RNA in Regulation

RNA 的调控

• 批准号: 9042624
• 负责人: Stephen J Tapscott
• 金额: $ 12.89万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2015-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9042624
• 关键词: 4q35; Affect; Alleles; Binding; Binding Sites; Biological Assay; Boundary Elements; CCCTC-binding factor; Cells; Chromatin; Chromatin Structure; Chromosomes; Collaborations; D4Z4; DNA Methylation; Development; Disease; Epigenetic Process; Facioscapulohumeral Muscular Dystrophy; Fibroblasts; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; Human; In Vitro; Individual; Knockout Mice; Lead; Maintenance; Maps; Mediating; Molecular Conformation; Muscle Cells; Nuclear; Pathogenesis; RNA; Regulation; Repression; Role; Site; Small Interfering RNA; Techniques; Testing; Transcript; Transcription Coactivator; Transgenic Mice; Undifferentiated; chromatin immunoprecipitation; chromatin modification; embryonic stem cell; gene repression; induced pluripotent stem cell; knock-down; method development; mouse model; novel therapeutics; prevent; stem cell differentiation

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.

项目3 CTCF 在 FSHD 4qD4Z4 位点染色质和核组织中的作用 染色质绝缘体蛋白 CTCF 已被证明可以保护基因免受表观遗传沉默。 CTCF 结合通常受到 DNA 甲基化的抑制，并且 CTCF 可以防止 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域紧密结合在一起。 顺式和反式的接近度。我们与 van der Maarel 博士合作，绘制了 CTCF 结合图谱 D4Z4 单元中的位点，并证明 CTCF 与部分缺失的致病性的结合增强 FSHD 细胞中的 D4Z4 等位基因降低了这些 CTCF 结合位点的 DNA 甲基化。而且， 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 假设 FSHD 中 D4Z4 单元中不适当的 CTCF 结合可能会干扰 建立或维持发育调节的表观遗传抑制并导致 转录或核定位不当。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 转录。在我们的目标 1 中，我们将确定 D4Z4 单元中的 CTCF 位点是否充当 绝缘体、转录调节因子或染色质边界元件，以及 CTCF 是否是必需的 调节 D4Z4 染色质结构和转录。这些研究将使用 siRNA 介导的方法完成 在对照和 FSHD 人类肌肉细胞以及 D4Z4 小鼠模型中 CTCF 的敲低。在目标 2 中，我们将 确定从对照和 FSHD 成纤维细胞产生的人 IPS 细胞是否准确地再现了 ES细胞体外分化前和分化过程中D4Z4的染色质结构以及CTCF是否结合 和/或 D4Z4 亚基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 染色质免疫沉淀技术以确定 CTCF、染色质背景和/或 发育状态介导 4q D4Z4 位点的染色体内或染色体间相互作用，以及是否 这些相互作用在 FSHD 细胞中发生了改变。