Monday, May 6, 2024
5/6/2024
Project Summary ATRX is a chromatin remodeler with diverse and essential roles in gene regulation and genomic stability. Mutations in ATRX are associated with neurodevelopmental disease and glioma. ATRX regulates several epigenetic pathways that each have the potential to impact localization of CTCF, a key chromatin architecture protein. ATRX suppresses R-loops, DNA:RNA hybrid structures that form co-transcriptionally and that co-localize with CTCF at gene terminator regions. ATRX also regulates DNA methylation, which can inhibit CTCF binding, and together with DAXX deposits the heterochromatic histone variant H3.3, which co-localizes with CTCF at imprinted genes. We have generated ATRX knockdown mouse embryonic stem cells (ATRX KD mESCs) and found that ATRX loss in these cells alters CTCF localization at hundreds of sites genome-wide. We propose to test the hypothesis that ATRX loss impacts CTCF localization through the dysregulation of multiple epigenetic pathways. We will test this hypothesis in two aims, using a number of genome-wide sequencing methods to reveal how epigenetic pathways are altered upon ATRX loss. In Aim 1, we will examine the consequences of ATRX loss on R-loop formation and gene expression. This aim will use BisMapR, a novel technique that detects genome-wide R-loops with high resolution and strand specificity, and RNA-Seq to measure transcription. In Aim 2, we will elucidate the roles of DNA methylation and histone H3.3 in ATRX regulation of CTCF. This aim will use bisulfite pyrosequencing and enhanced reduced representation bisulfite sequencing to assay DNA methylation in ATRX and H3.3 knockdown cells, and CUT&RUN to detect CTCF binding in H3.3 knockdown cells. We expect the results of this study to illuminate how the epigenetic functions of ATRX contribute to a novel role in CTCF regulation, and further understanding of the consequences of ATRX loss in disease.
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