RESUMEN
Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain (Igh) and controlled expression of the gene encoding the transcription factor c-Myc (Myc) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage-specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.
Asunto(s)
Linfocitos B/inmunología , Proliferación Celular , ADN Helicasas/inmunología , Elementos de Facilitación Genéticos/inmunología , Proteínas Nucleares/inmunología , Factores de Transcripción/inmunología , Animales , Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/inmunología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Elementos de Facilitación Genéticos/genética , Citometría de Flujo , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Hibridación Fluorescente in Situ , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
