Functional analysis of trans-acting factor binding to imprinting control region.

近年来，人们对染色体15q11-q13 AS/PWS结构域的印迹机制进行了深入的研究。我们之前已经在SNURF-SNRPN上游区域发现了AS和PWS印记控制中心（IC）。在雌性生殖系和早期胚胎发生中，双侧IC具有在母体染色体上建立母体表观遗传型和在PWS-IC体细胞中维持父系染色体上的父系表观遗传型的功能。通过对多例具有印迹缺陷的家族性AS或PWS患者的遗传模式、AS/PWS模型小鼠分析和转基因小鼠分析，预测了该功能。为了阐明IC的功能，我们分析了PWS-IC区域，并鉴定了一些反式作用因子结合位点。由于人类PWS-IC和小鼠Snurf-Snrpn启动子是基于敲除小鼠的几个证据的同源区域，因此进行了系统发育分析，在PWS-IC区域发现了几个保守序列。在H19/Igf2区，CTCF结合位点位于H19启动子区。这些位点起到绝缘体的作用，阻止igf2启动子的增强子活性。在系统发育分析中，PWS-IC和h19启动子中存在与CTCF结合位点、部分转录结合位点相似的保守序列。为了确认转录结合位点的功能，用几种DNA结构进行了瞬时转染实验，包括结合位点的突变。在实验中，CTCF样结合位点未表现出任何绝缘子活性，而其他转录因子结合位点则表现出阳性的表达活性。因此，CTCF样结合位点可能不具有绝缘子的功能，而其他转录结合位点可能具有一定的印迹维持功能。这项研究目前正在进行中。此外，还分析了几种印迹基因。

The mechanism of imprinting in chromosome 15q11-q13, AS/PWS domain has been analyzed continuously from several years ago. We have previously identified AS and PWS imprinting control center (IC) located at SNURF-SNRPN upstream region. The bipartite IC is supposed to have a function to establish maternal epigenotype on maternal chromosome in female germ line and early embryogenesis with AS-IC, and maintenance of the paternal epigenotype on paternal chromosome in somatic cells with PWS-IC. This function was predicted from inherited pattern of several familial AS or PWS patients with imprinting defect, AS/PWS model mouse analysis, and transgenic mouse analysis. Th elucidate the IC function, we analyzed the PWS-IC region with identification of some trans-acting factor binding sites. Since the human PWS-IC and mouse Snurf-Snrpn promoter are ortholog region based on several evidence of the knock out mouse, phylogenetic analysis was performed, and several conserved sequence were found in the PWS-IC region.In H19/Igf2 region, CTCF binding sites were found in the H19 promoter region. These sites are working as an insulator to prevent enhancer activity to the Igf2promoter.In the phylogenetic analysis, there are conserved sequences that have similar with the CTCF binding site, and some transcription binding sites in the PWS-IC as well as the H 19 promoter. To confirm the function of the transcription binding sites, transient transfection assay was performed with several kind of DNA construct including mutation at the binding site. In the assay, CTCF like binding site didn't show any insulator activity, and other transcription factor binding site show a positive activity for the expression. Therefore, the CTCF like binding site may not have a function of the insulator, and other transcription-binding site could have some function for imprinting maintenance. This research is currently ongoing. Furthermore, several imprinting genes were also analyzed.