
17.
Proc Natl Acad Sci U S A ; 108(23): 9566-71, 2011 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-21606361

RESUMO

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the V(H) region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5' of DFL16 and the 3' regulatory region, and also the intronic enhancer (Eµ), creating a D(H)-J(H)-Eµ-C(H) domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D(H) region and parts of the V(H) locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação/genética , Western Blotting , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , DNA Antissenso/genética , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Interferência de RNA , RNA Antissenso/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Coesinas
18.
Nat Immunol ; 11(7): 635-43, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20543837

RESUMO

It is now established that the transcription factors E2A, EBF1 and Foxo1 have critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1, as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate.


Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Redes Reguladoras de Genes , Células Precursoras de Linfócitos B/metabolismo , Fatores de Transcrição TCF/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem da Célula , Células Cultivadas , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Linfopoese/genética , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/patologia , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição TCF/genética , Transativadores/genética , Transativadores/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição
19.
Mol Cell ; 38(4): 576-89, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20513432

RESUMO

Genome-scale studies have revealed extensive, cell type-specific colocalization of transcription factors, but the mechanisms underlying this phenomenon remain poorly understood. Here, we demonstrate in macrophages and B cells that collaborative interactions of the common factor PU.1 with small sets of macrophage- or B cell lineage-determining transcription factors establish cell-specific binding sites that are associated with the majority of promoter-distal H3K4me1-marked genomic regions. PU.1 binding initiates nucleosome remodeling, followed by H3K4 monomethylation at large numbers of genomic regions associated with both broadly and specifically expressed genes. These locations serve as beacons for additional factors, exemplified by liver X receptors, which drive both cell-specific gene expression and signal-dependent responses. Together with analyses of transcription factor binding and H3K4me1 patterns in other cell types, these studies suggest that simple combinations of lineage-determining transcription factors can specify the genomic sites ultimately responsible for both cell identity and cell type-specific responses to diverse signaling inputs.


Assuntos
Linfócitos B/metabolismo , Linhagem da Célula , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Elementos Reguladores de Transcrição/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/citologia , Sítios de Ligação , Linhagem da Célula/genética , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Fatores de Transcrição/genética
20.
Nucleic Acids Res ; 33(9): 3072-81, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15917439

RESUMO

TFIIE and TFIIH are essential for the promoter opening and escape that occurs as RNA polymerase II transits into early elongation. XPB, a subunit of TFIIH, contains an ATP-dependent helicase activity that is used in both of these processes. Here, we show that the smaller beta subunit of TFIIE stimulates the XPB helicase and ATPase activities. The larger alpha subunit can use its known inhibitory activity to moderate the stimulation by the beta subunit. Regions of TFIIE beta required for the helicase stimulation were identified. Mutants were constructed that are defective in stimulating the XPB helicase but still allow intact TFIIE to bind and recruit XPB and TFIIH to form the pre-initiation complex. In a test for the functional significance of the stimulatory effect of TFIIE beta, these mutant forms of TFIIE were shown to be defective in a transcription assay on linear DNA. The data suggest that the beta subunit of TFIIE is an ATPase and helicase co-factor that can assist the XPB subunit of TFIIH during transcription initiation and the transition to early elongation, enhancing the potential diversity of regulatory targets.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição TFII/fisiologia , Transcrição Gênica , Células HeLa , Humanos , Mutação , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/genética
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