Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.780437
Title: Role of the chromatin remodeler ATRX in the regulation of gene expression
Author: Truch, Julia
ISNI:       0000 0004 7966 081X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Abstract:
The ATR-X syndrome is an inherited, X-linked condition associated with alpha-thalassaemia and a variety of developmental abnormalities including profound intellectual disabilities. It results from mutations in a chromatin remodeling factor (called ATRX) which has been shown to perturb a wide range of nuclear activities including replication, transcription and DNA methylation. Although ATRX is known to play a role in the turnover of the histone variant H3.3, the precise role of ATRX in each of these nuclear activities and how ATRX mutations affect phenotype, are poorly understood. Previous studies have shown that ATRX is associated with repeated regions, including pericentric heterochromatin, telomeres, and rDNA repeats, some of which have the propensity to form G-quadruplexes. However, within euchromatin very little is known about ATRX target genes and how their expression is affected by this protein. The two major obstacles to answering these questions are first, the poor quality of ATRX chromatin immunoprecipitation sequencing (ChIP-seq) protocols; and second the lack of a well-defined, tractable cell model to investigate the role of ATRX on the expression of its targets in normal and affected individuals. In this thesis I studied the role of ATRX in lymphoblastoid cell lines (LCL) as they are readily available from patients with ATR-X syndrome. First, I successfully optimised the protocol for ATRX ChIP-seq. This showed that ATRX is also found at regions of open chromatin including a subset of active promoters and enhancers. Second, using LCL from ATR-X patients and unaffected controls, I identified more than a hundred of candidate genes whose expression is affected by ATRX mutations. Intersecting these two datasets I concluded that more than fifty genes whose expression is altered by mutations in ATRX are probably primary targets of this protein including zinc finger transcription factors. I next assessed these findings using erythroid cells, which are of relevance since mutations in ATRX cause alpha-thalassaemia; a red cell phenotype caused by downregulation of alpha globin expression. Using differentiated human CD34+ cells, I confirmed the binding of ATRX at repeats within the alpha globin cluster but, reflecting the findings in LCL, I also identified ATRX enrichment at HBA1 and HBA2 genes and their associated enhancers suggesting that ATRX at these newly identified sites may normally contribute to the regulation of alpha globin expression. In conclusion, these high quality ATRX-ChIP datasets allowed me to identify new ATRX targets in human samples. The new set of genes affected by ATRX mutations, within their associated chromatin environment, offers the opportunity to further understand the role of ATRX in heath and disease. The findings show for the first time that ATRX is significantly bound at active euchromatic regions with rapid nucleosome turnover, including active enhancers, in human cells.
Supervisor: Gibbons, Richard ; Higgs, Douglas Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.780437  DOI: Not available
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