Biochemical and functional analyses of histone modifications within the nucleosome context

人类基因组是由等量的DNA和蛋白质组成的，这些蛋白质被称为组蛋白。这种被称为染色质的组织不仅有助于压缩基因组的大小，而且组蛋白还控制底层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功能。组蛋白经历许多类型的翻译后修饰（PTM）。一些修饰，如磷酸化和乙酰化，作为快速开关来打开或关闭基因，而其他PTM，如甲基化，提供关于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）的长期指令。 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）。例如，我们发现H3磷酸化以生长因子可诱导的方式发生在间期细胞中，并且其功能是激活促生长基因的转录。相反，在有丝分裂期间发生相同的H3磷酸化，以在细胞分裂之前浓缩染色体。这种相同的组蛋白修饰如何在不同的细胞周期阶段执行对比功能尚不清楚，这使我们假设H3磷酸化与其他组蛋白PTM结合起作用，以指定不同的下游效应。事实上，我们以前开发了一种新的技术来纯化磷酸化的含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的两个拷贝周围的DNA），并发现H3磷酸化触发下游组蛋白乙酰化，并且它们一起激活间期细胞中的转录。使用同样的技术，我们现在将测试和鉴定其他组蛋白PTM，它们可能在有丝分裂期间与H3磷酸化协同作用，介导染色体凝聚。此外，我们将采用这种技术来纯化具有两个拷贝的磷酸化H3（对称修饰）或仅具有一个磷酸化H3（不对称修饰）的核小体用于生化分析。最近的研究表明，对称与不对称组蛋白PTM可能具有不同的功能，我们将直接测试这是否也适用于H3磷酸化，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H3磷酸化是否也影响有丝分裂H3功能。 组蛋白PTM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PTM的功能是染色质调控的一个新的和几乎完全未知的方面。我们提出的解决这一新的研究领域的工作将产生显着的和重要的新知识，这将提高我们对染色质和组蛋白PTM机制的理解。

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.