Epigenetic Mechanisms of Retrotransposon Silencing

项目总结/摘要 反转录转座子是我们遗传密码的古老组成部分，在数百万年的时间里进行了自我复制 现在占了我们基因组的45%反转录转座子激活对细胞活力有严重影响- 反转录转座本质上是致突变的，甚至“死亡”的反转录转座子的表达也可以引发先天性的 引起炎症的免疫反应。因此，细胞已经进化出复杂的表观遗传机制， 通过将反转录转座子隔离在组成型异染色质中来沉默它们的表达， 标记了H3 K9甲基化和DNA CpG甲基化的表观遗传修饰。H3 K9失调 甲基化和CpG甲基化引起反转录转座子的广泛再激活，并在多种细胞中发生。 人类疾病。然而，很少有人知道的精确分子机制，启动 逆转录转座子沉默或维持逆转录转座子处于沉默状态。这项研究的主要目标是 该计划的目的是确定细胞用来抑制反转录转座子的精确分子机制 表情细胞逆转录转座子沉默机制的一个关键组成部分是组蛋白赖氨酸 甲基转移酶Setdb-1，其在反转录转座子处沉积H3 K9甲基标记。由于其核心作用， Setdb-1的活性受到一系列复杂分子的严格调控，这些分子控制着setdb-1的催化活性， 逆转录转座子序列的招募及其在整个长度上传播组蛋白甲基化的能力 反转录转座子Setdb-1活性的适当控制对于反转录转座子沉默是至关重要的，但许多 Setdb-1的监管方面仍然是个谜。我们将使用体外生化重组，冷冻EM 和基因组学来破译Setbd-1调控的精确机制。概述的工作 在这个建议中将解释Setdb-1是如何催化激活逆转录转座子沉默，Setdb-1如何 读取反转录转座子上的表观遗传密码，以及Setdb-1如何被招募到反转录转座子序列中， 建立H3 K9甲基化模式。

Project Summary/Abstract Retrotransposons are ancient components of our genetic code that have self-replicated over millions of years and now make up 45% of our genome. Retrotransposon activation has serious implications for cell viability – retrotransposition is mutagenic by its nature and even expression of “dead” retrotransposons can trigger innate immune responses that cause inflammation. Therefore, the cell has evolved intricate epigenetic mechanisms to silence expression of retrotransposons by sequestering them in constitutive heterochromatin, which is marked the epigenetic modifications of H3K9 methylation and DNA CpG methylation. Dysregulation of H3K9 methylation and CpG methylation causes widespread re-activation of retrotransposons and occurs in multiple human diseases. However, very little is known about the precise molecular mechanisms that initiate retrotransposon silencing or maintain retrotransposons in a silent state. The broad goal of this research program is to define the precise molecular mechanisms the cell uses to suppress retrotransposon expression. A key component of the cells retrotransposon silencing machinery is the histone lysine methyltransferase Setdb-1, which deposits H3K9 methyl marks at retrotransposons. Because of its central role, Setdb-1 activity is tightly regulated by a complex cast of molecular players that control its catalytic activation, recruitment to retrotransposon sequences and its ability to spread histone methylation across the length of retrotransposons. Appropriate control of Setdb-1 activity is critical for retrotransposon silencing, but many aspects of Setdb-1 regulation remain enigmatic. We will use in vitro biochemical reconstitutions, Cryo-EM and genomics to decipher the precise mechanisms that underly regulation of Setbd-1. The work outlined in this proposal will explain how Setdb-1 is catalytically activated for retrotransposon silencing, how Setdb-1 reads the epigenetic code on retrotransposons and how Setdb-1 is recruited to retrotransposon sequences to establish H3K9 methylation patterns.