In females, one X chromosome undergoes random X chromosome inactivation (XCI), silencing ~85–90% of its genes through epigenetic mechanisms to balance gene dosage between the sexes. Dysregulation of XCI in immune cells has been implicated in the aberrant dosage increase of immune-regulating genes, providing a potential basis for the female bias in autoimmune diseases. However, the molecular mechanisms governing XCI during normal immune cell development remain poorly understood, limiting our insight into how XCI becomes disrupted in autoimmune diseases.
To shed light on the molecular mechanisms governing the stability of XCI, we used RNA FISH to assess the localization of XIST during normal human and mouse B cell development. CRISPR interference in GM12878 B cells was used to understand expression dysregulation with loss of XIST and key XCI regulators (MATR3, SPEN and FIRRE) using allele-specific RNA-seq.
Contrary to the long-held assumption that XIST expression and localisation are stable in adult somatic cells, our preliminary RNA FISH data reveal dynamic changes in XIST localisation throughout human B cell development, beginning at the pro-B cell stage. This finding mirrors observations in murine bone marrow using a distinct probe design. Additionally, RNA-seq expression analysis indicates that XIST levels increase relative to the hematopoietic stem cell stage, where distinct XIST foci are observed. Together, these results suggest that both XIST expression and its localisation to the inactive X (Xi) are critical for maintaining XCI, highlighting XIST tethering to the Xi as a potential therapeutic target.
Secondly, allele-specific expression analysis reveals chromosome-wide reactivation of Xi-linked genes with loss of XIST expression. Notably, MATR3 and FIRRE are downregulated at the pro-B cell stage, identifying them as promising candidates for stabilising XCI maintenance in vivo. Furthermore, the expression patterns resulting from these perturbations may serve as a reference for comparison with autoimmune patient data.