RESUMO
Pax5 is a critical regulator of B-cell commitment. Here, we identified direct Pax5 target genes by streptavidin-mediated ChIP-chip analysis of pro-B cells expressing in vivo biotinylated Pax5. By binding to promoters and enhancers, Pax5 directly regulates the expression of multiple transcription factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer Pax5-dependent B-cell-specific activity to the Nedd9 gene controlling B-cell trafficking. Profiling of histone modifications in Pax5-deficient and wild-type pro-B cells demonstrated that Pax5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro-B cells. Pax5 rapidly induces these chromatin and transcription changes by recruiting chromatin-remodelling, histone-modifying and basal transcription factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which Pax5 controls B-cell commitment.
Assuntos
Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Marcação de Genes , Fator de Transcrição PAX5/fisiologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Técnicas de Introdução de Genes , Marcação de Genes/métodos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator de Transcrição PAX5/genética , Ligação Proteica/genética , Transporte Proteico/genética , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
V(H)-DJ(H) recombination of the immunoglobulin heavy chain (Igh) locus is temporally and spatially controlled during early B cell development, and yet no regulatory elements other than the V(H) gene promoters have been identified throughout the entire V(H) gene cluster. Here, we discovered regulatory sequences that are interspersed in the distal V(H) gene region. These conserved repeat elements were characterized by the presence of Pax5 transcription factor-dependent active chromatin by binding of the regulators Pax5, E2A, CTCF, and Rad21, as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B cell development. The pro-B cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal V(H)-DJ(H) recombination at the Igh locus.
Assuntos
Cromatina/genética , DNA Intergênico/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Fator de Transcrição PAX5/fisiologia , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Sítios de Ligação , Fator de Ligação a CCCTC , Imunoprecipitação da Cromatina , Sequência Conservada , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Transcrição PAX5/deficiência , Fator de Transcrição PAX5/genética , Células Precursoras de Linfócitos B/metabolismo , RNA Antissenso/biossíntese , RNA Antissenso/genética , Proteínas Repressoras/fisiologia , Transcrição GênicaRESUMO
Early B cell development depends on a network of transcription factors, whereby E2A and EBF1 regulate B cell specification and Pax5 controls B-lineage commitment. In contrast, activation of the transcription factor STAT5 in response to IL-7R signaling promotes cell survival by activating the prosurvival gene Mcl1 and orders immunoglobulin gene rearrangement by repressing Igk recombination in pro-B cells. Subsequently, it cooperates with the pre-B cell receptor to facilitate pre-B cell expansion. STAT5 also plays a key role in the generation of B cell precursor acute lymphoblastic leukemia, whereby the BCR-ABL1 translocation or the collaboration of JAK2 mutations with overexpression of the thymic stromal lymphopoietin receptor CRLF2 results in constitutive STAT5 activation leading to cytokine-independent survival and growth of leukemic cells.
Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Fator de Transcrição STAT5/imunologia , Animais , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Regulação Leucêmica da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/imunologia , Humanos , Interleucina-7/imunologia , Linfopoese/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
STAT5 and interleukin 7 (IL-7) signaling are thought to control B lymphopoiesis by regulating the expression of key transcription factors and by activating variable (V(H)) gene segments at the immunoglobulin heavy-chain (Igh) locus. Using conditional mutagenesis to delete the gene encoding the transcription factor STAT5, we demonstrate that the development of pro-B cells was restored by transgenic expression of the prosurvival protein Bcl-2, which compensated for loss of the antiapoptotic protein Mcl-1. Expression of the genes encoding the B cell-specification factor EBF1 and the B cell-commitment protein Pax5 as well as V(H) gene recombination were normal in STAT5- or IL-7 receptor alpha-chain (IL-7Ralpha)-deficient pro-B cells rescued by Bcl-2. STAT5-expressing pro-B cells contained little or no active chromatin at most V(H) genes. In contrast, rearrangements of the immunoglobulin-kappa light-chain locus (Igk) were more abundant in STAT5- or IL-7Ralpha-deficient pro-B cells. Hence, STAT5 and IL-7 signaling control cell survival and the developmental ordering of immunoglobulin gene rearrangements by suppressing premature Igk recombination in pro-B cells.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/genética , Células Progenitoras Linfoides/citologia , Linfopoese/genética , Fator de Transcrição STAT5/genética , Transdução de Sinais/imunologia , Animais , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Rearranjo Gênico/genética , Rearranjo Gênico/imunologia , Genes de Imunoglobulinas/genética , Genes de Imunoglobulinas/imunologia , Interleucina-7/genética , Interleucina-7/imunologia , Células Progenitoras Linfoides/imunologia , Linfopoese/imunologia , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/imunologiaRESUMO
Pax5 is an essential regulator of B cell identity and function. Here, we used transgenesis and deletion mapping to identify a potent enhancer in intron 5 of the Pax5 locus. This enhancer in combination with the promoter region was sufficient to recapitulate the B lymphoid expression of Pax5. The enhancer was silenced by DNA methylation in embryonic stem cells, but became activated in multipotent hematopoietic progenitors. It contained functional binding sites for the transcription factors PU.1, IRF4, IRF8, and NF-kappaB, suggesting that these regulators contribute to sequential enhancer activation in hematopoietic progenitors and during B cell development. In contrast, the promoter region was repressed by Polycomb group proteins in non-B cells and was activated only at the onset of pro-B cell development through induction of chromatin remodeling by the transcription factor EBF1. These experiments demonstrate a stepwise activation of Pax5 in early lymphopoiesis and provide mechanistic insights into the process of B cell commitment.
Assuntos
Linfócitos B/imunologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Linfopoese/fisiologia , Fator de Transcrição PAX5 , Regiões Promotoras Genéticas , Transgenes/genética , Animais , Linfócitos B/citologia , Sequência de Bases , Cromossomos Artificiais Bacterianos/genética , Citometria de Fluxo , Humanos , Camundongos , Dados de Sequência Molecular , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Transativadores/genética , Regulação para CimaRESUMO
The transcription factor Pax5 represses B lineage-inappropriate genes and activates B cell-specific genes in B lymphocytes. Here we have identified 170 Pax5-activated genes. Conditional mutagenesis demonstrated that the Pax5-regulated genes require continuous Pax5 activity for normal expression in pro-B and mature B cells. Expression of half of the Pax5-activated genes is either absent or substantially reduced upon Pax5 loss in plasma cells. Direct Pax5 target genes were identified based on their protein synthesis-independent activation by a Pax5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of Pax5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Pax5 directly activates the chromatin at promoters or putative enhancers of Pax5 target genes. The Pax5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation, and germinal-center B cell formation, thus revealing a complex regulatory network that is activated by Pax5 to control B cell development and function.
