Chromatin remodelling and gene expression

Supervisors:  Prof Richard Gibbons and Prof Doug Higgs

Project

ATRX, a chromatin remodeling protein which is involved in inserting the histone variant H3.3 into repetitive DNA plays an important role in regulating gene expression. Mutations in ATRX lead give rise to a complex human genetic disease characterized by severe learning difficulties, a characteristic facial appearance abnormal sexual development and a form of anaemia called alpha thalassaemia. This anaemia results from reduced expression of alpha globin, a component of haemoglobin. We have recently discovered that ATRX binds to tandem repeats many of which are G-rich including rDNA, telomeres and interstitial repeats (Law et al Cell 2010). Many of these sequences have the capacity to fold into G-quadruplex (G4) secondary structures in vitro to which ATRX protein binds. When ATRX is mutated there are changes in the expression of genes close to the interstitial repeats. The longer the repeat the greater the effect on gene expression and the closer the gene to the repeat the greater the effect. The underlying cause for this phenomenon is unknown but we have recently shown that ATRX is required for DNA replication through these G-rich repetitive sequences and in its absence replication is stalled and a DNA damage response is generated (Clynes et al PLoS One 2014). Our working hypothesis is that G quadruplex (G4) structures are the impediment to replication. Work by others has shown how DNA damage can lead to perturbed expression of adjacent genes (Shanbhag et al Cell 2010; Sarkies et al Mol Cell 2010); it is possible the effect on gene expression is secondary to nearby DNA damage. The aim of this project is to develop a cellular system to reproduce the in vivo observation and to determine the manner by which gene expression is perturbed in this genetic disease. The project will use patient-derived hematopoietic progenitors (CD34+) and iPS cells, CRISP/Cas9 edited CD34+ cells, and immortalized erythroid progenitor cell lines as models to recapitulate this phenomenon. These cells will be assayed to see if the G-rich interstitial repeats are associated with markers of DNA damage as well as characterizing the epigenetic profile of the nearby genes to determine the mechanism by which expression is perturbed. 

Training opportunities

This project offers an opportunity to learn how to induce lineage specific differentiation of CD34+ cells and iPS cells, FACS analysis and sorting, CRISP/Cas9 gene editing, epigenetic profiling including mapping G4 and R-loops in the genome, next generation sequencing, assaying replicative stress and DNA damage. Students will be trained in bioinformatics to facilitate the analysis of their genome-wide data.

References:

  1. Law MJ et al (2010) Cell 143:367-378 (work from our lab on ATRX targeting tandem repeats and recognizing G4)

  2. Clynes D et al (2014) PLoS One e92915 (work from our lab showing haow ATRX is required for the replication of G-rich repetitive DNA)

  3. Shanbhag N et al (2010) Cell 141:970-981 (gene silencing in cis to DNA double strand breaks)

  4. Sarkies P et al (2010) Mol Cell 40:703-713 (gene silencing associated with defective replication of structured DNA)

  5. Nguyen DT et al (2017) EMBO Rep 18:914-928 (work from our lab showing that ATRX suppresses R-loops in telomeric repeats)

For further information please contact: Prof Richard Gibbons