Exploration of the NBDY Gene and the Significance of XIST for X Inactivation

Select silencing of gene expression in one X chromosome in female mammals provides a means to overcome X-linked gene expression imbalances with males. Central to establishing gene silencing on the inactivated X chromosome are the actions of the long non-coding RNA XIST that triggers the repackaging of the chosen X into facultative heterochromatin. Additionally, microscopic and high throughput sequence-based studies have established that the mammalian inactive X chromosome (Xi) adopts an alternate conformation relative to the active X chromosome. Organized into several multi-megabase chromatin loops called superloops, the human Xi bears anchors at the base of these loops that are composed of large tandem repeat DNA, including DXZ4, Functional Intergenic Repeating RNA Element (FIRRE), and Inactive-X CTCF-binding Contact Element (ICCE). Furthermore, ICCE is nestled within the intronic region of a micropetide expressing gene established as NBDY, a novel protein that has been implicated to interact with components of nonsense mediated decay (NMD). Little else is known about the properties or origins of ICCE or NBDY and the role that XIST plays in maintaining chromatin at the human inactive X chromosome (Xi) has remained elusive. Here, we used bioinformatical tools to elucidate the nature of the ICCE repeat and observe the localization of NDBY with fluorescent microscopy under lentivirally induced overexpression conditions. With genome engineering, we were able to delete the promotor of XIST to knockout expression in non-cancerous diploid human somatic cells. We find that primary DNA sequence conservation of ICCE is only retained in higher primates, but that ICCE orthologs exist beyond the primate lineage. Like DXZ4, what is conserved is organization of the underlying DNA into a large tandem repeat, the physical location within the NBDY locus and short DNA sequences corresponding to specific CTCF and Yin Yang 1 binding motifs that correlate with female-specific DNA hypomethylation. Unlike DXZ4, ICCE is not common to all eutherian mammals, but analysis of certain ICCE CTCF motifs reveal striking similarity with the DXZ4 motif. Further examination of NBDY itself led to successful overexpression of the gene in RPE1 cells which was visualized as a diffuse signal in the nucleus and as pinprick cytoplasmic foci through fluorescent microscopy. Additionally lentiviral induction time trials revealed a shift in NBDY localization from the cytoplasmic to nuclear at the six-hour mark, with a strong signal detected overlapping with promyelocytic leukemia protein (PML) bodies. In XIST knockout cells, some heterochromatin features exhibit limited change at the Xi, but two of those assessed showed significant reductions including histone H2A monoubiquitylation at lysine 119 and histone H3 trimethylation at lysine 27, both of which are covalent histone modifications catalyzed by the polycomb repressive complexes 1 and 2 respectively. Coupled with these reductions, we observed an occasional gain of euchromatin signatures on Xp, but despite these signs of chromatin instability, we did not observe appreciable changes in the reactivation of genes from the Xi. Collectively, these data reveal several novel insights about the NBDY gene and ICCE; the findings imply a further role for NBDY in PML body association and senescence and support an evolutionary relationship between DXZ4 and ICCE. Additionally, the data compiled on the absence of XIST are consistent with maintenance of dosage compensation at the Xi involving multiple redundant layers of gene silencing.