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 4 qD 4 Z4位点染色质和细胞核结构中的作用 染色质绝缘子蛋白CTCF已被证明可以保护基因免受表观遗传沉默。 CTCF结合通常被DNA甲基化抑制，CTCF防止DNA甲基化的扩散。 最近的证据表明，CTCF也可以发挥作用，使空间分离的染色质结构域紧密结合， 在与货车der Maarel博士的合作中，我们绘制了CTCF结合图， 位点的D4 Z4单位，并证明了增强结合CTCF的部分删除的致病性 D4 Z4等位基因，并降低这些CTCF结合位点的DNA甲基化。此外，委员会认为， 在未分化的ES细胞中也观察到CTCF与D4 Z4的结合增强。这些发现导致了 假设FSHD中D4 Z4单位中不适当的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 不适当的转录或核定位。因此，我们的长期目标是确定 CTCF结合在疾病相关等位基因上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D4 Z4单元中的CTCF位点是否作为 绝缘子，转录调节因子或染色质边界元件，以及CTCF是否是必要的， 调节D4 Z4染色质结构和转录。这些研究将使用siRNA介导的 CTCF在对照和FSHD人肌肉细胞和D4 Z4小鼠模型中的敲低。在目标2中，我们将 确定由对照和FSHD成纤维细胞产生的人IPS细胞是否准确地再现了 D4 Z4在ES细胞体外分化前和分化过程中的染色质结构，以及CTCF是否结合 和/或缺失D4 Z4亚基阻止了发育调节的染色质介导的抑制， D4 Z4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 在4 q D4 Z4位点，发育状态介导染色体内或染色体间的相互作用， 这些相互作用在FSHD细胞中改变。