XIST RNA and architecture of the inactive X chromosome: implications for the repeat genome

Abstract

XIST RNA paints and induces silencing of one X chromosome in mammalian female cells, providing a powerful model to investigate long-range chromosomal regulation. This chapter focuses on events downstream from the spread of XIST RNA across the interphase chromosome, to consider how this large noncoding RNA interacts with and silences a whole chromosome. Several lines of evidence are summarized that point to the involvement of repeat sequences in different aspects of the X-inactivation process. Although the "repeat genome" comprises close to half of the human genome, the potential for abundant repeats to contribute to genome regulation has been largely overlooked and may be underestimated. X inactivation has the potential to reveal roles of interspersed and other repeats in the genome. For example, evidence indicates that XIST RNA acts at the architectural level of the whole chromosome to induce formation of a silent core enriched for nongenic and repetitive (Cot-1) DNA, which corresponds to the DAPI-dense Barr body. Expression of repeat RNAs may contribute to chromosome remodeling, and evidence suggests that other types of repeat elements may be involved in escape from X inactivation. Despite great progress in decoding the rest of the genome, we suggest that the repeat genome may contain meaningful but complex language that remains to be better studied and understood.

FIGURE 1. XIST RNA paints the inactive X-chromosome (Xi) altering its chromatin composition, structure, and nuclear organization

Note that most images show the Xi in the peripheral heterochromatic compartment of the nucleus in these human somatic cells. A) Histone H4K20 methylation, and B) histone H3K27 methylation are enriched on the Xi. C) DAPI DNA stain reveals the condensed Barr Body (arrow) at the periphery of the nucleus. D) Ubiquitinated H2A K119 is also enriched on the Xi. E) XIST RNA paints the Xi, and repeat RNA (Cot-1 RNA) is present throughout the non-nucleolar nucleoplasm, but absent from the Xi (F: arrow) and other regions of heterochromatin.

FIGURE 2. AURKB chromatin phosphorylation affects the binding of XIST RNA to multiple anchor points which we suggest bridge chromatin and insoluble nuclear structure

A) In normal cells (left) XIST RNA paints the chromosomes territory at interphase and releases from the chromosome early in mitosis. In cells in which AURKB is manipulated (right), use of an inhibitor of PP1 (which releases repression of AURKB) causes XIST RNA to release from the interphase chromosome, whereas RNAi or inhibition of AURKB causes XIST RNA to be retained on metaphase chromosomes. B) Model of XIST RNA interaction with the interphase chromosome proposes that XIST RNA is anchored at multiple points and bridges chromatin with insoluble scaffold (matrix) proteins of the nucleus. To release XIST RNA in interphase all anchor points must be abrogated, but retention of one anchor point may be sufficient to force chromosomal retention of XIST RNA at mitosis. C) Manipulation of XIST RNA binding in live cells provides a strategy to determine which chromatin proteins or modifications most closely parallel XIST behavior. In this analysis, only histone ubiquitination followed the same pattern as XIST RNA under all conditions. Figures adapted from Hall et al., 2009.

FIGURE 3. L1 LINE density versus gene density across all human chromosomes and the XE escape domain

The silenced region of the X-chromosome (XS) has the highest LINE 1 (L1) density compared to each chromosome in the human genome as well as to the escape region of the X (XE). Note that autosomes show significant variations in the density of L1 elements, but this data shows the XS has significantly more than the most enriched autosome (Chr 4) which has similar gene density. Since LINE content is known to correlate with gene density, it was important to show that the difference is not simply explained by gene density. This data also shows that the Y chromosome is more enriched in L1s than autosomes, consistent with the possibility that some of the evolutionary accumulation of LINE elements on the sex chromosomes may have to do with their lower recombination frequency. Figure reproduced from McNeil et al., 2006.

FIGURE 4. The pseudoautosomal region, which fully escapes inactivation on the Xi, exhibits a striking 11 fold enrichment in the GATA repeat sequence

Right) Shows the distribution of GATA repeats (and CTAT) along the X chromosome determined by word count analysis, and a close-up view of the pseudoautosomal and XE region that escapes inactivation (yellow). Importantly, small repeats of (GATA)n were widely dispersed at very many sites throughout this ~10 Mb segment, and were not found in large blocks. Left) DNA FISH using a GATA DNA oligo probe labels the pseudoautosomal region of the X-chromosome strongly, confirming that it is unique in the genome for this striking sequence feature.

FIGURE 5. An unanticipated finding that inactive X-linked genes are predominantly not located within the heterochromatic Barr body (BB)

A) Two theoretical models of gene organization on Xi (left) and the model supported by empirical results (right). Initially, it was presumed that either (Model A) escape from silencing is controlled at the local level and all genes are located within the BB or (Model B) genes escape inactivation by positioning outside XIST RNA and the Barr Body with silenced genes well within the heterochromatic BB. However, the data supports the surprising result that all genes positioned with high probability on the outer border of the XIST RNA territory, outside the Barr Body. B) Examples of gene organization relative to the Xi and Xa DNA chromosome territory (left), Barr Body (middle), and XIST RNA (right). Left shows one gene at a peripheral position relative to the DNA territories of Xi and to a lesser extent Xa. Note: the densely packed interior region of the Xi is evident in contrast to a more extended conformation of the Xa. Middle shows three X-linked genes located just outside the DAPI dense BB. Right shows a 3D still-shot of a video showing four X-linked genes at the outer edge of the XIST RNA paint (from Clemson et al., 2006). C) The linescan on the left, through the Xi of one nucleus, shows relative sizes of the BB (DAPI), the XIST RNA painted region, and the X-chromosome DNA territory (X-paint). The BB comprises 62% of the X-territory and 87% of the XIST RNA paint, while the XIST RNA region covers 80% of the X-territory. The linescan on the right shows the position of an X-linked gene (MIC-2) just outside the BB and at the outer edge of the X DNA territory. Adapted from Clemson et al., 2006.

FIGURE 6. Results and model for the relationship of Cot-1 repeat RNA to active and inactive X chromosomes

A) Cot-1 repeat RNA (green) is present throughout the nucleoplasm of active chromosomes but is essentially absent within the interior core of the Xi, even though Cot-1 repeat DNA (red) is detectable within DAPI dense Barr Body (white). Thus the Xi has a core of silent repeat elements. The linescans through the Xi in a normal female fibroblast quantifies the sharp distinction between Cot-1 DNA enrichment and lack of Cot-1 RNA. B) Model for the loosely peripheral organization of genes on Xa, which on Xi becomes more striking and surrounds a compacted and silent inner core. We suggest that Cot-1 RNA may be expressed throughout the central regions of euchromatic interphase chromosome territories even though evidence indicates the central regions have fewer genes.