Abnormal epigenetic regulation leads to defective silencing of transposable elements (TEs). The most abundant class of TEs are long interspersed nuclear elements-1 (L1), which cover approximately 20% of the human genome and 17% of the murine genome (Hemberger, 2002; Lander et al., 2001; Mouse Genome Sequencing Consortium et al., 2002). Full-length L1s can “jump” within the genome by a mechanism called retrotransposition (Heffron et al., 1979). Despite several silencing mechanisms, L1s are still active during development and in the brain (Muotri et al., 2005). Several factors are known to be involved in L1 silencing, however this remains a poorly understood field. Therefore, I will establish a screen to detect LINE-1 regulators.
I am establishing embryonic stem cell (ESC) reporter cell lines, where loss of L1 silencing is indicated by GFP expression. Endogenous silencing mechanisms will lead to prompt silencing of the L1 promoter. I will identify genes that reactivate the L1 promoter by either the addition of a hairpin library which targets approximately 1,200 known epigenetic factors and controls or a whole-genome CRISPR library in duel complementary screens. Some gene knockdowns or knockouts will lead to loss of L1 silencing, which I will detect by flow cytometry for GFP positive cells, followed by DNA sequencing to identify pertinent shRNAs or gRNAs.
This approach will identify regulators of L1 retroelements. Following up these hits will shine light onto fundamental epigenetic silencing mechanisms and will give insights into the mechanisms by which L1 may be mis-regulated in disease, potentially leading to treatments for L1-driven diseases.