RESUMO
OBF1, also known as Bob.1 or OCA-B, is a B lymphocyte-specific transcription factor which coactivates Oct1 and Oct2 on B cell specific promoters. So far, the function of OBF1 has been mainly identified in late stage B cell populations. The central defect of OBF1 deficient mice is a severely reduced immune response to T cell-dependent antigens and a lack of germinal center formation in the spleen. Relatively little is known about a potential function of OBF1 in developing B cells. Here we have generated transgenic mice overexpressing OBF1 in B cells under the control of the immunoglobulin heavy chain promoter and enhancer. Surprisingly, these mice have greatly reduced numbers of follicular B cells in the periphery and have a compromised immune response. Furthermore, B cell differentiation is impaired at an early stage in the bone marrow: a first block is observed during B cell commitment and a second differentiation block is seen at the large preB2 cell stage. The cells that succeed to escape the block and to differentiate into mature B cells have post-translationally downregulated the expression of transgene, indicating that expression of OBF1 beyond the normal level early in B cell development is deleterious. Transcriptome analysis identified genes deregulated in these mice and Id2 and Id3, two known negative regulators of B cell differentiation, were found to be upregulated in the EPLM and preB cells of the transgenic mice. Furthermore, the Id2 and Id3 promoters contain octamer-like sites, to which OBF1 can bind. These results provide evidence that tight regulation of OBF1 expression in early B cells is essential to allow efficient B lymphocyte differentiation.
Assuntos
Linfócitos B/fisiologia , Diferenciação Celular/genética , Desenvolvimento Embrionário/imunologia , Transativadores/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese Extramedular/genética , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/citologia , Baço/imunologia , Baço/fisiologia , Fatores de Tempo , Transativadores/metabolismo , Transativadores/fisiologia , Transfecção , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Circadian oscillation of clock-controlled gene expression is mainly regulated at the transcriptional level. Heterodimers of CLOCK and BMAL1 act as activators of target gene transcription; however, interactions of PER and CRY proteins with the heterodimer abolish its transcriptional activation capacity. PER and CRY are therefore referred to as negative regulators of the circadian clock. To further elucidate the mechanism how positive and negative components of the clock interplay, we characterized the interactions of PER2, CRY1 and CRY2 with BMAL1 and CLOCK using a mammalian two-hybrid system and co-immunoprecipitation assays. RESULTS: Both PER2 and the CRY proteins were found to interact with BMAL1 whereas only PER2 interacts with CLOCK. CRY proteins seem to have a higher affinity to BMAL1 than PER2. Moreover, we provide evidence that PER2, CRY1 and CRY2 bind to different domains in the BMAL1 protein. CONCLUSION: The regulators of clock-controlled transcription PER2, CRY1 and CRY2 differ in their capacity to interact with each single component of the BMAL1-CLOCK heterodimer and, in the case of BMAL1, also in their interaction sites. Our data supports the hypothesis that CRY proteins, especially CRY1, are stronger repressors than PER proteins.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ritmo Circadiano , Flavoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Proteínas CLOCK , Células COS , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Criptocromos , Flavoproteínas/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Circadianas Period , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
OBF-1 (Bob.1, OCA-B) is a lymphoid-specific transcriptional coactivator that associates with the transcription factors Oct-1 or Oct-2 on the conserved octamer element present in the promoters of several ubiquitous and lymphoid-specific genes. OBF-1-deficient mice have B cell-intrinsic defects, lack germinal centers, and have severely impaired immune responses to T cell-dependent antigens. Crucial genes that are regulated by OBF-1 and that might explain the observed phenotype of OBF-1 deficiency have remained elusive to date. Here we have generated transgenic mice expressing OBF-1 specifically in T cells and examined these together with mice lacking OBF-1 to discover transcriptional targets of this coactivator. Using microarray analysis, we have identified the Ets transcription factor Spi-B as a direct target gene critically regulated by OBF-1 that can help explain the phenotype of OBF-1-deficient mice. Spi-B has been implicated in signaling pathways downstream of the B cell receptor and is essential for germinal center formation and maintenance. The present findings establish a hierarchy between these two factors and provide a molecular link between OBF-1 and B cell receptor signaling.
