In order to dosage compensate X-linked genes between males and females, one of the two X-chromosomes in female cells is almost completely silenced. XIST RNA is a large non-coding RNA (15kb mature transcript) that is transcribed exclusively from the inactive X-chromosome and “paints” the full length of the chromosome in cis (it never leaves the chromosome it is transcribed from and is not seen in the cytoplasm). XIST RNA then initiates a cascade of events that leads to the epigenetic silencing of one of the two X-chromosomes in most of the cells in female mammalian embryos.
|XIST RNA paints the X-chromosome territory in interphase human female cells|
|The inactive chromosome exhibits numerous epigenetic hallmarks of silenced heterochromatin.|
|The heterochromatic marks on the inactive chromosome delineate the heterochromatic Barr Body seen by DNA stain (DAPI)|
Manipulating XIST RNA’s interaction with the chromosome: Top images show XIST RNA localization in normal interphase and its natural release at mitosis. Bottom two images show induced interphase release or induced metaphase retention of XIST RNA.
Our previous studies have shown that the XIST RNA does not bind exclusively via DNA/RNA binding. If DNA is removed from a nucleus in situ (by DNAse), XIST RNA remains tightly bound to the remaining protein component of the cell. XIST RNA naturally falls off the inactive chromosome during mitosis, suggesting mitotic protein modifications may play a role. Here we find that manipulating the activity of the mitotic kinase Aurora B (AURKB) in interphase or mitosis can affect the binding of XIST to the chromosome. This suggests that XIST RNA binding to the chromosome is affected by phosphorylation of an AURKB substrate.
Only inhibition of protein phosphatase 1 (PP1) inappropriately activates AURKB in interphase and affects XIST binding.
|A new approach to study XIST RNA binding in vivo, in relationship to chromatin modifications
Strategy to narrow the candidate factors involved in XIST RNA binding.The ability to manipulate XIST RNA behavior in vivo provides a strategy to determine which chromosome/ chromatin associated proteins most closely mirror the binding pattern of XIST RNA. Of the ten chromosome-associated proteins examined here, only four show the requisite fluctuations in mitosis when XIST RNA is released (H3S10 & S28ph, HP1 and ubiquitin). Of these four, only ubiquitin was differentially affected in AURKB RNAi treated cells coincident with XIST RNA (asterisks indicate completely concordant patterns).
Model for multiple XIST RNA anchor points impacting its localization to chromatin.Interphase: In normal interphase cells, XIST RNA is tightly bound to the chromosome due to protein interactions at multiple anchor points. The loss of a single anchor point may not be sufficient to release XIST RNA from the chromosome. Mitosis: The normal loss of all anchor points at prophase in human cells releases XIST RNA from the chromosome. Inducing the retention of a single anchor point is sufficient to significantly retain XIST RNA on the chromosome. This study shows that at least one of the anchor points involved in human XIST RNA binding is regulated by Aurora B kinase.