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Abstract

X-chromosome inactivation (XCI) is an essential process in female mammals ensuring dosage compensation between the sexes by silencing one X chromosome. Almost chromosome-wide gene repression, which is accompanied by heterochromatin formation on the inactive X chromosome (Xi), is maintained throughout life. While the factors involved in establishing gene silencing on the Xi are largely known, which of those are necessary for maintenance of XCI remains unclear. Specifically, the roles of repressive histone modifications such as H2AK119ub and H3K27me3, established by Polycomb repressive complexes (PRC) 1 and 2, respectively, in maintaining this repression, are not fully understood. Monoallelic autosomal expression (MAE) is another example of epigenetically regulated gene repression on only one of two alleles. While certain repressive factors are known to be associated with the 'inactive' allele at some MAE loci, the molecular mechanisms underlying stable monoallelic gene repression and the role of PRC1/H2AK119ub in maintaining monoallelic repression is yet to be determined. This thesis explores the role of PRC1/H2AK119ub in maintaining gene repression in XCI and MAE. Using an auxin-inducible degron system, RING1B, a core PRC1 component, was acutely depleted in a Ring1a-/- background in both dividing neural progenitor cells (NPCs) and non- dividing astrocytes. Allele-specific CUT&RUN and RNA sequencing were used to analyze the effects of RING1B depletion. The findings reveal that RING1/H2AK119ub is crucial for regulating a subset of X-linked genes, including both repressed and active genes on Xi and Xa. It is also essential for maintaining the silencing of certain MAE genes, ensuring monoallelic expression. Some MAE genes required RING1 for repression of the 'inactive' allele and fine-tuning expression from the 'active' allele. Initial changes in gene expression due to RING1B depletion were dependent on RING1/H2AK119ub but independent of H3K27me3, which was only depleted at later timepoints upon RING1B depletion. Astrocytes exhibited a different dependency on RING1 for gene silencing, with smaller changes in gene expression compared to NPCs, suggesting varying requirements for RING1 in maintaining monoallelic gene expression across cell types. This work highlights the critical role of RING1 in maintaining gene repression and regulating allele-specific gene expression in XCI and MAE, providing new insights into the mechanisms of monoallelic expression and associated epigenetic processes.