Characterization of factors required for expression of DNA methylated genes

项目摘要 这一建议旨在了解DNA甲基化基因的表达机制。脱氧核糖核酸 甲基化是大多数真核生物基因调控的关键机制。它通常与基因有关。 在启动子区域发现沉默。然而，DNA甲基化并不总是导致完全 基因沉默。在某些情况下，DNA甲基化基因可以被活跃地转录，这表明 反沉默因素的存在。 我进行了一次基因筛查，寻找这种抗沉默因素。我发现MED12，MED13， 和SAC3B能够抑制DNA甲基化GFP报告基因的表达。所有这三种蛋白质 以前发现，在基因调控中，通过一个确定的网络，各种因素共同发挥作用。此外，缺陷 在MED12和MED13之前已经被证明会导致人类的严重疾病，如癌症和 心血管疾病。对SAC3B的初步研究表明，SAC3B优先影响 DNA甲基化基因。因此，假设从屏幕上分离的基因是反沉默的 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说，在MED12/13和SAC3B中DNA甲基化的重要性- 介导的基因表达将通过使用相同的GFP报告来确定，而不是DNA甲基化。在……里面 此外，DNA甲基化相关基因表达对MED12/13和MED12/13的全基因组依赖性 SAC3B将通过使用DNA甲基转移酶突变体和DNA甲基转移酶抑制剂进行测试。它是 预计某些基因的表达只会受到这些反沉默因子的影响，当它们 携带DNA甲基化。接下来，我计划确定DNA甲基化的什么序列上下文(CG、CHG或 CHH，其中H不是G)优先与这一现象相关。除了DNA甲基化，还有两种 其他类型的表观遗传特征也可能促进上述反沉默的招募 各种因素。一种是组蛋白修饰，另一种是染色质可及性。我会对整个基因组进行分析 这些抗沉默因子的结合部位，并检查哪些其他组蛋白标记在这些部位富含 结合部位。将进行atac-seq以确定其基因组周围染色质的可及性。 结合部位。最后，我将把这些抗沉默因子与甲基化和沉默的内源DNA捆绑在一起 使用经过良好测试的锌指系统进行定位。如果我的假设是正确的，预计被拴住的反- 沉默蛋白将在不影响DNA甲基化的情况下激活转录。最后，免疫沉淀 结合质谱学分析抗沉默蛋白的组成。 复合体。

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.