Chromatin remodeling and gene expression
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.
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.
As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques.
Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.
The department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.
|1||Shanbhag NM, Rafalska-Metcalf IU, Balane-Bolivar C, Janicki SM, Greenberg RA. 2010. ATM-dependent chromatin changes silence transcription in cis to DNA double-strand breaks.Cell, 141 (6), pp. 970-81. - http://www.ncbi.nlm.nih.gov/pubmed/20550933|
|2||Law MJ, Lower KM, Voon HP, Hughes JR, Garrick D, Viprakasit V, Mitson M, De Gobbi M, Marra M, Morris A, Abbott A, Wilder SP, Taylor S, Santos GM, Cross J, Ayyub H, Jones S, Ragoussis J, Rhodes D, Dunham I, Higgs DR, Gibbons RJ. 2010. ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner.Cell, 143 (3), pp. 367-78. - http://www.ncbi.nlm.nih.gov/pubmed/21029860|
|3||Sarkies P, Reams C, Simpson LJ, Sale JE. 2010. Epigenetic instability due to defective replication of structured DNA.Mol. Cell, 40 (5), pp. 703-13. - http://www.ncbi.nlm.nih.gov/pubmed/21145480|
|4||Clynes D, Jelinska C, Xella B, Ayyub H, Taylor S, Mitson M, Bachrati CZ, Higgs DR, Gibbons RJ. 2014. ATRX dysfunction induces replication defects in primary mouse cells.PLoS ONE, 9 (3), pp. e92915. - http://www.ncbi.nlm.nih.gov/pubmed/24651726|
Nguyen DT, Voon HPJ, Xella B, Scott C, Clynes D, Babbs C, Ayyub H, Kerry J, Sharpe JA, Sloane-Stanley JA, Butler S, Fisher CA, Gray NE, Jenuwein T, Higgs DR, Gibbons RJ. 2017 The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats. EMBO Rep. 2017 Jun;18(6):914-928. -https://www.ncbi.nlm.nih.gov/pubmed/28487353