Yoshida Wataru, Tomikawa Junko, Inaki Makoto, Kimura Hiroshi, Onodera Masafumi, Hata Kenichiro, Nakabayashi Kazuhiko
Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Setagaya, Tokyo, Japan.
Department of Human Genetics, National Research Institute for Child Health and Development, Setagaya, Tokyo, Japan.
PLoS One. 2015 Jun 25;10(6):e0131204. doi: 10.1371/journal.pone.0131204. eCollection 2015.
Insulators are cis-elements that control the direction of enhancer and silencer activities (enhancer-blocking) and protect genes from silencing by heterochromatinization (barrier activity). Understanding insulators is critical to elucidate gene regulatory mechanisms at chromosomal domain levels. Here, we focused on a genomic region upstream of the mouse Ccnb1ip1 (cyclin B1 interacting protein 1) gene that was methylated in E9.5 embryos of the C57BL/6 strain, but unmethylated in those of the 129X1/SvJ and JF1/Ms strains. We hypothesized the existence of an insulator-type element that prevents the spread of DNA methylation within the 1.8 kbp segment, and actually identified a 242-bp and a 185-bp fragments that were located adjacent to each other and showed insulator and enhancer activities, respectively, in reporter assays. We designated these genomic regions as the Ccnb1ip1 insulator and the Ccnb1ip1 enhancer. The Ccnb1ip1 insulator showed enhancer-blocking activity in the luciferase assays and barrier activity in the colony formation assays. Further examination of the Ccnb1ip1 locus in other mammalian species revealed that the insulator and enhancer are highly conserved among a wide variety of species, and are located immediately upstream of the transcriptional start site of Ccnb1ip1. These newly identified cis-elements may be involved in transcriptional regulation of Ccnb1ip1, which is important in meiotic crossing-over and G2/M transition of the mitotic cell cycle.
绝缘子是顺式作用元件，可控制增强子和沉默子的作用方向（增强子阻断），并通过异染色质化保护基因不被沉默（屏障活性）。了解绝缘子对于阐明染色体结构域水平的基因调控机制至关重要。在此，我们聚焦于小鼠Ccnb1ip1（细胞周期蛋白B1相互作用蛋白1）基因上游的一个基因组区域，该区域在C57BL/6品系的E9.5胚胎中发生甲基化，但在129X1/SvJ和JF1/Ms品系的胚胎中未甲基化。我们推测存在一种绝缘子样元件，可阻止DNA甲基化在1.8 kbp片段内扩散，并且实际上在报告基因检测中鉴定出两个彼此相邻的片段，分别为242 bp和185 bp，它们分别具有绝缘子和增强子活性。我们将这些基因组区域分别命名为Ccnb1ip1绝缘子和Ccnb1ip1增强子。Ccnb1ip1绝缘子在荧光素酶检测中表现出增强子阻断活性，在集落形成检测中表现出屏障活性。对其他哺乳动物物种中Ccnb1ip1基因座的进一步研究表明，该绝缘子和增强子在多种物种中高度保守，且位于Ccnb1ip1转录起始位点的紧邻上游。这些新鉴定的顺式作用元件可能参与了Ccnb1ip1的转录调控，而Ccnb1ip1在减数分裂交叉互换和有丝分裂细胞周期的G2/M转换中具有重要作用。
