Epigenic factors such as DNA methylation and post-translational modification of the tails of histone proteins have been investigated with repect to regulatinggenome organisation throughout development. We, and others, have shown that loss of methylation can redirect Polycomb Repressive Complex 2 (PRC2) away from its target sites and redeposit its mark - H3K27me3 at the hypomethylated pericentromeric heterchromatic (PCH) regions within the nucleus. The aim of the project is to investigate the dynamic relationship between Polycomb targeting to PCH and the loss and gain of DNA methylation at satellite heterochromatin. Particularly in the context of epigenetic modifiers such as H3K9me3 and H2AK119Ub, in what order do these marks move to and from PCH and what other components participate in the targeting of polycomb to hypomethylated heterochromatin?

    The main tool for this project is a DNMT1tet/tet OFF - doxycycline responsive mES cell line (CH9 cell line) which reversibly represses the expression of the maintenance DNA methyltransferase (Dnmt1) and subsequently DNA methylationin the presence of dox. Furthermore, in vitro systems, such as different culturing media and cell lines with various levels of DNA methylation, alongside the CH9 cells, will be used to dissect the dynamics of Polycomb localisation in mouse embryonic cells. Moving away from fixed cells, reporter constructs will be generated to track the histone modifications in live cells using single molecule localisation techniques such as STORM. Additionally, STED techniques will be employed to attempt to resolve the structure of PCH upon modulation of DNA methylation. The work will then extend to look at in vivo models - early blastocyst (which is hypomethylated) to see if the observations made in vitro recapitulate what is happening in vivo.

 

DNA Methylation

                                             Hypothetical model of Polycomb Targeting to PCH.

          In the presence of DNA methylation, both Polycomb marks are at their target loci in the genome and H3K9me3 is located in PCH to maintain a repressive state. In the absence of DNA methylation Polycomb marks are found in PCH and H3K9me3 has been displaced. The intermediate state is one where all three histone marks are in PCH after DNA methylation has been lost.

 

Conferences:

ESRIC interdisciplinary symposium 01- 2018  -  Poster presentation

CamBioScience - Emerging epigenetics: Detecting and Modifying Epigenetics Marks  03 - 2018

High Content Imaging of Stem Cells Meeting  03 - 2018

UCL Genetics Institute - Epigenetics Sympoium  05 - 2018

EMBL in the UK  05 - 2018

RMS - European Light Microscopy initiative  06 - 2018

UoE - Chromatin, Epigenetics and Transcriptional Reugulation Meeting 2017 - 2018