"The impact of dynamic Xist RNA localization on spatial organization of the inactive X chromosome in female B cells upon cell cycle re-entry"

Isabel Sierra, Son Nguyen, Han-Seul Ryu, Zachary Beethem, Jennifer Phillips-Cremins, Eric Joyce and Montserrat Anguera

Chromatin is organized in a complex hierarchical manner in order to regulate gene expression. The inactive X-chromosome (Xi), unlike the autosomes and the active X, has a unique bipartite structure and is tethered to the nuclear periphery. The Xi is formed during early development by a process called X-Chromosome Inactivation (XCI), with Xist upregulation and retention of Xist RNA transcripts at the Xi. XCI maintenance silences most, but not all, X-linked genes on the Xi, and XCI ‘escape’ genes exhibit cell and tissue-specific expression. In most somatic cells, silencing and structure of the Xi is maintained by persistent association of Xist RNA and heterochromatic marks. However, female lymphocytes have ‘dynamic’ XCI maintenance, where Xist RNA and heterochromatic modifications are absent from the Xi in naïve lymphocytes, but re-localize after in-vitro activation. Quantification of Xi transcription and the nuclear organization of the Xi during ‘dynamic’ XCI maintenance has not been examined. To address this, we used an F1 mouse model of skewed XCI to perform allele-specific RNAseq analyses of gene expression and nuclear organization in splenic B-cells cultured with CpG to induce activation and cell cycle re-entry. We found that the Xi is dosage compensated in naïve lymphocytes despite the cytological absence of Xist RNA at the Xi, and we identified immunity-related genes that specifically escape XCI in naïve versus activated lymphocytes. We used quantitative allele-specific DNA FISH and allele-specific Hi-C to determine how the Xi chromatin architecture is organized in naïve and in-vitro activated B-cells. We found that the global Xi structure does not change with cell cycle re-entry, yet there are significant new DNA interactions and compartments across the Xi in activated B-cells. Our results suggest small-scale changes combined with stable Xi ultrastructure allow for highly precise gene expression changes without compromising dosage compensation during ‘dynamic’ XCI in lymphocytes.