Histone demethylases and regulation of chromatin and transcription in eukaryotes

描述（由申请人提供）： 组蛋白的N-末端被大量的共价修饰修饰，包括甲基化、乙酰化、磷酸化、类小泛素化和泛素化。这些修饰共同作用，为许多重要的核事件（包括转录、DNA复制、重组和修复）产生适当的染色质模板。DNA损伤信号和修复机制的缺陷可能导致癌症的发展，因此了解控制这一途径的机制具有重要的健康意义。近年来，虽然我们对组蛋白修饰在转录调控中的理解取得了重大进展，但对这些修饰如何影响DNA损伤反应（DDR）以及组蛋白修饰酶调节DDR途径的精确分子机制知之甚少。在过去的资助期间，我们发现组蛋白去甲基化酶LSD 1，迄今为止被认为只作为一个转录调节因子，也发挥了进化保守的作用，在DDR。LSD 1的缺失不影响修复基因的表达;此外，染色质免疫沉淀将LSD 1置于DNA损伤位点，表明LSD 1直接参与了这一过程。重要的是，LSD 1的缺失导致组蛋白H2 A/H2A.X泛素化受损，随后53 BP 1募集的缺失，53 BP 1是DNA损伤修复的重要分子。在本申请中，我们提出通过鉴定LSD 1调节组蛋白H2 A/H2 A. X泛素化的分子机制来验证我们的假设，即组蛋白H3 K4去甲基化和H2 A/H2 A. X泛素化之间存在对DDR重要的新串扰。考虑到LSD 1介导单甲基化和二甲基化组蛋白H3 K4的去甲基化，我们还将通过确定三甲基化H3 K4的去甲基化酶以及H3 K4甲基转移酶的宿主是否也在DDR中发挥作用来研究H3 K4甲基化动力学对于DDR是否通常重要。最后，我们将开始解决的问题，是否有一个特定的星座的组蛋白修饰在DNA损伤位点，诱导一个独特的染色质结构域的DNA修复，通过描绘组蛋白修饰景观和负责的酶在DNA损伤位点。我们预计，新的范例出现从拟议的研究，显着影响我们的理解表观遗传调控的DDR。

DESCRIPTION (provided by applicant): The N-terminal tails of histone proteins are heavily decorated by a plethora of covalent modifications including methylation, acetylation, phosphorylation, sumoylation and ubiquitylation. These modifications work together to generate appropriate chromatin templates for a host of important nuclear events, including transcription, DNA replication, recombination and repair. A defect in the DNA damage signaling and repair machinery can lead to the development of cancer, and therefore understanding the mechanisms that control this pathway has important health implications. In recent years, although significant progress has been made in our understanding of histone modifications in transcriptional regulation, much less is known about how these modifications impact the DNA damage response (DDR) and the precise molecular mechanisms by which histone- modifying enzymes regulate the DDR pathway. In the past funding period, we discovered that the histone demethylase LSD1, which thus far is thought to function only as a transcriptional regulator, also plays an evolutionarily conserved role in the DDR. Loss of LSD1 does not affect the expression of repair genes; furthermore, chromatin immunoprecipitation places LSD1 at DNA damage sites, suggesting a direct involvement of LSD1 in this process. Importantly, loss of LSD1 results in impaired histone H2A/H2A.X ubiquitylation, with subsequent loss of 53BP1 recruitment, a molecule important for DNA damage repair. In this application, we propose to test our hypothesis that a novel crosstalk exists between histone H3K4 demethylation and H2A/H2A.X ubiquitylation important for the DDR by identifying molecular mechanisms by which LSD1 regulates histone H2A/H2A.X ubiquitylation. Given that LSD1 mediates demethylation of mono- and di-methylated histone H3K4, we will also investigate whether H3K4 methylation dynamics is generally important for the DDR by determining whether demethylases for the tri-methylated H3K4 as well as a host of H3K4 methyltransferases also play a role in the DDR. Lastly we will begin to address the question of whether there is a specific constellation of histone modifications at DNA damage sites that induce a distinct chromatin domain for DNA repair, by delineating the histone modification landscape and the responsible enzymes at DNA damage sites. We anticipate that new paradigms to emerge from the proposed studies, significantly impacting our understanding of epigenetic regulation in the DDR.