Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria.
Max Perutz Laboratories, University of Vienna, Vienna BioCenter (VBC), Vienna, Austria.
EMBO J. 2023 Aug 1;42(15):e112741. doi: 10.15252/embj.2022112741. Epub 2023 Jun 20.
While extended loop extrusion across the entire Igh locus controls V -DJ recombination, local regulatory sequences, such as the PAIR elements, may also activate V gene recombination in pro-B-cells. Here, we show that PAIR-associated V 8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted 890 kb containing all 14 PAIRs in the Igh 5' region, which reduced distal V gene recombination over a 100-kb distance on either side of the deletion. Reconstitution by insertion of PAIR4-V8.7E strongly activated distal V gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro-B-cell-specific activity of PAIR4 depends on CTCF, as mutation of its CTCF-binding site led to sustained PAIR4 activity in pre-B and immature B-cells and to PAIR4 activation in T-cells. Notably, insertion of V8.8E was sufficient to activate V gene recombination. Hence, enhancers of the PAIR4-V8.7E module and V8.8E element activate distal V gene recombination and thus contribute to the diversification of the BCR repertoire in the context of loop extrusion.
虽然整个 Igh 基因座的延伸环挤压控制 V-DJ 重组，但局部调节序列，如 PAIR 元件，也可能在 pro-B 细胞中激活 V 基因重组。在这里，我们表明 PAIR 相关的 V8 基因在其下游序列中含有保守的假定调节元件 (V8E)。为了研究 PAIR4 及其 V8.7E 的功能，我们删除了 Igh5' 区域中包含所有 14 个 PAIR 的 890kb 区域，这导致缺失两侧 100kb 距离的远端 V 基因重组减少。插入 PAIR4-V8.7E 的重建强烈激活了远端 V 基因重组。PAIR4 本身导致重组的诱导降低，表明 PAIR4 和 V8.7E 作为一个调节单元起作用。PAIR4 的 pro-B 细胞特异性活性取决于 CTCF，因为其 CTCF 结合位点的突变导致 PAIR4 在 pre-B 和未成熟 B 细胞中的持续活性以及在 T 细胞中的 PAIR4 激活。值得注意的是，V8.8E 的插入足以激活 V 基因重组。因此，PAIR4-V8.7E 模块和 V8.8E 元件的增强子激活远端 V 基因重组，从而有助于在环挤压的背景下 BCR 库的多样化。
