RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Proteínas Represoras/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , ADN sin Sentido/genética , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos/genética , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Interferencia de ARN , ARN sin Sentido/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , CohesinasRESUMEN
Contraction of the large Igh and Igkappa loci brings all V genes, spanning >2.5 Mb in each locus, in proximity to DJ(H) or J(kappa) genes. CCCTC-binding factor (CTCF) is a transcription factor that regulates gene expression by long-range chromosomal looping. We therefore hypothesized that CTCF may be crucial for the contraction of the Ig loci, but no CTCF sites have been described in any V loci. Using ChIP-chip, we demonstrated many CTCF sites in the V(H) and V(kappa) regions. However, CTCF enrichment in the Igh locus, but not the Igkappa locus, was largely unchanged throughout differentiation, suggesting that CTCF binding alone cannot be responsible for stage-specific looping. Because cohesin can colocalize with CTCF, we performed chromatin immunoprecipitation for the cohesin subunit Rad21 and found lineage and stage-specific Rad21 recruitment to CTCF in all Ig loci. The differential binding of cohesin to CTCF sites may promote multiple loop formation and thus effective V(D)J recombination.
