Molecular mechanisms of gene silencing by H4 methylation

描述(申请人提供)：组蛋白蛋白负责DNA在细胞核内的紧凑和功能组织。这些小的、进化保守的蛋白质是一系列不同的翻译后修饰的宿主，每一种修饰通常与特定的DNA模板化过程有关，例如基因调控。我们最近发现了一种新的哺乳动物基因沉默途径，该途径由PR-Set7酶介导，特异性地单甲基化组蛋白H4赖氨酸20(H4K20)。我们的新发现表明，这种沉默途径针对的是某些基因的启动子，这些基因在生长和分化程序中发挥作用。基于这些发现，我们的中心假设是，H4K20单甲基化的一个主要作用是通过抑制促进生长和分化的特定基因集来维持细胞身份。这项建议的目的是剖析这一基本的基因沉默途径的分子机制，长期目标是确定这一途径在建立和维持细胞同一性方面的作用。我们最近发现PR-Set7与一个新的组蛋白H3赖氨酸9(H3K9)甲基转移酶结合，在基因启动子处产生一个抑制单甲基-H4K20和H3K9的“反式尾部组蛋白密码”。在目标1中，我们将使用现有的分子和生化方法来鉴定和表征该酶，并确定其在体内对基因抑制的作用。我们还发现了第一个已知的H4K20单甲基结合蛋白，L(3)MBT，并确定这种相互作用是基因抑制所必需的。在目标2中，我们将定义L(3)MBT与单甲基化H4K20结合和基因抑制所需的区域和氨基酸。在目标3中，我们将继续识别受该沉默途径调控的其他基因。这些基因作为体内模型，剖析了这一途径的每一个组成部分对体内基因抑制的单独贡献。总而言之，这一提议将阐明哺乳动物参与关键生物学过程的基本基因调控途径的分子机制，并可能对人类健康和疾病产生深远而广泛的影响。

DESCRIPTION (provided by applicant): The histone proteins are responsible for the compaction and functional organization of DMA in the nucleus. These small, evolutionary conserved proteins are host to a diverse array of post-translational modifications, each of which is typically associated with a specific DNA-templated process, such as gene regulation. We recently discovered a novel mammalian gene silencing pathway mediated by the PR-Set7 enzyme that specifically monomethylates histone H4 lysine 20 (H4K20). Our new findings indicate that this silencing pathway is targeted to the promoters of certain genes that function in growth and differentiation programs. Based on these findings, our central hypothesis is that one major role of H4K20 monomethylation is to maintain cellular identity by repressing specific sets of genes that promote growth and differentiation. The goal of this proposal is to dissect the molecular mechanisms of this fundamental gene silencing pathway with the long range goal of determining the role of this pathway in establishing and maintaining cellular identity. We recently discovered that PR-Set7 associates with a novel histone H3 lysine 9 (H3K9) methyltransferase to create a repressive monomethyl-H4K20 and H3K9 "trans-tail histone code" at gene promoters. In Aim 1 we will use established molecular and biochemical methods to identify and characterize this enzyme and determine its in vivo role in gene repression. We have also discovered the first known H4K20 monomethyl- binding protein, L(3)MBT, and determined that this interaction is essential for gene repression. In Aim 2 we will define the regions and amino acids of L(3)MBT required for binding monomethylated H4K20 and gene repression. In Aim 3 we will continue to identify additional genes regulated by this silencing pathway. These genes serve as the in vivo models to dissect apart the individual contributions of each of the components of this pathway on in vivo gene repression. Collectively, this proposal will illuminate the molecular mechanisms of a fundamental mammalian gene regulation pathway involved in critical biological processes and will likely have far-reaching and wide-spread impacts on human health and disease.