Characterization of factors required for expression of DNA methylated genes

DNA 甲基化基因表达所需因子的表征

• 批准号: 9329310
• 负责人: Qikun Liu
• 金额: $ 5.71万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9329310
• 关键词: ATAC-seq; Affect; Binding; Binding Sites; Biochemical; Cardiovascular Diseases; Chemicals; Chimeric Proteins; Chromatin; Complex; Cytosine; DNA; DNA Methylation; DNA Methyltransferase Inhibitor; DNA Modification Methylases; DNA Modification Process; DNA Transposable Elements; Defect; Dependence; Disease; Epigenetic Process; Eukaryota; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomics; Histones; Human; Immunoprecipitation; Investigation; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Methylation; Modification; Mutation; Play; Promoter Regions; Proteins; Recruitment Activity; Regulator Genes; Reporter; Role; Series; System; Testing; Time; Transcriptional Activation; Work; Zinc Fingers; genome-wide; genomic profiles; histone modification; human disease; mutant; protein complex

Project Summary This proposal aims at an understanding of the mechanism of expression of DNA methylated genes. DNA methylation is a key mechanism for gene regulation in most eukaryotes. It is generally associated with gene silencing when found in promoter regions. However, DNA methylation does not always cause complete silencing of a gene. In some cases DNA methylated genes can be actively transcribed suggesting the presence of anti-silencing factors. I performed a genetic screen to look for such anti-silencing factors. I found that mutations in MED12, MED13, and SAC3B were able to repress the expression of a DNA methylated GFP reporter. All of the three protein factors were found previously to work together in gene regulation through a defined network. Moreover, defects in MED12 and MED13 have been previously shown to cause severe diseases in humans, such as cancers and cardiovascular diseases. Preliminary study of SAC3B suggested that it preferentially affects the expression of DNA methylated genes. It is therefore hypothesized that genes isolated from the screen are anti-silencing factors preferentially required for the expression of DNA methylated genes. To provide further support for this hypothesis, the importance of DNA methylation in MED12/13- and SAC3B- mediated gene expression will be determined by using the same GFP reporter without DNA methylation. In addition, the genome-wide dependence of DNA methylation related gene expression on MED12/13 and SAC3B will be tested by using DNA methyltransferase mutants and DNA methyltransferase inhibitors. It is expected that the expression of certain genes will be only affected by these anti-silencing factors when they carry DNA methylation. Next, I plan to determine what sequence context of DNA methylation (CG, CHG, or CHH, where H is not G) is preferentially associated with this phenomenon. In addition to DNA methylation, two other types of epigenetic features may also facilitate the recruitment of the above-mentioned anti-silencing factors. One is histone modification, and the other is chromatin accessibility. I will profile the genome-wide binding sites of these anti-silencing factors, and examine what other histone marks are enriched at these binding sites. ATAC-seq will be performed to determine the chromatin accessibility around their genomic binding sites. Finally, I will tether these anti-silencing factors to an endogenous DNA methylated and silenced locus using a well-tested zinc finger system. If my hypothesis is correct, it is expected that the tethered anti- silencing proteins will activate transcription without affecting DNA methylation. Finally, immunoprecipitation combined with mass spectrometry will be performed to dissect the composition of anti-silencing protein complexes.

项目摘要 该建议旨在理解DNA甲基化基因表达的机制。DNA 甲基化是大多数真核生物中基因调控的关键机制。它通常与基因有关。 当在启动子区域中发现时沉默。然而，DNA甲基化并不总是导致完全的 基因的沉默。在某些情况下，DNA甲基化基因可以活跃地转录，这表明 抗沉默因子的存在。 我做了一个基因筛选来寻找这种抗沉默因子。我发现MED 12，MED 13， 和SAC 3B能够抑制DNA甲基化GFP报告基因的表达。三种蛋白质 以前发现，这些因子通过一个确定的网络在基因调控中共同起作用。此外，缺陷 在MED 12和MED 13中，先前已经显示出在人类中引起严重的疾病，例如癌症， 心血管疾病SAC 3B的初步研究表明，它优先影响 DNA甲基化基因。因此假设从筛选中分离的基因是抗沉默的 DNA甲基化基因表达优先需要的因子。 为了进一步支持这一假设，我们研究了MED 12/13-和SAC 3B-中DNA甲基化的重要性。 介导的基因表达将通过使用没有DNA甲基化的相同GFP报告基因来确定。在 此外，DNA甲基化相关基因表达对MED 12/13的全基因组依赖性， 将使用DNA甲基转移酶突变体和DNA甲基转移酶抑制剂检测SAC 3B。是 预期某些基因的表达将只受这些抗沉默因子的影响， 携带DNA甲基化。接下来，我计划确定DNA甲基化（CG，CHG或 CHH，其中H不是G）优先与这种现象相关。除了DNA甲基化， 其它类型的表观遗传特征也可促进上述抗沉默的募集 因素一个是组蛋白修饰，另一个是染色质可及性。我会在全基因组范围内 这些抗沉默因子的结合位点，并检查哪些其他组蛋白标记在这些位点富集 结合位点。将进行ATAC-seq以确定其基因组周围的染色质可及性。 结合位点。最后，我将把这些抗沉默因子与甲基化和沉默的内源性DNA连接起来， 基因座使用一个良好的测试锌指系统。如果我的假设是正确的，预计系留反- 沉默蛋白将激活转录而不影响DNA甲基化。最后，免疫沉淀 结合质谱技术分析抗沉默蛋白的组成 配合物