ERRBS-Seq of XpY*x and XmY*x murine CD4+ T cells

Most autoimmune diseases have a higher incidence in women than men due to differences in sex hormones, sex chromosomes, or both. Female (XX) and male (XY) sex chromosome complements differ in parent-of-origin of the X chromosome, with females inheriting one each from the mother and father, while males inherit only one from the mother2. Here, we discovered that the paternal X (Xp) had higher DNA methylation than the maternal X (Xm) in autoantigen specific CD4+ T lymphocytes, potentially suppressing X gene expression in XX. Using the “Four Core Genotypes” model, the transcriptomes of autoantigen specific CD4+ T lymphocytes showed higher expression of many X genes in XY- than XX. Expression of toll-like receptor 7 (Tlr7) and forkhead box P3 (FoxP3), two X chromosome genes important in autoimmunity, was higher in XY- than XX, confirming genome wide methylation and transcription data. Thus, parent-of-origin differences in DNA methylation of X genes can lead to sexual dimorphisms in gene expression during autoimmunity. Enhanced reduced representation bisulfite sequencing of autoantigen specific CD4+ T cells from paternally imprinted XpY*x and maternally imprinted XmY*x mice on SJL background. Mice were induced at 8-12 weeks of age by subcutaneous injection of PLP peptide 139-151 (200 ug/mouse, Mimotopes) emulsified in Complete Freund’s adjuvant, supplemented with Mycobacterium tuberculosis H37Ra (200ug/mouse; Difco Laboratories), over draining auxillary and inguinal lymph nodes one time on Day 0. Mice were sacrificed 12 days after induction. Auxillary, brachial, and inguinal lymph nodes were collected and processed into single cell suspensions. Single cell suspensions were resuspended in autoMACS Buffer (90uL/ 107 cells, Miltenyi Biotec). The cells were incubated with CD3 and CD4 microbeads (10 uL/10^7 cells, Miltenyi Biotec) for 15 min at 4°C. Cells were washed with and resuspended in autoMACS buffer (500 uL/10^8cells) then sorted on an autoMACSTM Separator (Miltenyi Biotec) by positive selection. Samples were quick frozen in liquid nitrogen and stored at -80°C until DNA isolation. Genomic DNA was isolated using AllPrep DNA/RNA Mini Kit (Qiagen).