Department of Life Science, College of Science, Rikkyo University, Toshima-ku, Tokyo, Japan.
Gene. 2011 Oct 15;486(1-2):1-7. doi: 10.1016/j.gene.2011.06.032. Epub 2011 Jul 2.
Previously, we used homologous recombination to delete six groups of cell-type-specific DNase I hypersensitive sites (DHSs), potential transcriptional and epigenetic regulators, scattered in and around the Ig-β gene from their natural context in B-lymphocyte-derived chicken DT40 cells. Simultaneous deletion of all six groups completely shut down transcription and epigenetic regulation of the Ig-β gene; therefore, the cooperation of the scattered regulatory regions was essential for transcription and epigenetic regulation. In this study, we regrouped the cell-type-specific DHSs of Ig-β, those in the original six deletions and three additional ones, into three larger regional groups-the long upstream region, the intron, and the long downstream region-and deleted these groups individually or in combination. Combinatorial deletion of all three regional groups decreased Ig-β mRNA levels to 0.4% of the control, which was significantly higher than <0.1%, the level resulting from deletion of all six smaller groups. Histone H3 and H4 acetylation and H3K4 dimethylation levels at the Ig-β promoter were low in cells carrying deletions of all six smaller groups, but intermediate levels of acetylation and enhanced H3K4 dimethylation were observed in cells carrying deletions of all three larger groups. While CG methylation was definitely present at the Ig-β promoter in cells carrying all six smaller deletions, it was nearly absent from the Ig-β promoter in cells carrying all three larger deletions. Thus, combinatorial deletion of larger regulatory regions had less effect on transcription and epigenetic regulation at the chicken Ig-β gene than combinatorial deletion of shorter ones. Analysis of several combinatorial deletions, where combinations included two larger deletions and one smaller deletion, revealed the relative effects of each deletion on transcription of the Ig-β gene. Investigation of the CG methylation status at the Ig-β promoter in one combinatorial deletion demonstrated that USI was involved in the maintenance of CG methylation.
先前，我们使用同源重组的方法从鸡 DT40 细胞的天然环境中删除了散布在 Ig-β 基因周围和内部的六组细胞类型特异性 DNA 酶 I 超敏位点（DHSs），这些 DHSs 可能是转录和表观遗传调控因子。同时删除这六组 DHSs 完全关闭了 Ig-β 基因的转录和表观遗传调控；因此，分散的调控区域的协同作用对于转录和表观遗传调控至关重要。在这项研究中，我们将 Ig-β 的细胞类型特异性 DHSs 重新分组，将原始的六个缺失组和另外三个缺失组中的 DHSs 分为三个较大的区域组——长上游区、内含子区和长下游区——然后单独或组合删除这些区域组。三个区域组的组合删除将 Ig-βmRNA 水平降低至对照的 0.4%，显著高于六个较小缺失组的 <0.1%。在缺失所有六个较小缺失组的细胞中，Ig-β 启动子处的组蛋白 H3 和 H4 乙酰化和 H3K4 二甲基化水平较低，但在缺失所有三个较大缺失组的细胞中观察到中间水平的乙酰化和增强的 H3K4 二甲基化。虽然在缺失所有六个较小缺失组的细胞中 Ig-β 启动子处确实存在 CG 甲基化，但在缺失所有三个较大缺失组的细胞中，Ig-β 启动子处几乎不存在 CG 甲基化。因此，与较短缺失的组合相比，较大调控区域的组合缺失对鸡 Ig-β 基因的转录和表观遗传调控的影响较小。对几种组合缺失的分析，其中组合包括两个较大缺失和一个较小缺失，揭示了每个缺失对 Ig-β 基因转录的相对影响。对一个组合缺失中 Ig-β 启动子的 CG 甲基化状态的研究表明，USI 参与了 CG 甲基化的维持。
