Imprinted methylation and its effect on expression of the mouse Zfp127 gene.

The Zfp127 gene is located on mouse chromosome 7 in an imprinted region that is homologous to the 2-Mb Prader-Willi and Angelman Syndromes region on human chromosome 15q11-q13. Here, we show that the gene is differentially methylated, the maternal allele being methylated and the paternal allele being unmethylated. This maternal methylation is established promptly after fertilization prior to syngamy. We also provide data that demonstrate the significance of methylation in the paternal expression of the gene. The expression of the Zfp127 gene in methyltransferase-deficient mice is significantly higher, suggesting that the gene is biallelically expressed in these mice. The data presented here will help to understand the mechanism by which the monoallelic expression of the entire 2-Mb Prader-Willi and Angelman Syndrome region is regulated.     

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.     

Parental origin of germ-line and somatic mutations in the retinoblastoma gene

Segregation analysis of polymorphic sites within the retinoblastoma (RB) gene and on chromosome 13, as well as the parental origin of the lost allele in the tumor, were analyzed in 24 families with RB patients. Four mutant alleles transmitted through the germ-line and seven de novo germ-line mutant alleles were identified in 11 patients with hereditary RB. Segregation analysis within the RB gene and on chromosome 13 was useful for DNA diagnosis of susceptibility to RB in relatives of hereditary patients, even if mutations were not identified. All seven de novo germ-line mutant alleles were paternally derived. The bias toward the paternal allele for de novo germ-line mutations of the RB gene was statistically significant. Seven paternal alleles and six maternal alleles were lost in 13 non-hereditary RB tumors with no bias in the parental origin of the somatic allele loss. These results suggest that the physical environment or a deficiency in DNA repair during spermatogenesis may be associated with significant risk factors for de novo germ-line mutations.     

The 5' flank of mouse H19 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication

BACKGROUND: During mouse prenatal development, the neighbouring insulin-like growth factor II (Igf2) and H19 loci are expressed monoallelically from the paternal and maternal alleles, respectively. Identical spatiotemporal expression patterns and enhancer deletion experiments show that the Igf2 and H19 genes share a common set of enhancers. Deletion of a differentially methylated region in the 5' flank of the H19 gene partially relieves the repression of the maternal Igf2 and paternal H19 alleles in the soma. The mechanisms underlying the function of the 5' flank of the H19 gene are, however, unknown. RESULTS: Chromatin analysis showed that the 5' flank of the mouse H19 gene contains maternal-specific, multiple nuclease hypersensitive sites that map to linker regions between positioned nucleosomes. These features could be recapitulated in an episomal-based H19 minigene, which was propagated in human somatic cells. Although the 5' flank of the H19 promoter has no intrinsic silencer activity under these conditions, it unidirectionally extinguished promoter-enhancer communications in a position-dependent manner, without directly affecting the enhancer function. CONCLUSIONS: The unmethylated 5' flank of the H19 gene adopts an unusual and maternal-specific chromatin conformation in somatic cells and regulates enhancer-promoter communications, thereby providing an explanation for its role in manifesting the repressed state of the maternally inherited Igf2 allele.     

Refined localization of a gene for pediatric gastroesophageal reflux makes HTR2A an unlikely candidate gene

Our objective in this study was to determine whether mutations in the gene for the 5-hydroxytryptamine receptor 2A (HTR2A) cause the autosomal dominant form of severe pediatric gastroesophageal reflux (GER), which we had previously mapped to a 21-cM region at chromosome 13q14. Direct sequencing of the HTR2A gene was carried out on DNA from affected and unaffected members of families with severe pediatric GER displaying genetic linkage to the HTR2A locus. In addition, we performed high-resolution linkage mapping within the GER gene region using additional polymorphic markers closely linked to HTR2A. Several previously reported polymorphisms in the HTR2A gene were identified in three families affected with GER. In addition, we identified a novel polymorphism at nucleotide -1273 in the HTR2A promoter. No mutant allele cosegregated exclusively with the GER phenotype in any family. Linkage analysis using additional polymorphic markers narrowed the region of the GER gene to a 9 cM interval between markers D13S263 and CAGR1, formally excluding HTR2A as a candidate gene. In conclusion, sequence analysis of HTR2A and linkage analysis argue against mutations in HTR2A being a cause of severe pediatric GER.     

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.     

Evidence consistent with human L1 retrotransposition in maternal meiosis I

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.     

Exclusion of the retinoblastoma gene and chromosome 13q as the site of a primary lesion for human breast cancer.

Chromosome 13q has been suggested as the site of a gene predisposing to human breast cancer, because loss of heterozygosity of alleles on this chromosome has been observed in some ductal breast tumors and because two breast cancer lines are altered at the retinoblastoma gene (RB1) at 13q14. To test this possibility, linkage of breast cancer susceptibility to 14 loci on chromosome 13q loci was assessed in extended families in which breast cancer is apparently inherited as an autosomal dominant trait. RB1 was excluded as the site of a breast cancer gene by a lod score of Z = -7.60 at close linkage for 13 families. Multipoint analysis yielded negative lod scores throughout the region between 13q12 and 13q34; over most of this distance, Z less than -2.0. Therefore, chromosome 13q appears to be excluded as the site of primary lesion for breast cancer in these families. In addition, comparison of tumor versus normal tissues of nonfamilial breast cancer patients revealed an alteration at the 5' end of RB1 in a mucoid carcinoma but no alterations of RB1 in five informative ductal adenocarcinomas. Linkage data and comparisons of tumor and normal tissues suggest that changes in the RBI locus either are secondary alterations associated with progression of some tumors or occur by chance.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.     

Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia.

A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.