Role of CTCF in Chromatin and Nuclear Organization at the FSHD 4qD4Z4Locus

项目3 CTCF在FSHD 4qD4Z4基因座染色质和核组织中的作用 染色质绝缘蛋白CTCF已被证明可以保护基因免受表观遗传沉默的影响。 CTCF结合通常被DNA甲基化抑制，而CTCF防止DNA甲基化的扩散。 最近的证据表明，CTCF还可以使空间上分离的染色质结构域接近 接近性，无论是顺式还是反式。在与van der Maarel博士的合作下，我们已经绘制了CTCF绑定 D4Z4单位中的位点，并显示CTCF与部分缺失的致病病毒的结合增强 FSHD细胞中存在D4Z4等位基因，并降低了这些CTCF结合位点的DNA甲基化。此外， 在未分化的ES细胞中，也观察到CTCF与D4Z4的结合增强。这些发现导致了 假设FSHD D4Z4单位中不适当的CTCF结合可能干扰 建立或维持发育调节的表观遗传抑制并导致 不适当的转录或核定位。因此，我们的长期目标是确定是否 CTCF与疾病相关等位基因的结合调节区域染色质结构和/或RNA 抄写。在我们的Aim1中，我们将确定D4Z4单元中的CTCF站点是否起到 绝缘体、转录调节因子或染色质边界元件，以及CTCF是否对 调控D4Z4染色质结构和转录。这些研究将使用siRNA介导法进行。 CTCF在对照组、FSHD人肌细胞和D4Z4小鼠模型中的表达下调。在目标2中，我们将 确定对照组和FSHD成纤维细胞产生的人IPS细胞是否准确地概括了 D4Z4在ES细胞体外分化前后的染色质结构及CTCF是否结合 和/或D4Z4亚单位的缺失可防止发育调节的染色质介导的抑制 D4Z4转录本。最后，在目标3中，我们将结合使用染色质构象捕获和 染色质免疫沉淀技术，以确定CTCF、染色质上下文和/或 发育状态调节4Q D4Z4基因座的染色体内或染色体间的相互作用 这些相互作用在FSHD细胞中发生了改变。

PROJECT 3 ROLE OF CTCF IN CHROMATIN AND NUCLEAR ORGANIZATION AT THE FSHD 4qD4Z4 LOCUS The chromatin insulator protein, CTCF, has been shown to protect genes from epigenetic silencing. CTCF binding is generally inhibited by DNA methylation and CTCF prevents spreading of DNA methylation. Recent evidence suggests that CTCF can also act to bring spatially separated chromatin domains in close proximity, both in cis and in trans. In collaboration with Dr. van der Maarel, we have mapped CTCF binding sites in the D4Z4 unit and demonstrated both enhanced binding of CTCF to the partially deleted pathogenic D4Z4 allele in FSHD cells and decreased DNA methylation at these CTCF binding sites. Moreover, enhanced CTCF binding to D4Z4 was also observed in undifferentiated ES cells. These findings lead to the hypothesis that inappropriate CTCF binding in the D4Z4 units in FSHD might interfere with the establishment or maintenance of developmentally regulated epigenetic repression and result in inappropriate transcription or nuclear localization. Therefore, our long-term goal is to determine whether CTCF binding on the disease associated allele regulates regional chromatin structure and/or RNA transcription. In our Aim1, we will determine whether the CTCF sites in the D4Z4 units function as insulators, transcriptional regulators, or chromatin boundary elements, and whether CTCF is necessary for regulating D4Z4 chromatin structure and transcription. These studies will be done using siRNA-mediated knock-down of CTCF in control and FSHD human muscle cells and D4Z4 mouse models. In Aim 2, we will detennine whether human IPS cells generated from control and FSHD fibroblasts accurately recapitulate the chromatin structure of D4Z4 in ES cells before and during in vitro differentiation, and whether CTCF binding and/or deletion of D4Z4 subunits prevent a developmentally regulated chromatin-mediated repression of D4Z4 transcripts. Finally, in Aim 3, we will use chromatin conformation capture in combination with chromatin immunoprecipitation techniques to determine whether CTCF, chromatin context, and/or developmental state mediate intra- or inter-chromosomal interactions at the 4q D4Z4 locus and whether these interactions are altered in FSHD cells.