HUSH-mediated epigenetic regulation of retroelements

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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.