부산대학교 도서관

ATRX is a chromatin remodeller involved in a wide range of nuclear processes, including transcription. Germline mutations in ATRX give rise to alpha- thalassaemia/intellectual disability X-linked (ATR-X) syndrome and downregulate alpha globin expression through a mechanism which is poorly understood. To understand how ATRX regulates gene expression, here I use alpha thalassaemia as an exemplar to study how ATRX loss downregulates alpha globin expression, which would provide insights into understanding the general gene regulation mechanism of ATRX. Specifically, I aim to recapitulate the alpha thalassaemia phenotype observed in ATR-X syndrome patients in an ATRX deficient erythroid cell system, and then to determine the molecular mechanisms that cause perturbed alpha globin expression. To this end, an ATRX deficient CD34+ haematopoietic stem cell model was generated via CRISPR-Cas9. However, the cells did not show distinct alpha thalassaemia phenotype at the bulk level when differentiated into erythrocytes. Further gene expression analysis in single erythroid colony and single erythroid cell RNA-seq revealed that ATRX deficiency led to alpha thalassaemia in only a subset of cells. Differential gene expression analysis indicated the downregulation of alpha-like globin was likely associated with replicative stress/DNA damage, in particular the activated pATM-RNF168-ubiquitination pathway. To validate the mechanisms, an erythroid progenitor cell line containing a dTAG-13 inducible ATRX degron system was generated, in which the alpha-like globin gene was tagged with a fluorescent reporter for the enrichment of the affected cell subpopulation. This cell model recapitulated the alpha thalassaemia phenotype and was used to investigate the function and gene regulation mechanism of ATRX. It has been shown that ATRX mainly regulates alpha globin expression at the basophilic erythroblast stage, and ATRX loss-induced alpha globin downregulation is not associated with DNA methylation. As a prime candidate for causing replicative stress/DNA damage in the absence of ATRX, a putative G4 DNA-forming region ψζ VNTR in the alpha globin cluster was studied to explore its contribution to the gene regulation. It is demonstrated that ATRX binds to this region in a gene expression dependent manner. G4 stabilizer treatment downregulated α-globin gene expression and deletion of this region in erythroid cells abolished the effect of ATRX depletion on alpha globin expression, indicating ATRX regulates alpha globin expression through binding to the G-rich ψζ VNTR.