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

染色体15q11-q13、AS/PWS结构域印记的机制早在几年前就被不断地分析。我们此前已经确定了位于SNURF-Snrpn上游区域的和PWS印花控制中心(IC)。二分体IC在雌性生殖系母体染色体上建立母系表型和AS-IC的早期胚胎发生，以及在pWS-IC的体细胞中维持父系染色体上的父系表型。从几个有印记缺陷的家族性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中预测了该功能。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部位。由于人PWS-IC和小鼠SNurf-Snrpn启动子是基于多种基因敲除小鼠证据的同源区域，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区域，在H19启动子区域发现了CTCF结合位点。在系统发育分析中，在pWS-IC和H19启动子中有一些与CTCF结合位点相似的保守序列，也有一些转录结合位点。为了证实转录结合位点的功能，对包括结合位点突变在内的几种DNA构建物进行了瞬时转染实验。在实验中，CTCF样结合部位没有显示任何绝缘子活性，其他转录因子结合部位显示出阳性表达活性。因此，CTCF样结合位点可能不具有绝缘体的功能，而其他转录结合位点可能具有印迹维持功能。这项研究目前正在进行中。此外，还对几个印记基因进行了分析。

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.