How do some X-linked genes escape silencing by XIST RNA?
It was long believed that the heterochromatic Barr Body visible in the center of the inactive X territory was the manifestation of the silencing of the protein coding genes on the X-chromosome, and that silenced genes were located within this heterochromatic structure. However, it was unknown whether the genes that escaped inactivation were also within the Barr Body but resistant to its effects (model A), or were located outside the Barr Body (model B). We were surprised to find that all genes regardless of their activity were located outside the heterochromatic Barr Body!
Repeat DNA is enriched in the Barr Body and is silenced
If genes are not within the Barr Body, then this then begs the question what is inside. By using the Cot-1 DNA fraction of the genome (all the highly repetitive sequences) as a probe for RNA/DNA FISH, we found that the Barr Body was made up of the repeat fraction of the chromosome, and that this DNA was transcriptionally silent. However, Cot-1 RNA was found transcribed throughout the rest of the euchromatic regions on the nucleus, and across the active X-chromosome territory.
Surprisingly, even the silenced genes are not within the heterochromatic Barr body
Genes (small green dots) are located outside the Barr Body (black), but genes (red) are seen at the inner edge of the XIST RNA territory (green).
Are intergenic repeats (50% of genome) widely expressed on active chromosomes and silenced on the Xi?
Genes located within the outer region of a chromosome territory have been reported for other autosomes in the literature and we confirmed this structure on the active X-chr. We postulate that most active chromosome territories contain at their center a repeat rich core, which is silenced and converted into the heterochromatic Barr Body during X-inactivation.
Bioinformatic analysis implicates the “junk” of the genome in chromosome regulation
We are interested in studying the potential involvement of specific DNA sequence elements in XIST RNA binding and silencing of the chromosome. X;autosome translocations suggest autosomal chromatin has a reduced capacity for inactivation by XIST RNA. In addition, a region on the human X wholly escapes inactivation, suggesting sequence specificity in this process. Analysis of all the 9-mer “words” in the human genome suggest LINE and simple sequence repeats are selectively more enriched across the X chromosome. Surprisingly, a singular enrichment of GATA tandem repeats delineate the 8 Mb region that escapes inactivation, suggesting a new paradigm whereby the Xp segment escapes inactivation due to the presence of elements that exclude heterochromatinization, rather than lack of elements that promote it.
LINE1 repeats may facilitate silencing on the X-chromosome
The GATA repeat may facilitate escape from silencing within the pseudoautosomal region of the X-chromosome
GATA repeats are 11 fold increased on the region that escapes inactivation compared to the rest of the X-chromosome that is largely silenced. This enrichment is also found in other eutherians that contain a pseudoautosomal region on their X-chromosome.
GATA enrichment can be seen by FISH.
Using DNA FISH on chromosomes, the escape region is strikingly labeled with the GATA probe (red).
| || |