Program > Browse abstracts by speaker > Michael David C.

Transposable elements (TEs) comprise about half of the mammalian genome and constitute the most abundant class of non-coding genomic elements known to date. TEs are mostly expressed in the early stages of development and are believed to play roles in recruiting pluripotency-associated factors. Due to their highly repetitive nature, several studies have investigated TEs only within the context of families and subfamilies. However, the challenge with this approach is that it leaves us with little or no insight into the unique role of individual TE loci. This study uses an epigenetic editing technology called iCRUSH to specifically knock down the expression of two candidate TE loci in E14 mouse embryonic stem cells (mESCs) to better understand how individual TE locus regulate nearby genes as well as distal genes critical to cell fate choices. iCRUSH is a doxycycline-inducible piggyBac system that uses an array of GCN4 repeats to recruit up to five KRAB and DNMT3A/3L effectors unto a target locus to induce H3K9me3 deposition and DNA methylation respectively. After iCRUSH targeting of 2 TE loci, we assessed the specificity and reliability of DNA methylation on the specific loci versus other loci with near sequence identity. Furthermore, we measured the enrichment of H3K9me3 by CUT&RUN qPCR of the targeted TE loci. A novel method, CELLO-seq, which uses unique molecular identifiers (UMI) based on single-cell RNA sequencing has been optimised to target TE expression to a particular locus. By utilising CELLO-seq, changes in nearby gene expression levels within 2-10kb were assessed. In the future CELLO-seq will be used to assess whether TEs impact distal pluripotency genes such as Pou5F1, Nanog, and Sox2. This will give us further insight into the role of locus-specific TEs in proximal and distal gene expression regulation.