Clcn4-2 genomic structure differs between the X locus in Mus spretus and the autosomal locus in Mus musculus: AT motif enrichment on the X

Abstract

In Mus spretus, the chloride channel 4 gene Clcn4-2 is X-linked and dosage compensated by X up-regulation and X inactivation, while in the closely related mouse species Mus musculus, Clcn4-2 has been translocated to chromosome 7. We sequenced Clcn4-2 in M. spretus and identified the breakpoints of the evolutionary translocation in the Mus lineage. Genetic and epigenetic differences were observed between the 59ends of the autosomal and X-linked loci. Remarkably, Clcn4-2 introns have been truncated on chromosome 7 in M. musculus as compared with the X-linked loci from seven other eutherian mammals. Intron sequences specifically preserved in the X-linked loci were significantly enriched in AT-rich oligomers. Genome-wide analyses showed an overall enrichment in AT motifs unique to the eutherian X (except for genes that escape X inactivation), suggesting a role for these motifs in regulation of the X chromosome.[Supplemental material is available for this article. The sequencing data from this study have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) under accession nos. HM053970 and HM053971.] In mammals, X-linked genes are regulated by special epigenetic mechanisms, because females have two X chromosomes and males only have one, while autosomes are present in two copies. These regulatory mechanisms are (1) X up-regulation in both sexes to balance expression between X-linked and autosomal genes (Gupta et al. 2006;Nguyen and Disteche 2006) and (2) X inactivation in females (Lyon 1961). Ohno (1967) predicted that due to these unique regulatory mechanisms the gene content of the X chromosome would be highly conserved between mammalian species. The chloride channel 4 gene (hereafter called Clcn4 when considering multiple mammalian species) illustrates a rare exception to this conservation, since it is X-linked in most mammals including human, primates, dog, cow, and horse (CLCN4), as well as rat (Clcn4-2), but located on chromosome 7 in the laboratory mouse (Clcn4-2) (derived from a mixture of M. musculus musculus and M. musculus domesticus and thereafter referred to as M. musculus) (Palmer et al. 1995;Rugarli et al. 1995;Flicek et al. 2010).Clcn4-2 is X-linked in the wild-derived mouse M. spretus, suggesting that it was translocated to an autosome in one branch of Mus (musculus) during evolution (Palmer et al. 1995;Rugarli et al. 1995). We have previously used F1 mice from crosses between Mus species to show that Clcn4-2 is subject to X inactivation (Rugarli et al. 1995) and that its expression is doubled on the active X from M. spretus compared with each autosomal allele from M. musculus (Adler et al. 1997). Thus, Clcn4-2 is subject to both types of dosage-compensation mechanisms, X up-regulation, and X inactivation. The different location of this gene in closely related mouse species provides a system to explore whether X-linked genes differ from autosomal genes in terms of DNA sequence and epigenetic modifications. Our hypothesis based on studies in other organisms is that specific sequence moti...

Genes that escape from X inactivation

“…Wang et al (2006) reported that the majority of 3-mers and 5-mers enriched around human genes subject to X inactivation, but not around escape genes, are AT-rich (Wang et al 2006). This is consistent with our recent observations that mouse escape genes contain fewer AT-rich motifs than genes subject to X inactivation (Nguyen et al 2011). The role of AT-rich motifs in the process of silencing by X inactivation is supported by findings that SATB1, a protein that specifically binds AT-rich DNA, both binds to the Xi and causes disruption in the silencing function of Xist if mutated in mouse cells (Agrelo et al 2009).…”

“…Xist cis-spreading along the X is still not well understood, but specific motifs may be involved. Notably, repeat elements such as interspersed repetitive elements (LINE-1) are critical for the formation of a silenced compartment and AT-rich motifs may also be involved (Agrelo et al 2009; Chow et al 2010; Nguyen et al 2011). Xist recruits the polycomb complex PRC2, which mediates the initial histone changes (e.g., methylation of lysine 27 of histone H3) on the inactive X (Plath et al 2003).…”

Dosage Compensation of the Sex Chromosomes

“…At the autosomal locus, the size of Clcn4-2 is dramatically reduced by deletions of large portions of its introns. AT-rich sequence motifs deleted on the autosome but preserved on the X are abundant throughout the entire X (118). Whether these motifs are implicated in either X upregulation and/or X inactivation remains to be investigated.…”

KIBRA: In the brain and beyond

“…While fishes encode only two WWC genes, most other vertebrates including frog, rat and human have all three WWC members (3). Notably, due to a chromosomal translocation event in the evolution of the mouse lineage, Mus musculus expresses only KIBRA/WWC1 and WWC2 but lacks WWC3 (4). Whether there is functional interplay among the WWC proteins is almost completely unknown.…”