Comparative sequence analysis of the X-inactivation center region in mouse, human, and bovine

Abstract

We have sequenced to high levels of accuracy 714-kb and 233-kb regions of the mouse and bovine X-inactivation centers (Xic), respectively, centered on the Xist gene. This has provided the basis for a fully annotated comparative analysis of the mouse Xic with the 2.3-Mb orthologous region in human and has allowed a three-way species comparison of the core central region, including the Xist gene. These comparisons have revealed conserved genes, both coding and noncoding, conserved CpG islands and, more surprisingly, conserved pseudogenes. The distribution of repeated elements, especially LINE repeats, in the mouse Xic region when compared to the rest of the genome does not support the hypothesis of a role for these repeat elements in the spreading of X inactivation. Interestingly, an asymmetric distribution of LINE elements on the two DNA strands was observed in the three species, not only within introns but also in intergenic regions. This feature is suggestive of important transcriptional activity within these intergenic regions. In silico prediction followed by experimental analysis has allowed four new genes, Cnbp2, Ftx, Jpx, and Ppnx, to be identified and novel, widespread, complex, and apparently noncoding transcriptional activity to be characterized in a region 5' of Xist that was recently shown to attract histone modification early after the onset of X inactivation.

Figure 1

Comparative map of Xic region in mouse and human.

Figure 2

Comparison of mouse and human genomic sequences in Xic region. Genomic sequences were first analyzed with RepeatMasker to identify and mask repeated elements and then aligned with SIM. Conserved blocks with a similarity score >30 are displayed (see Methods). An electronic version of this figure is available at http://pbil.univ-lyon1.fr/datasets/Xic2002/data.html. Because the alignment is very long and because of an inversion of the Xpct gene, it was not possible to display the whole human–mouse comparison in a single continuous line. The alignment, therefore, is displayed in six overlapping fragments. The overlaps are designed to allow the continuity of the fragments to be better appreciated.

Figure 3

Three-way comparison of mouse, human, and bovine genomic sequences around Xist. See Fig. 2 legend. An electronic version of this figure is available at http://pbil.univ-lyon1.fr/datasets/Xic2002/data.html.

Figure 4

Cloning and characterization of Ppnx and Jpx genes. (A) Jpx gene. Schematic diagram showing the three exons (light grey boxes) of the murine Jpx gene, at positions 343745–343379 (exon 1), 340888–340796 (exon 2), 334465–334364 (exon 3), as defined by the three ESTs represented as dark grey boxes above the map. ESTs are identified by their GenBank accession number. The presence of an alternative splice at position 343626 nt in exon 1 generates either a 315- or 562-nt transcript. The presence of a poly(A) tail in EST BE994494 identifies the 3′ end of the gene. The first exon may correspond to the 5′ end of the gene, as a CpG island (diagonally hatched box) lies in the vicinity. The two last exons are LTR/MaLR-repeat elements (white boxes). (B) Northern analysis using a PCR probe corresponding to exon 1 of the Jpx gene after 5 d exposure. (C) Ppnx gene. Schematic diagram illustrating the six exons of the murine Ppnx gene. All exons and introns are represented to scale with the exception of introns 2 and 5 that are very large (21707 bp and >45389 bp, respectively). Each exon (grey) is identified by its coordinates: exon 1, 591535–591619; exon 2, 592146–592314; exon 3, 614021–614268; exon 4, 615349–615488; exon 5, 618116–>619619 (with three alternative splice donor sites at position 618262, 618719, and 618758), and exon 6, 665008–665306. The black boxes represent two pseudogenes predicted by GENSCAN; the small black arrow shows an antisense transcription detected in intron 2. The single-exon NapIl2 gene is shown diagonally hatched. Three independent clones A, B, and C obtained after screening a testis cDNA library are shown below the genomic structure. Clone A does not contain either a poly(A) consensus signal or a poly(A) track and has an in-frame stop codon present in its most 3′ part. The end of the 3′ UTR track is likely 300 bp downstream of the stop codon (unpubl.). Clones B and C possess a stop codon in exon 6 followed by a 3′ UTR of 177 bp, containing both a poly(A) signal and a poly(A) tail. Three of the RT-PCR products suggesting the existence of alternative splice donor sites in exon 5 are also represented (1, 2, and 3). (D) Northern analysis of Ppnx expression. A mouse multiple tissue Northern blot was hybridized with a probe corresponding to the third exon of Ppnx. The Ppnx hybridization signals were obtained after 8 d exposure.

Figure 5

Comparison of Xist gene intron–exon structure in mouse, bovine, and human. Introns and the first exon are not drawn to scale. Conserved regions corresponding to known exons (in at least one species) are indicated. Consensus splice signals that align with boundaries of known exons are in capitals. In mouse and human, 8 exons have been identified (Brown et al. 1992; Sheardown et al. 1997). Note, however, that some exons in mouse have no counterpart in the human and vice versa. To avoid ambiguities in exon numbering, we used the prefix “h” to identify human exons, and “m” to identify mouse exons. Mouse exons m1, m3, m4, m6, m7, and m8 are conserved and correspond to human exons h1, h3, h4, h5, h6, and h8, respectively. In both species, we identified an alternative donor splice site in exon m7–h6, although it is not located exactly at the same position in human and mouse. With the exception of this splice site, splice signals (donor and acceptor) of these six exons, as well as the polyadenylation signal in the last exon, are perfectly conserved in mouse, man, and bovine (which suggests that they also correspond to true exons in the latter species). Human exon h2 is located in a region that is not conserved in mouse or bovine. Mouse exon m2 is conserved in the three species, with correct splice signals, except in human where the splice acceptor is mutated (AT instead of AG), which suggests that this exon is no longer functional in human. Mouse exon m5 is conserved in the three species with correct splice signals and might correspond to an unidentified alternative exon in humans. Human exon h7 is located in a region that is conserved in the three species, but consensus splice signals are not found in mouse and bovine. a, mouse Xist cDNA (GenBank accession number L04961; Brockdorff et al. 1992); b, mouse ESTs (BE626785, BE632200, and R74734); c, mouse exon m7a reported by Hong et al. (1999); d, human XIST cDNA (M97168; Brown et al. 1992); e, human exon h6a reported by Hong et al. (2000); f, human ESTs (AV699347, AV700119, and AV700677). Exon positions in mouse (bp): m1, 354096…363614; m2, 366392…366482; m3, 366632…366763; m4, 367512…367722; m5, 367867…368013; m6, 368341…368495; m7a, 369277…376940; m7b, 369277…371600; and m8, 376603…376940. In human, exon positions are indicated relative to the XIST genomic sequence (U80460): h1, 18317…29688; h2, 33567…33630; h3, 37696…37832; h4, 39796…40004; h5, 41840…42003; h6a, 43087…50396; h6b, 43087…44956; h7, 48855…49000; and h8, 50042…50396.