Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
J Immunol. 2012 Jun 1;188(11):5547-60. doi: 10.4049/jimmunol.1002346. Epub 2012 Apr 27.
V(D)J recombination of Ig and TCR genes is strictly regulated in a lineage- and stage-specific manner by the accessibility of target gene chromatin to the recombinases RAG1 and RAG2. It has been shown that enforced expression of the basic helix-loop-helix protein, E2A, together with RAG1/2 in a nonlymphoid cell line BOSC23 can induce V(D)J recombination in endogenous Igκ and TCR loci by increasing chromatin accessibility of target gene segments. In this study, we demonstrate that ectopically expressed E2A proteins in BOSC23 cells have the ability to bind directly to the promoter and recombination signal sequence of Vκ genes and to recruit histone acetyltransferase CBP/p300. Overexpression of CBP/p300 in conjunction with E2A results in enhancement of E2A-induced histone acetylation, germline transcription, and Igκ rearrangement. Conversely, knockdown of endogenous CBP/p300 expression by small interfering RNA leads to a decrease in histone acetylation, germline transcription and Igκ rearrangement. Furthermore, analyses using a mouse pre-B cell line revealed that endogenous E2A proteins also bind to a distinct set of Vκ genes and regulatory regions in the mouse Igκ locus and act to increase histone acetylation by recruiting p300, confirming the similar findings observed with BOSC23 cells. These observations indicate that E2A plays critical roles in inducing Igκ rearrangement by directly binding to and increasing chromatin accessibility at target gene segments.
免疫球蛋白（Ig）和 T 细胞受体（TCR）基因的 V(D)J 重组在谱系和阶段特异性上受到 RAG1 和 RAG2 重组酶对靶基因染色质可及性的严格调控。已经表明，在非淋巴样细胞系 BOSC23 中强制表达基本螺旋-环-螺旋蛋白 E2A 与 RAG1/2 一起，可以通过增加靶基因片段的染色质可及性来诱导内源性 Igκ 和 TCR 基因座的 V(D)J 重组。在本研究中，我们证明 BOSC23 细胞中异位表达的 E2A 蛋白能够直接结合 Vκ 基因的启动子和重组信号序列，并募集组蛋白乙酰转移酶 CBP/p300。与 E2A 共表达 CBP/p300 会导致 E2A 诱导的组蛋白乙酰化、生殖系转录和 Igκ 重排增强。相反，通过小干扰 RNA 敲低内源性 CBP/p300 表达会导致组蛋白乙酰化、生殖系转录和 Igκ 重排减少。此外，使用小鼠前 B 细胞系的分析表明，内源性 E2A 蛋白也结合到小鼠 Igκ 基因座中的一组独特的 Vκ 基因和调控区域，并通过募集 p300 来增加组蛋白乙酰化，证实了与 BOSC23 细胞观察到的相似发现。这些观察结果表明，E2A 通过直接结合靶基因片段并增加染色质可及性，在诱导 Igκ 重排中发挥关键作用。
