Biochemical and functional analyses of histone modifications within the nucleosome context

核小体背景下组蛋白修饰的生化和功能分析

• 批准号: RGPIN-2019-06277
• 负责人: Cheung, Peter
• 金额: $ 2.62万
• 依托单位: York University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738814
• 关键词: Biochemical; functional; analyses; histone; modifications

The human genome is made up of equal parts DNA and proteins called histones. This organization, known as chromatin, not only helps to compact the genome in size, but the histone proteins also control accessibility of the underlying DNA and play important regulatory functions in major biological processes such as transcription and replication. Histones are subjected to many types of post-translational modifications (PTMs). Some modifications, such as phosphorylation and acetylation, act as quick on-off switches to turn genes on or off, whereas other PTMs, such as methylation, provide long term instructions on how to package chromatin (loosely or tightly) at different parts of the genome over many cell divisions. My lab's has had a long standing interest in elucidating intricate mechanisms involving histone PTMs and how they regulate gene expression (e.g., transcriptional activation or repression). For example, we found that H3 phosphorylation occurs in a growth factor-inducible manner in interphase cells and this functions to activate transcription of growth-promoting genes. In contrast, the same H3 phosphorylation occurs during mitosis to condense chromosomes prior to cell division. How this same histone modification performs contrasting functions at different cell cycle stages is still unclear and led us to hypothesize that H3 phosphorylation works in combination with other histone PTMs to specify distinct downstream effects. Indeed, we previously developed a novel technique to purify phosphorylated H3-containing nucleosomes (the physiological form of chromatin comprising DNA wrapped around two copies each of histones H2A, H2B, H3 and H4) and found that H3 phosphorylation triggers downstream histone acetylation, and together they activate transcription in interphase cells. Using this same technique, we will now test and identify other histone PTMs that might synergize with H3 phosphorylation during mitosis to mediate chromosome condensation. In addition, we will adapt this technique to purify nucleosomes that either have two copies of phosphorylated H3 (symmetrically modified) or with only one H3 that is phosphorylated (asymmetrically modified) for biochemical analyses. Recent studies suggested symmetric vs. asymmetric histone PTMs may have distinct functions, and we will directly test whether this is true for H3 phosphorylation as well, particularly in the context of H3 phosphorylation-coupled histone acetylation and transcriptional activation. Finally, we will test whether asymmetric H3 phosphorylation also affects the mitotic H3 function as well. Histone PTMs play important roles in the regulation of many nuclear processes, and the functions of symmetric vs. asymmetric histone PTMs is a new and almost completely unknown aspect of chromatin regulation. Our proposed work addressing this new area of research will yield significant and important new knowledge that will enhance our understanding of chromatin and histone PTM mechanisms.

人类基因组由等量的DNA和称为组蛋白的蛋白质组成。这种被称为染色质的组织不仅有助于压缩基因组的大小，而且组蛋白还控制潜在DNA的可及性，并在转录和复制等主要生物过程中发挥重要的调节作用。组蛋白受到多种类型的翻译后修饰（ptm）。一些修饰，如磷酸化和乙酰化，就像打开或关闭基因的快速开关，而其他的PTMs，如甲基化，提供了如何在许多细胞分裂中在基因组的不同部分包装染色质（松散或紧密）的长期指导。我的实验室长期以来一直对阐明涉及组蛋白ptm的复杂机制以及它们如何调节基因表达（例如，转录激活或抑制）感兴趣。例如，我们发现H3磷酸化在间期细胞中以生长因子诱导的方式发生，其功能是激活生长促进基因的转录。相反，同样的H3磷酸化发生在有丝分裂期间，在细胞分裂之前凝聚染色体。这种相同的组蛋白修饰如何在不同的细胞周期阶段发挥不同的功能尚不清楚，这使我们假设H3磷酸化与其他组蛋白PTMs联合起作用，以指定不同的下游作用。事实上，我们之前开发了一种新技术来纯化磷酸化的含有H3的核小体（染色质的生理形式，包括包裹在组蛋白H2A， H2B， H3和H4的两个拷贝上的DNA），并发现H3磷酸化触发下游组蛋白乙酰化，它们一起激活间期细胞的转录。使用同样的技术，我们现在将测试和鉴定其他可能在有丝分裂期间与H3磷酸化协同介导染色体凝聚的组蛋白PTMs。此外，我们将采用该技术纯化具有两个磷酸化H3拷贝（对称修饰）或只有一个磷酸化H3拷贝（不对称修饰）的核小体，用于生化分析。最近的研究表明，对称与非对称组蛋白ptm可能具有不同的功能，我们将直接测试H3磷酸化是否也是如此，特别是在H3磷酸化偶联组蛋白乙酰化和转录激活的背景下。最后，我们将测试不对称的H3磷酸化是否也会影响有丝分裂的H3功能。组蛋白PTMs在许多核过程的调控中发挥重要作用，对称与不对称组蛋白PTMs的功能是染色质调控的一个新的几乎完全未知的方面。我们提出的研究这一新领域的工作将产生重要的新知识，这将增强我们对染色质和组蛋白PTM机制的理解。