Investigating the Role of Histone Modifications in Heterochromatin Formation

 描述（由申请人提供）：异染色质参与多种生物学过程的调节，包括重组、染色体分离、转座子抑制和基因表达。因此，在包括亨廷顿氏病和许多癌症在内的多种疾病中反复发现异染色质相关蛋白的突变。然而，这些突变在疾病进展中的作用知之甚少。更深入地了解异染色质调节和异染色质环境改变的后果对于理解疾病的潜在联系至关重要。异染色质调控的一个潜在机制是组蛋白的翻译后修饰（PTM），包括组蛋白H3上赖氨酸9（H3 K9 me）的甲基化。一些实验室试图通过突变建立这种修饰的酶来研究H3 K9 me的作用。然而，他们的研究结论是有限的，因为这些酶除了H3 K9之外还有几种非组蛋白底物。不幸的是，通过将该残基突变为不可修饰的氨基酸来直接测试H3 K9的作用在高等真核生物中是不可行的，这是由于工程化替换组蛋白基因的困难。由于这个原因，尽管H3 K9的修饰被认为是异染色质的重要调节剂已有十多年，但H3 K9在动物中的功能从未被直接测试过。通过使用我们实验室和合作者最近建立的果蝇组蛋白替换平台，我们将分析H3 K9通过用H3 K9突变组蛋白替换内源性组蛋白对异染色质调控的贡献。本研究的第一个目的是通过评估异染色质相关蛋白的募集和染色质致密化来确定突变H3 K9是否导致异染色质结构改变。考虑到H3 K9 me募集异染色质因子的能力，我们预计一些基因组位置会失去异染色质蛋白，从而形成更开放的染色质环境。我们将通过ChIP-seq和多线染色体细胞学检查异染色质蛋白1和异染色质相关组蛋白PTM的定位来测试这一想法。此外，我们将使用位置效应杂色测定法从遗传学上评估异染色质形成，并使用FAIRE-seq探索全基因组开放染色质谱，以询问H3 K9突变体中的染色质结构。该提案的第二个目的将解决H3 K9对于两个异染色质调节过程（转座子抑制和染色体分离）的功能重要性。我们假设，一个妥协异染色质环境将导致去阻遏转座因子和染色体分离的缺陷。我们将通过定量PCR分析转座子表达和共聚焦显微镜研究染色体分离来验证这些假设。最终，我们希望分析H3 K9在调节异染色质中的作用，以增强我们对染色质结构改变如何导致疾病的理解。

 DESCRIPTION (provided by applicant): Heterochromatin is implicated in the regulation of diverse biological processes including recombination, chromosome segregation, transposon repression, and gene expression. As such, mutations in heterochromatin associated proteins are recurrently found in a variety of diseases, including Huntington's disease and numerous cancers. However, the role of these mutations in the progression of disease is poorly understood. A deeper understanding of heterochromatin regulation and the consequences of altered heterochromatin environments will be essential for understanding potential connections to disease. One potential mechanism of heterochromatin regulation is post-translational modification (PTM) of histones, including methylation of lysine nine on histone H3 (H3K9me). Several labs have attempted to study the role of H3K9me by mutating the enzymes that establish this modification. However, conclusions from their studies are limited because these enzymes have several non-histone substrates in addition to H3K9. Unfortunately, directly testing the role of H3K9 by mutating this residue to a non-modifiable amino acid has been unfeasible in higher eukaryotes due to the difficulty of engineering replacement histone genes. For this reason, although modification of H3K9 has been considered an important regulator of heterochromatin for more than a decade, the function of H3K9 in animals has never been directly tested. By using a Drosophila histone replacement platform recently established by our lab and collaborators, we will analyze the contribution of H3K9 to heterochromatin regulation by replacing endogenous histones with H3K9 mutant histones. The first aim of this study seeks to determine if mutating H3K9 results in altered heterochromatin structure by assessing recruitment of heterochromatin associated proteins and chromatin compaction. Given the ability of H3K9me to recruit heterochromatin factors we expect some genomic locations to lose heterochromatic proteins and consequently form a more open chromatin environment. We will test this idea by examining localization of Heterochromatin Protein 1 and heterochromatin associated histone PTMs via ChIP-seq and polytene chromosome cytology. Moreover, we will genetically assess heterochromatin formation using Position-Effect Variegation assays and explore genome-wide open chromatin profiles using FAIRE-seq to interrogate chromatin structure in H3K9 mutants. The second aim of this proposal will address the functional importance of H3K9 for two heterochromatin regulated processes, transposon repression and chromosome segregation. We hypothesize that a compromised heterochromatic environment will lead to de-repression of transposable elements and defects in chromosome segregation. We will test these hypotheses by performing quantitative PCR to analyze transposon expression and confocal microscopy to investigate chromosome segregation. Ultimately, we expect analysis of H3K9's role in regulating heterochromatin to augment our understanding of how altered chromatin structure may lead to disease.