CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells
CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells
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DOI：
10.1073/pnas.1019391108
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发表时间：
2011-06-07
影响因子：
11.1
通讯作者：
Feeney, Ann J.
中科院分区：
文献类型：
--
作者：
Degner, Stephanie C.;Verma-Gaur, Jiyoti;Feeney, Ann J.
在 V(D)J 重排过程中，类似于 2.5-Mb Igh 基因座的压缩和循环对于使所有 V-H 基因与 D-H-J(H) 片段接近以创建多样化的抗体库至关重要，但直接负责此的蛋白质尚不清楚。由于 CCCTC 结合因子 (CTCF) 已被证明参与长程染色体相互作用，我们假设 CTCF 可能促进 Igh 基因座的收缩。对 pro-B 细胞进行 ChIP 测序，揭示 CTCF 和 Rad21 结合共定位于整个 V-H 区域的大约 60 个位点以及 Igh 基因座内的其他 2 个位点。这些众多的 CTCF/粘连蛋白位点可能形成 Igh 基因座处的多环玫瑰花结结构的基础，该结构在 Ig 重链重排过程中压缩。为了测试 CTCF 是否参与位点压缩，我们使用 3D-FISH 测量用 CTCF shRNA 逆转录病毒转导的 pro-B 细胞的压缩。 CTCF 结合的减少导致 Igh 基因座压缩的减少。通过染色体构象捕获测定测量了 Igh 位点内的长程相互作用，揭示了 DFL16 的 5' CTCF 位点和 3' 调节区以及内含子增强子 (E mu) 之间的直接相互作用，从而产生了 D-H-J(H)-E mu-C-H 结构域。 CTCF 的敲低还导致整个 D-H 区域和部分 V-H 基因座的反义转录增加，表明 CTCF 具有广泛的调节作用。总之，我们的研究结果表明 CTCF 在 Igh 基因座的 3D 结构和反义种系转录的调节中发挥着重要作用，并且它有助于 Igh 基因座的压缩。
Compaction and looping of the similar to 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 similar to 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 (E mu), creating a D-H-J(H)-E mu-C-H domain. Knock-down 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.