RESUMO
During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.
Assuntos
Eritropoese/fisiologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Globinas/genética , Substâncias Macromoleculares , Fator de Transcrição MafK , Camundongos , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteômica , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
T cells develop through distinct stages directed by a series of signals. We explored the roles of SWI/SNF-like BAF chromatin remodeling complexes in this process by progressive deletion of the ATPase subunit, Brg, through successive stages of early T cell development. Brg-deficient cells were blocked at each of the developmental transitions examined. Bcl-xL overexpression suppressed cell death without relieving the developmental blockades, leading to the accumulation of Brg-deleted cells that were unexpectedly cell cycle arrested. These defects resulted partly from the disruptions of pre-TCR and potentially Wnt signaling pathways controlling the expression of genes such as c-Kit and c-Myc critical for continued development. Our studies indicate that BAF complexes dynamically remodel chromatin to propel sequential developmental transitions in response to external signals.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , DNA Helicases , Genes myc , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Imunológicos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Desnaturação de Ácido Nucleico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteína bcl-XAssuntos
Cromatina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cromatina/química , Cromatina/genética , Fosfatos de Inositol/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro , Linfócitos T/metabolismoRESUMO
Thymic development produces two sub-lineages of T cells expressing either CD4 or CD8 co-receptors that assist antibody production and mediate cell killing, respectively. The mechanisms for mutually exclusive co-receptor expression remain poorly defined. We find that mutations in the high mobility group (HMG) domain of BAF57--a DNA-binding subunit of the mammalian SWI/SNF-like chromatin-remodelling BAF complexes--or in the BAF complex ATPase subunit Brg, impair both CD4 silencing and CD8 activation. Brg is haploinsufficient for CD8 activation, but not for CD4 silencing, whereas BAF57 mutations preferentially impair CD4 silencing, pointing to target- and subunit-specific mechanisms of chromatin remodelling. BAF complexes directly bind the CD4 silencer, but the BAF57 HMG domain is dispensable for tethering BAF complexes to the CD4 silencer or other chromatin loci in vivo, or for remodelling reconstituted templates in vitro, suggesting that chromatin remodelling in vivo requires HMG-dependent DNA bending. These results indicate that BAF complexes contribute to lineage bifurcation by reciprocally regulating lineage-specific genes, reminiscent of the role of the yeast SWI/SNF complex in mediating mating-type switching.
