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.     

The oocyte-specific methylated region of the U2afbp-rs/U2af1-rs1 gene is dispensable for its imprinted methylation

Imprinted genes harbor discrete regions which are differentially methylated in gametes; usually the final differential methylation patterns in adults are established during embryogenesis through modifications of the initial methylation patterns in gametes. Previous reports have shown that a 200-bp region termed region II within the CpG island of the mouse imprinted U2afbp-rs gene is methylated in oocytes but not in sperm, suggesting that this region is a center for the propagation of methylated CpGs on the maternal allele and is also a candidate for an imprinting control element. To determine whether region II is required for the imprinted methylation of this gene at the endogenous locus, we generated mice carrying a deletion of this region. We herein show that parental methylation differences still exist in the CpG island on the region II-deleted allele. These findings suggest that region II is dispensable for the imprinted methylation of the U2afbp-rs gene.     

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.     

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.     

Peg5/Neuronatin is an imprinted gene located on sub-distal chromosome 2 in the mouse.

We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5-7 , were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape and behavior in neonates.     

The magnetism responsive gene Ntan1 in mouse brain

We have previously identified Ntan1 as a magnetism response gene by differential display screening in cultured rat hippocampal neurons. Ntan1 mRNA was ubiquitously expressed in all the mouse tissues examined but relatively abundant in brain, retina and testis. Ntan1 mRNA expression was detectable in the embryonic 12-day mouse brain and gradually increased with ageing. In situ hybridization analysis showed high localization of Ntan1 mRNA in pyramidal cell layer of CA region and granular cell layer of dentate gyrus in the hippocampus, and Purkinje and granular cell layers in the cerebellum, respectively. Ntan1 mRNA expression was significantly increased about two-fold 12 h after brief exposure for 15 min to magnetism at 100 mT with a gradual decrease thereafter in cultured mouse hippocampal neurons. When embryonic 12-day-old or newborn mice were successively exposed to magnetic fields at 100 mT for 2 h, four times per day until the postnatal seventh day, Ntan1 mRNA was significantly increased about 1.5-2-fold in the hippocampus in vivo. The mice exposed to magnetic fields under the same condition showed significantly decreased locomotor activity. These results suggest that magnetic exposure affects higher order neural functions through modulation of genes expression.     

Inducible and neuron-specific gene expression in the adult mouse brain with the rtTA2S-M2 system

To achieve inducible and reversible gene expression in the adult mouse brain, we exploited an improved version of the tetracycline-controlled transactivator-based system (rtTA2(S)-M2, rtTA2 hereafter) and combined it with the forebrain-specific CaMKIIalpha promoter. Several independent lines of transgenic mice carrying the CaMKIIalpha promoter-rtTA2 gene were generated and examined for anatomical profile, doxycycline (dox)-dependence, time course, and reversibility of gene expression using several lacZ reporter lines. In two independent rtTA2-expressing lines, dox-treatment in the diet induced lacZ reporter expression in neurons of several forebrain structures including cortex, striatum, hippocampus, amygdala, and olfactory bulb. Gene expression was dose-dependent and was fully reversible. Further, a similar pattern of expression was obtained in three independent reporter lines, indicating the consistency of gene expression. Transgene expression could also be activated in the developing brain (P0) by dox-treatment of gestating females. These new rtTA2-expressing mice allowing inducible and reversible gene expression in the adult or developing forebrain represent useful models for future genetic studies of brain functions.     

Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.