Influence of DNA methylation changes on gene expression from the inactive X chromosome in B cells

Project Abstract 80% of patients afflicted with autoimmune diseases are female, and most autoimmune diseases have no cure. Patients with female-biased autoimmune diseases such as systemic lupus erythematosus (SLE) aberrantly express X-linked genes, suggesting that impairments in X chromosome inactivation (XCI) may impact disease. XCI transcriptionally silences one X chromosome in XX females via enrichment of repressive epigenetic marks including Xist RNA, heterochromatic histone marks, and DNA methylation (DNAm) to equalize X-linked gene expression to XY males. While in vitro stimulated B cells exhibit association of all these repressive epigenetic marks to the Xi, our laboratory found that Xist RNA and heterochromatic histone modifications are delocalized from the Xi in naïve B cells. Preliminary allele-specific RNA-seq data indicate that majority of the inactive X remains ‘continuously silent’ in naïve and stimulated cells, indicating that the inactive X in B cells remains silent even in the absence of Xist RNA and heterochromatic marks. This suggests the existence of Xist RNA independent mechanisms regulating Xi-linked gene expression in female B cells. Allele-specific RNA-seq data reveal that of the 42 genes which are expressed from the inactive X in naïve cells, some are ‘continuously expressed’ in stimulated cells. In addition, some genes become ‘newly silent’ or ‘newly expressed’ in stimulated cells. Allele-specific whole genome bisulfite sequencing (WGBS) data indicate that the Xi remains highly enriched for DNAm compared to the active X and autosomes in both naïve and stimulated cells, indicating that it may be responsible for regulating gene expression from the inactive X independently of Xist RNA. As the conversion of repressive 5’-methylcytosine (5mC) to 5’-hydroxymethylcytosine (5hmC) associates with gene expression and WGBS alone does not differentiate them, I distinguished between these two forms of DNAm using the novel technique bisulfite-assisted APOEBEC-coupled epigenetic sequencing (bACE-seq) alongside WGBS. My preliminary data indicate that 5hmC is present at CpG dinucleotides within promoters of some expressed X-linked genes, and that B cell stimulation alters its enrichment levels. DNA methyltransferases (DNMT) deposit 5mC, whereas Ten Eleven Translocation (TET) enzymes remove it by generating 5hmC. I will test my central hypothesis that DNMT and TET enzymes regulate gene expression from the inactive X in stimulated B cells in an Xist RNA-independent manner. In Aim 1, I will perturb DNMT1 (Aim 1a), the DNMT responsible for 5mC maintenance during cell replication, and DNMT3a/3b (Aim 1b), the DNMTs responsible for novel deposition of 5mC, and assess the impact on 5mC enrichment and gene expression from the Xi. In Aim 2a, I will perturb TET2/3, the most highly expressed TETs in B cells, and assess the impact of perturbed 5hmC enrichment on Xi-linked gene expression. In Aim 2b, I will perturb Xist RNA expression and assess whether DNAm modulates Xi-linked gene expression independently of Xist RNA. Elucidating this mechanism will reveal how X-linked gene expression can become perturbed in female-biased autoimmune diseases.