Investigating the spread of escape from X-chromosome inactivation in human cells

X-chromosome inactivation (XCI) is a developmental epigenetic process that silences supernumerary X chromosomes in mammalian cells to maintain gene dosage between XX females and XY males. After initiation by the long non-coding RNA XIST, silencing of the inactive X (Xi) is maintained through somatic cell divisions. Some genes are still expressed from the Xi and ‘escape’ XCI. ‘Escapees’ are important contributors to sexual dimorphism in development and disease, and understanding how genes escape silencing on the Xi provides a model system for selective epigenetic regulation. This thesis focuses on how XCI status can spread, which could have implications for disease states and the loosening of epigenetic regulation with age. Bioinformatic studies have identified DNA elements that are associated with spread of or protection from silencing by XCI, yet functional validation studies are still rare. Most XCI experiments are performed in mouse models, which are readily manipulated but have fewer escapees; as such, findings may not reflect human biology. Here, a candidate escape-regulatory element between an escapee and a subject gene was identified and then deleted with CRISPR/Cas9 in human somatic cells. After finding that this region was resilient to changes in escape status during XCI maintenance, the candidate element was examined in a mouse embryonic stem cell model system to assess its role during XCI initiation. This element could act as an insulator to protect a gene from silencing through XCI initiation, but was dispensable if another insulator was present, suggesting that similar compensatory features may exist in somatic cells. Separately, to test whether silencing can spread into an active promoter during maintenance, transgenes were integrated at two evolutionarily distinct silenced regions of the Xi. Transgenes were silenced by XCI at both loci, indicating that ongoing transcription is insufficient to maintain escape during XCI maintenance. Notably, only the transgenes silenced on the short arm of the X chromosome gained promoter DNA methylation. Overall, this thesis provides functional insights into how XCI escape status can spread or be constrained in human somatic cells.