Understanding the regulation of chromatin accessibility by KRAB-ZFPs through high-resolution profiling and rapid depletion of their co-factor TRIM28

PhD project strategic theme: Understanding the rules of life

Kruppel-associated box domain zinc finger proteins (KRAB-ZFPs) are a large family of DNA binding factors found exclusively in higher vertebrates. They are emerging rapidly through evolution, with new members of the family found at every phylogenetic branch. They target transposable elements (TEs) and have been implicated in their transcriptional repression. This function is mediated by the KRAB domain which interacts with TRIM28, resulting in the local establishment of heterochromatin. So far, the best described function of KRAB-ZFPs is epigenetic silencing of transposable elements in embryonic stem cells. Nevertheless, comparative gene expression analysis indicates that members of the family display specific transcription patterns in various somatic cell types. It is hypothesized that they play a role in controlling chromatin accessibility of TE-embedded enhancers in a cell-type specific fashion, selectively preventing transcription factors from binding and modulating the expression of nearby genes. Therefore, understanding the transcriptional outcomes of KRAB-ZFP activity in different cell types is of fundamental interest to decipher their contribution to gene regulation.

This project aims to improve our understanding of KRAB-ZFPs through the study of their cofactor TRIM28. First, we plan to perform ChIP-exo experiments on TRIM28 in multiple different cell types. Although there are a few public datasets available in the literature which show differential patterns of TRIM28 activity. They have been performed by different labs using different antibodies and sequenced with short read lengths, which is a problem with transposable elements. A TRIM28 ChIP protocol was recently optimised by the Imbeault lab to yield high-quality results using the commercially available TRIM28 antibody. ChIP-exo is an updated version of the chromatin immunoprecipitation method which combines an exonuclease digestion to yield single-base pair resolution of protein binding sites on the genome, which will allow us to precisely identify the KRAB-ZFPs responsible for the recruitment of TRIM28 at any given site of interest.

The second component of this project will use the dTAG (degradation tag) system to rapidly deplete TRIM28 at the protein level so we can distinguish early, middle and late transcriptional events triggered by the loss of the functional cofactor of all KRAB-ZFPs. Past studies perturbed TRIM28 expression using shRNA technologies, allowing for an initial evaluation of the transcriptional function of the KRAB/TRIM28 system. However, TRIM28 depletion is lethal to most cell types at the assayed time-points (4 days, when the protein is finally degraded following the shRNA-induced depletion of the mRNA). Inducing rapid degradation (~1h) of TRIM28 at the protein level using the dTAG system will help circumvent this obstacle and allow us to fully reconstruct the timeline of gene expression events triggered by TRIM28 depletion in various cell types.

This information will allow us to understand how KRAB-ZFPs regulate epigenetic silencing between cell types and how evolution has shaped our genome to make use of regulatory regions contained within transposable elements. This may also allow us to further annotate the non-coding genome which will help in understanding the functional impact of mutations found outside genes and how these may lead to disease.