HUSH-mediated epigenetic regulation of retroelements

所有细胞都需要沉默外来的DNA，因为外来核酸的入侵威胁到了它们的完整性。一个特别的危险是移动的遗传元件或“跳跃基因”的不受控制的活动，它们可以插入宿主DNA并改变遗传信息。虽然这些元素在宿主基因组中不受控制的传播可能是灾难性的，但从进化的角度来看，这些遗传改变也可能是有益的。事实上，移动的遗传元件有助于遗传变异，是基因组进化的强大驱动力。因此，它们的活动必须受到严格管制。在人类细胞中，LINE-1或L1（长散置核元件-1）是唯一已知的移动的元件，并受到其宿主的严格控制。了解这些控制机制具有生物学和临床重要性，因为越来越多的证据表明LINE-1活性异常是从遗传改变到衰老和肿瘤等疾病的重要原因。抑制移动的元件的一个关键机制是在DNA整合位点组装抑制性异染色质。在人类细胞中，Lehner实验室最近发现了HUSH（人类沉默枢纽）转录表观遗传阻遏物复合物，该复合物招募SETDB 1甲基转移酶，以存款抑制性异染色质的典型组蛋白标记H3 K9 me 3进入DNA。HUSH沉默来自逆转录病毒（包括HIV）的整合DNA，逆转录病毒从外部攻击细胞，或逆转录转座子（LINE-1）从内部攻击细胞。同样的机制是否被用来沉默病毒和转座子还不清楚。一个无法解释的重要差异是，HUSH的病毒沉默是“基因组位置依赖性的”，因为H3 K9 me 3从相邻的异染色质传播，而LINE-1沉默似乎是DNA序列依赖性的。事实上，HUSH调节基因组中任何地方的L1整合，无论这发生在异染色质或缺乏H3 K9 me 3的转录活性常染色质中。HUSH如何促进H3 K9 me 3组装以抑制LINE-1仍然知之甚少，这种序列特异性沉默可能需要额外的基因产物的活性。我的项目的目的是研究HUSH介导的病毒和LINE-1限制的机制。我将使用去卷积的LINE-1报告基因进行CRISPR介导的全基因组正向遗传筛选，以识别LINE-1元件序列特异性沉默所需的新基因。虽然这不是一项微不足道的任务，但Lehner实验室已经建立了执行这些屏幕的专业知识。我的阳性对照将鉴定HUSH复合物的三种组分，以及MORC 2和SETDB 1。我希望能发现参与这种序列特异性沉默的新基因。新鉴定的基因最初将用不同的报告基因和细胞系进行验证，以确认它们在HUSH介导的沉默中的作用，并进一步表征它们的作用机制。

All cells need to silence incoming DNA as invasion by foreign nucleic acid threatens their very integrity. A particular danger is the uncontrolled activity of mobile genetic elements or 'jumping genes', which can insert into the host DNA and alter genetic information. While the uncontrolled spread of such elements in the host genome is potentially catastrophic, from an evolutionary standpoint these genetic alterations may also be beneficial. Indeed, mobile genetic elements contribute to genetic variability and are powerful drivers of genome evolution. Their activity must therefore be highly regulated. In human cells, LINE-1s or L1s (Long interspersed nuclear element-1) are the only known mobile elements and are tightly controlled by their host. Understanding these control mechanisms is of biological and clinical importance as increasing evidence implicates aberrant LINE-1 activity as an important cause of disease, from genetic alterations, to senescence and tumours. One key mechanism to repress mobile elements is to assemble repressive heterochromatin at the site of DNA integration. In human cells, the Lehner lab recently discovered the HUSH (Human Silencing Hub) transcriptional epigenetic repressor complex which recruits the SETDB1 methyltransferase to deposit the canonical histone mark of repressive heterochromatin, H3K9me3 on incoming DNA. HUSH silences integrated DNA derived from either retroviruses (including HIV), which attack the cell from the outside, or retrotransposons (LINE-1s) which attack the cell from within. Whether the identical mechanism is used to silence viruses and transposons is unclear. An unexplained and important difference is that viral silencing by HUSH is 'genome position-dependent' as H3K9me3 spreads from neighboring heterochromatin, while LINE-1 silencing appears to be DNA-sequence dependent. In fact HUSH regulates L1 integrations anywhere in the genome, whether this occurs within heterochromatin or transcriptionally active euchromatin that lacks H3K9me3. How HUSH promotes H3K9me3 assembly for LINE-1 repression remains poorly understood, and this sequence-specific silencing likely requires the activity of additional gene products. The aim of my project is to investigate the mechanism(s) of HUSH-mediated viral and LINE-1 restriction. I will use a deconvoluted LINE-1 reporter to perform a CRISPR-mediated genome-wide forward genetic screen to identify novel genes required for the sequence-specific silencing of LINE-1 elements. While this is not a trivial undertaking, the Lehner lab has established expertise in the execution of these screens. My positive controls will be the identification of the three components of the HUSH complex, together with MORC2 and SETDB1. I hope to identify novel genes involved in this sequence-specific silencing. Newly identified genes will initially be validated with different reporters and cell lines to confirm their role in HUSH-mediated silencing, and further characterized for their mechanism of action.