Elucidating the role of XIST domains in maintenance of X-chromosome inactivation

X-inactive specific transcript (XIST) long non-coding RNA (lncRNA) is essential for initiation and establishment of X-chromosome inactivation (XCI). The role of individual human XIST domains in maintaining gene silencing remains poorly understood. My thesis work investigated the impact of CRISPR/Cas9-mediated deletions of tandem repeat and exonic regions of XIST in hTERT RPE-1 cells, by evaluating changes in XIST expression, localization, and XCI maintenance. Deletion of tandem repeat A-C completely ablated XIST expression. RNA sequencing of XIST knockout (KO) clones revealed altered expression of X-linked genes, including reactivation of MED14, variable expression changes in USP9X, and no change in MAGED1. Autosomal gene expression was also impacted, suggesting a broader XIST influence beyond X-linked silencing. Lack of MED14 CpG methylation in wild type (WT) indicates that XIST-dependent genes may rely on XIST for maintaining silencing in the absence of methylation. Similarly, tandem repeat A deletion showed loss of XIST expression, while individual deletions of regions containing tandem repeat F, Exon 4 (E4) and tandem repeat E significantly reduced XIST expression compared to WT. RT-qPCR data suggests formation of a unique splice variant following tandem repeat F deletion. RNA-IF demonstrated complete XIST delocalization following tandem repeat E deletion, indicating its role in tethering XIST to the Xi. Pyrosequencing revealed that deletions of regions containing tandem repeat A, F, BC, and E affected the allelic expression of some X-linked genes highlighting domain-specific contributions in maintaining XCI. RNA-IF FISH tagging full-length XIST and CDKN1A- interacting zinc finger protein 1 (CIZ1) showed a complete colocalization of XIST with CIZ1in WT and deletion clones except for A, AC, and E. Double Immunofluorescence tagging CIZ1 and H3K9me3 revealed distinct and adjacent domains at the Xi. Distinct domain formation of H3K9me3 and CIZ1, along with partial Xi-reactivation post XIST KO suggests the presence of both XIST-dependent and XIST-independent pathways for maintenance of gene silencing on the Xi. While XIST appears essential for silencing of certain regions others may rely on alternative pathways, possibly involving H3K9me3. Future work should focus on combinatorial XIST domain deletions and cross-referencing data with H3K9me3 enrichment to dissect XIST-independent pathways responsible for XCI maintenance.