XIST RNA and X inactivation

XIST RNA coats the inactive X-chromosome and induces epigenetic silencing.

The inactive chromosome exhibits numerous epigenetic hallmarks. For example, methylation of histones H4K20 and H3K27, H2AK119 ubiquitination and condensation into a compact structure called the Barr body that stains densely with the DNA stain DAPI.

Selected references:

XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol. 1996 Feb;132(3):259-75.

Hall LL, Lawrence JB. The cell biology of a novel chromosomal RNA: chromosome painting by XIST/Xist RNA initiates a remodeling cascade. Semin Cell Dev Biol. 2003 Dec;14(6):369-78.

 XIST lncRNA condenses cytoarchitecture before widespread gene silencing

XIST modifies cytoarchitecture before widespread gene silencing. In just 2–4 h, barely visible transcripts populate the large “sparse zone” surrounding the smaller “dense zone”; importantly, density zones exhibit different chromatin impacts. Sparse transcripts immediately trigger immunofluorescence for H2AK119ub and CIZ1, a matrix protein. H3K27me3 appears hours later in the dense zone, which enlarges with chromosome condensation. Genes examined are silenced after compaction of the RNA/DNA territory. Insights into this come from the findings that the A-repeat alone can silence genes and rapidly, but only where dense RNA supports sustained histone deacetylation. We propose that sparse XIST RNA quickly impacts architectural elements to condense the largely non-coding chromosome, coalescing RNA density that facilitates an unstable, A-repeat-dependent step required for gene silencing.

Reference:

Early chromosome condensation by XIST builds A-repeat RNA density that facilitates gene silencing. Cell Rep. 2023 Jul 25;42(7):112686. 

How the Barr Body is organized

Genes are outside the Barr body.

Left image: genes (red) are seen at the inner edge of the XIST RNA territory (green).

Right image: A pool of 3 X-linked genes (small green dots) are located outside the Barr body (black).

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.

 Select references:

XIST RNA and architecture of the inactive X chromosome: implications for the repeat genome. Cold Spring Harb Symp Quant Biol. 2010;75:345-56.

The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences. Proc Natl Acad Sci U S A. 2006 May 16;103(20):7688-93.

How XIST binds to chromatin

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. 

AURKB mediated chromatin modifications regulate XIST RNA binding.

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.

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.

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. 

XIST binding to chromatin is complex.

The protein SAF-A has been suggested to connect XIST to DNA through its RNA- and DNA-binding domains. We tested the role of SAF-A in localizing human XIST RNA in different cell types and found that it was generally not essential to maintain XIST RNA localization in normal cells such as Tig-1 fibroblasts and kidney cells whereas loss of SAF-A resulted in more mislocalization in transformed cells (HEK293, Neuro2A).

Our results suggest that SAF-A is one of multiple redundant anchors in primary somatic cells and that other anchors are often lost or compromised in many transformed cells. We also found that human SAF-A’s putative RNA-binding (RGG) domain was not required to maintain localized XIST RNA, further suggesting that XIST RNA is not simply tethered to chromatin by a single-molecule SAF-A “bridge.”

Select references:

AURKB-mediated effects on chromatin regulate binding versus release of XIST RNA to the inactive chromosome. J Cell Biol. 2009 Aug 24;186(4):491-507.

SAF-A Requirement in Anchoring XIST RNA to Chromatin Varies in Transformed and Primary Cells. Dev Cell. 2016 Oct 10;39(1):9-10.

XIST RNA: a window into the broader role of RNA in nuclear chromosome architecture. Philos Trans R Soc Lond B Biol Sci. 2017 Nov 5;372(1733)