Novel CpG-free vertebrate insulator: role for YY1

描述(由申请人提供)：绝缘体是形成基因边界并保护基因免受邻近染色质影响的调节性DNA序列。绝缘体蛋白与基因组中的绝缘体DNA元件结合，并将这些区域拴在亚核结构上，从而形成染色质环，使得一个基因的增强子/沉默元件可能无法接触到邻近基因的启动子。考虑到人类基因组的复杂性，人们预计会有各种绝缘体和绝缘体蛋白调节大约35,000个基因的转录。例如，仅在果蝇中，就有四种类型的绝缘体和结合蛋白被报道。相比之下，到目前为止，在脊椎动物中只有一种类型的绝缘体(鸡珠蛋白HS4)和绝缘体蛋白(CTCF)被报道。CTCF介导的绝缘子活性受DNA甲基化的调节。我们已经鉴定了一种不含CpG、不依赖CTCF的脊椎动物绝缘子(SP-10绝缘子)，该绝缘子来自睾丸特异基因。SP-10绝缘子在小鼠和人之间是保守的。初步突变研究表明，转录因子YY1可能介导了SP-10绝缘子的功能。在拟议的初步研究中，我们将研究YY1是否在体外(使用EMSA)和生理环境(使用CHIP)中与SP-10绝缘体相互作用(特定目标1)；并在细胞培养模型和YY1条件性基因敲除小鼠模型中使用RNAi解决YY1对SP-10绝缘体功能的需求(特定目标2)。这项可行性研究是一个小型的自给自足的研究项目，有可能导致更大的假设驱动的研究。最近的一项研究表明，YY1对精子发生是必不可少的。目前的研究将把YY1的作用扩大到睾丸特异的基因转录，并在长期内对男性不育起作用。 与公共卫生有关：不孕不育的发病率正在上升；据估计，在美国，每六对处于生育年龄的夫妇中就有一对无法怀孕。男性因素占不育病例的50%。由于对控制精子产生(精子发生)的机制了解不深，男性的治疗选择有限。在这里，我们建议研究转录因子YY1在调节睾丸特异基因转录中的作用。由于最近的小鼠模型表明，男性生殖细胞中YY1的缺失会导致精子发生缺陷，因此，关于YY1在睾丸中的功能的研究对男性不育具有非常重要的意义，因此具有很高的公共卫生意义。

DESCRIPTION (provided by applicant): Insulators are regulatory DNA sequences which form gene boundaries and protect genes from the influence of neighboring chromatin. Insulator-proteins bind to insulator DNA elements in the genome and tether those regions to subnuclear structures thus forming chromatin loops such that enhancer/silencer elements of one gene may not gain access to the promoter of a neighboring gene. Given the complexity of the human genome, one would expect that a variety of insulators and insulator- proteins regulate transcription of the estimated 35,000 genes. For example, in Drosophila alone, four types of insulators and binding proteins have been reported. In contrast, only one type of insulator (chicken globin HS4) and insulator-protein (CTCF) has been reported so far in vertebrates. CTCF mediated insulator activity is modulated by methylation of DNA. We have characterized a CpG-free, CTCF-independent vertebrate insulator (SP-10 insulator) from a testis-specific gene. The SP-10 insulator is conserved between mice and men. Preliminary mutation studies have indicated that transcription factor YY1 may mediate the SP-10 insulator function. In the proposed pilot study, we will investigate if YY1 interacts with the SP-10 insulator in vitro (using EMSA) and in a physiological context (using ChIP) (Specific Aim 1); and address the requirement of YY1 for SP-10 insulator function using RNAi in a cell culture model as well as a YY1 conditional knockout mouse model (Specific Aim 2). This feasibility study is a small self-contained research project with the potential to lead to a larger hypothesis-driven study. A recent study has shown that YY1 is essential for spermatogenesis. The current study will expand the role of YY1 to testis-specific gene transcription and in the long-term to male infertility. PUBLIC HEALTH RELEVANCE: Incidence of infertility is on the rise; it has been estimated that in the United States one in six couples in the reproductive age group are unable to conceive. The male factor contributes to 50% of the infertility cases. Treatment options are limited for men owing to a poor understanding of the mechanisms controlling sperm production (spermatogenesis). Here we propose to study the role of a transcription factor YY1 in regulating testis-specific gene transcription. Since the mouse model has recently shown that absence of YY1 in male germ cells leads to defective spermatogenesis, the proposed studies on the function of YY1 in testis are highly important for male infertility and are therefore of high public health relevance.