Females generally exhibit a stronger immune response than males, which lowers infection risk but raises susceptibility to inflammatory diseases. Emerging evidence suggests that X chromosome inactivation (XCI) in female immune cells may be unstable, potentially contributing to sex differences in immunity. While XCI has been widely studied in embryonic development, little is known about its complexity in adult immune cells. This knowledge gap stems from (i) the former lack of single-cell, allele-specific techniques and (ii) assumptions that XCI is stable in post-embryonic cells.
To investigate this, we used allele-specific single-cell long-read RNA sequencing, along with whole-genome sequencing, B cell receptor sequencing and flow cytometry, to study XCI dynamics during normal B cell development in mouse and human bone marrow, as well as in rare auto-reactive B cells isolated from the peripheral blood of female patients with systemic lupus erythematosus (SLE).
Preliminary data analysis revealed the reactivation of several genes on the inactive X chromosome in normal pro-B cells in bone marrow. We identified a long non-coding RNA and an RNA-binding protein as possible regulators of unstable XCI. To confirm their roles, we are performing CRISPR interference in female cell lines K562 and GM12878, followed by allele-specific single-cell long-read RNA sequencing. In SLE, we found that X-linked genes upregulated in auto-reactive B cells depend on the long non-coding RNA XIST, suggesting dysregulation of XIST-mediated silencing in SLE.
Overall, this work suggests a role for unstable XCI and X-linked gene dosage in female immune cell development. Understanding female-specific epigenetic and transcriptional regulation will improve insights into autoimmune disease mechanisms and sex-based immunity differences.