The chromatin remodeller ATRX facilitates diverse nuclear processes, in a stochastic manner, in both heterochromatin and euchromatin

The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin

A recurrent de novo mutation in ACTG1 causes isolated ocular coloboma

Ocular coloboma (OC) is a defect in optic fissure closure and is a common cause of severe congenital visual impairment. Bilateral OC is primarily genetically determined and shows marked locus heterogeneity. Whole-exome sequencing (WES) was used to analyze 12 trios (child affected with OC and both unaffected parents). This identified de novo mutations in 10 different genes in eight probands. Three of these genes encoded proteins associated with actin cytoskeleton dynamics: ACTG1, TWF1, and LCP1. Proband-only WES identified a second unrelated individual with isolated OC carrying the same ACTG1 allele, encoding p.(Pro70Leu). Both individuals have normal neurodevelopment with no extra-ocular signs of Baraitser–Winter syndrome. We found this mutant protein to be incapable of incorporation into F-actin. The LCP1 and TWF1 variants each resulted in only minor disturbance of actin interactions, and no further plausibly causative variants were identified in these genes on resequencing 380 unrelated individuals with OC

Role of the chromatin remodeler ATRX in the regulation of gene expression

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.</p

Role of the chromatin remodeler ATRX in the regulation of gene expression

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.</p