Mouse U2af1-rs1 is a neomorphic imprinted gene.

The mouse U2af1-rs1 gene is an endogenous imprinted gene on the proximal region of chromosome 11. This gene is transcribed exclusively from the unmethylated paternal allele, while the methylated maternal allele is silent. An analysis of genome structure of this gene revealed that the whole gene is located in an intron of the Murr1 gene. Although none of the three human U2af1-related genes have been mapped to chromosome 2, the human homolog of Murr1 is assigned to chromosome 2. The mouse Murr1 gene is transcribed biallelically, and therefore it is not imprinted in neonatal mice. Allele-specific methylation is limited to a region around U2af1-rs1 in an intron of Murr1. These results suggest that in chromosomal homology and genomic imprinting, the U2af1-rs1 gene is distinct from the genome region surrounding it. We have proposed the neomorphic origin of the U2af1-rs1 gene by retrotransposition and the particular mechanism of genomic imprinting of ectopic genes.     

Comparative analyses of genomic imprinting and CpG island-methylation in mouse Murr1 and human MURR1 loci revealed a putative imprinting control region in mice

Human MURR1 is an orthologue of mouse Murr1 gene, which was previously reported to be imprinted only in adult brain with a maternal allele-predominant expression and to contain another imprinted gene, U2af1-rs1, in the first intron. Human MURR1 was found not to harbor the U2af1-rs1 orthologue and to be expressed biallelically in tissues, including adult brain. Three genes identified around Murr1 and their orthologues around MURR1 were expressed biallelically. These findings suggest that the mouse imprinting locus is limited to a small region and the introduction of U2af1-rs1 in mouse causes the imprinting of this locus. The CpG island (CGI) at U2af1-rs1 with maternal methylation was the only differentially methylated region among CGIs found in these loci. Detailed methylation analyses of the U2af1-rs1 CGI in germ cells led to identification of a region with oocyte-specific methylation. These results suggest that this region is the imprinting control region of the Murr1/U2af1-rs1 locus in mouse.     

DNA Methylation Is Linked to Deacetylation of Histone H3, but Not H4, on the Imprinted Genes Snrpn and U2af1-rs1

The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpn is explored by chromatin immunoprecipitation (ChIP) and resolution of parental alleles using single-strand conformational polymorphisms. The U2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5' DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5' untranslated region (5'-UTR) and 3'-UTR of U2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins.     

A case-by-case evolutionary analysis of four imprinted retrogenes

Retroposition is a widespread phenomenon resulting in the generation of new genes that are initially related to a parent gene via very high coding sequence similarity. We examine the evolutionary fate of four retrogenes generated by such an event; mouse Inpp5f_v2, Mcts2, Nap1l5, and U2af1-rs1. These genes are all subject to the epigenetic phenomenon of parental imprinting. We first provide new data on the age of these retrogene insertions. Using codon-based models of sequence evolution, we show these retrogenes have diverse evolutionary trajectories, including divergence from the parent coding sequence under positive selection pressure, purifying selection pressure maintaining parent-retrogene similarity, and neutral evolution. Examination of the expression pattern of retrogenes shows an atypical, broad pattern across multiple tissues. Protein 3D structure modeling reveals that a positively selected residue in U2af1-rs1, not shared by its parent, may influence protein conformation. Our case-by-case analysis of the evolution of four imprinted retrogenes reveals that this interesting class of imprinted genes, while similar in regulation and sequence characteristics, follow very varied evolutionary paths.     

Identification of Murr1 as a regulator of the human delta epithelial sodium channel

The human delta epithelial sodium channel (deltaENaC) subunit is related to the alpha-, beta-, and gammaENaC subunits that control salt homeostasis. DeltaENaC forms an amiloride-sensitive Na+ channel with the beta and gamma subunits. However, the in vivo function of deltaENaC is not known. To gain insight into the function of deltaENaC, a yeast two-hybrid screen of a human brain cDNA library was carried out using the C- and N-terminal domains of deltaENaC. A novel deltaENaC-interacting protein called Murr1 (mouse U2af1-rs1 region) was isolated in the C-terminal domain screen. Murr1 is a 21-kDa protein mutated in Bedlington terriers suffering from copper toxicosis. The interaction of Murr1 and deltaENaC was confirmed by glutathione S-transferase pulldown assay and coimmunoprecipitation. To test the functional significance of the interaction, Murr1 was coexpressed with deltabetagammaENaC in Xenopus oocytes. Murr1 inhibited amiloride-sensitive sodium current in a dose-dependent manner. In addition, deletion of the last 59 amino acids of deltaENaC abolished the inhibition. Murr1 also bound to the beta- and gammaENaC subunits and inhibited alphabetagammaENaC sodium current. Therefore, these results suggest that Murr1 is a novel regulator of ENaC.     

Inhibition of histone deacetylases alters allelic chromatin conformation at the imprinted U2af1-rs1 locus in mouse embryonic stem cells

Most loci that are regulated by genomic imprinting have differentially methylated regions (DMRs). Previously, we showed that the DMRs of the mouse Snrpn and U2af1-rs1 genes have paternal allele-specific patterns of acetylation on histones H3 and H4. To investigate the maintenance of acetylation at these DMRs, we performed chromatin immunoprecipitation on trichostatin-A (TSA)-treated and control cells. In embryonic stem (ES) cells and fibroblasts, brief (6-h) TSA treatment induces global hyperacetylation of H3 and H4. In ES cells only, TSA led to a selective increase in maternal acetylation at U2af1-rs1, at lysine 5 of H4 and at lysine 14 of H3. TSA treatment of ES cells did not affect DNA methylation or expression of U2af1-rs1, but was sufficient to increase DNase I sensitivity along the maternal allele to a level comparable with that of the paternal allele. In fibroblasts, TSA did not alter U2af1-rs1 acetylation, and the parental alleles retained their differential DNase I sensitivity. At Snrpn, no changes in acetylation were observed in the TSA-treated cells. Our data suggest that the mechanisms regulating histone acetylation at DMRs are locus and developmental stage-specific and are distinct from those effecting global levels of acetylation. Furthermore, it seems that the allelic U2af1-rs1 acetylation determines DNase I sensitivity/chromatin conformation.