The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences

Abstract

We investigated whether genes escape X chromosome inactivation by positioning outside of the territory defined by XIST RNA. Results reveal an unanticipated higher order organization of genes and noncoding sequences. All 15 X-linked genes, regardless of activity, position on the border of the XIST RNA territory, which resides outside of the DAPI-dense Barr body. Although more strictly delineated on the inactive X chromosome (Xi), all genes localized predominantly to the outer rim of the Xi and active X chromosome. This outer rim is decorated only by X chromosome DNA paints and is excluded from both the XIST RNA and dense DAPI staining. The only DNA found well within the Barr body and XIST RNA territory was centromeric and Cot-1 DNA; hence, the core of the X chromosome essentially excludes genes and is composed primarily of noncoding repeat-rich DNA. Moreover, we show that this core of repetitive sequences is expressed throughout the nucleus yet is silenced throughout Xi, providing direct evidence for chromosome-wide regulation of "junk" DNA transcription. Collective results suggest that the Barr body, long presumed to be the physical manifestation of silenced genes, is in fact composed of a core of silenced noncoding DNA. Instead of acting at a local gene level, XIST RNA appears to interact with and silence core architectural elements to effectively condense and shut down the Xi.

Fig. 1.

Examples of gene positions relative to XIST RNA. Gene signals were scored as occurring either IN, OUT, or on the BORDER of the RNA domain. (A) IN: X centromere is green, XIST RNA is red, and overlap appears yellow. (B) OUT: NFIB (red) was found outside of the XIST RNA (green) most of the time in cells with an X;9 translocation. Most of the genes fell into the BORDER category: (C) PGK-1 (red) is found mostly at the periphery of the XIST RNA domain (green). SLC16A2 (green) (D) and TIMP1 (red) (E) show the typical gene position bordering the XIST RNA territory. (F) The XIST gene identified with an intron probe (red) borders the mature XIST RNA (green) and the Barr body (black) (Inset). We subdivided the IN category further to include: (G) IN HOLE: SLC16A2 (green), like most genes, although rarely found inside of the XIST domain (red), when it was inside, it was often in a hole of the XIST signal (Inset). (H) GATA, a marker for the psuedoautosomal region (red), was found to primarily border the XIST RNA (green). (I) Three X-linked genes (green) simultaneously detected relative to the Barr body (black) show that genes are not usually embedded within the Barr body.

Fig. 2.

Genes border XIST RNA at interphase. Genomic probes for genes, α-satellite, and subtelomeric sequences were cohybridized with XIST RNA in normal diploid fibroblasts, WI38 (CCL 75) and TIG-1 (AG06173). The signals were scored relative to XIST RNA (see Fig. 1 A–C for examples of each category). Genes that escape X inactivation are denoted by an asterisk; TIMP1 escapes X inactivation weakly and is denoted by an ∗. (A) Clearly, all Xi-linked genes examined, regardless of activity and position on the metaphase chromosome, maintain a bordering relationship with XIST RNA the majority of the time, whereas the α-satellite sequences were found mostly inside of the XIST RNA domain. (B) By using a different protocol to preserve the 3D architecture of the nucleus (see Materials and Methods), the IN category was subdivided to include IN (Hole) (see Fig. 1G for an example). The genes were scored as touching the border of XIST RNA the majority of the time, whereas the α-satellite sequences were found mostly inside of the XIST RNA domain.

Fig. 3.

Genes are on the periphery of the X chromosome at interphase. (A) Although the genes generally bordered the X chromosome territory, they were more often found IN or IN HOLE than when scored against XIST RNA. Additionally, the genes were more often found inside of Xa than Xi. (B and C) Representative example of gene vs. X paint. (B) SLC16A2 (red) is inside the outer edge of the Xi territory [(Lower Inset) shows the Barr body by DAPI staining] but is found IN HOLE in the Xa Paint (Upper Inset) (see arrow). (C) The Xa is often more dispersed with irregular boundaries compared with Xi. The ZFX gene (red) is found IN HOLE (Inset) (see arrow) of DNA density at Xa, and just inside the Xi. (D–G) Pooled X probes show the characteristic positioning at the edge of the X territory. ZFX, MIC2, SL16A2, and HPRT (all in green) were pooled together and hybridized with X paint (red), examples of Xa are in D and E, and examples of Xi are in F and G.

Fig. 4.

The Barr body is composed of silent repetitive elements. (A) Cot-1 DNA (red) and Cot-1 RNA (green) were simultaneously detected in normal diploid fibroblasts (TIG-1). Cot-1 DNA (C) is found throughout the nucleoplasm but is more intense over the DAPI-stained Barr body (B). (D) Cot-1 RNA is also found throughout the nucleoplasm but is not transcribed from the Barr body. (E) A line scan through the Xi shows that the pixel intensity and location of the DAPI and Cot-1 DNA signal overlap, whereas the Cot-1 RNA signal is depleted throughout the Barr body. (F) Cot-1 DNA (red) and Cot-1 RNA (green) is detected inside the Barr body. (H) Cot-1 DNA is more intense over the DAPI stained Barr body [It is not clear exactly why the interphase Xi is stained differentially with DAPI. Some evidence suggests the intense staining of the Xi DNA represents differences in folding rather than just condensation (24) (G), whereas the Cot-1 RNA is depleted in this region (I). (J) A line scan through the Barr body shows that the Cot-1 DNA signal is more intense over the Barr body. (K–M) X paint (green) (L) vs. Cot-1 RNA (M). Although the Barr body identified by DAPI (K) (arrowhead) and chromosome painting (L) (arrowhead) is devoid of Cot-1 transcription (M) (arrowhead), Xa (L) (arrow) is actively transcribing Cot-1 RNA (M) (arrow). (N) Ongoing RNA Pol II-mediated transcription is shown using the H5 antibody against the elongating form of the RNA Pol II enzyme (red). The DAPI DNA fluorescence (P) reveals that the heterochromatic Barr body is deficient in RNA Pol II activity (O).

Fig. 5.

Models of higher level organization of the inactive X chromosome. Previously, we proposed two alternate views: (A) genes that escape inactivation (green) are interspersed rather than cytologically separate from the genes that are subject to inactivation (red); or (B) genes that escape X inactivation would be organized at the chromosome periphery, outside of the Barr body. Our work here supports a different model in which genes, regardless of activity, are found at the periphery at the chromosome territory (C). The genes are found outside of the XIST RNA (yellow), which extends beyond the Barr body, the visible manifestation of the heterochromatin.