Characterization of novel parent-specific epigenetic modifications upstream of the imprinted mouse H19 gene.
Genomic imprinting results in parent-specific monoallelic expression of a small number of genes in mammals. The identity of imprints is unknown, but much evidence points to a role for DNA methylation. The maternal alleles of the imprinted H19 gene are active and hypomethylated; the paternal alleles are inactive and hypermethylated. Roles for other epigenetic modifications are suggested by allele-specific differences in nuclease hypersensitivity at particular sites. To further analyze the possible epigenetic mechanisms determining monoallelic expression of H19, we have conducted in vivo dimethylsulfate and DNase I footprinting of regions upstream of the coding sequence in parthenogenetic and androgenetic embryonic stem cells. These cells carry only maternally and paternally derived alleles, respectively. We observed the presence of maternal-allele-specific dimethylsulfate and DNase I footprints at the promoter indicative of protein-DNA interactions at a CCAAT box and at binding sites for transcription factors Sp1 and AP-2. Also, at the boundary of a region further upstream for which existent differential methylation has been suggested to constitute an imprint, we observed a number of strand-specific dimethylsulfate reactivity differences specific to the maternal allele, along with an unusual chromatin structure in that both strands of maternally derived DNA were strongly hypersensitive to DNase I cutting over a distance of 100 nucleotides. We therefore reveal the existence of novel parent-specific epigenetic modifications, which in addition to DNA methylation, could constitute imprints or maintain monoallelic expression of H19.
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
P E
PE
Szabó
G P
GP
Pfeifer
J R
JR
Mann
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000737:chemistry; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D018392:Genomic Imprinting / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D011033:Podophyllin / Q000031:analogs & derivatives / Q000235:genetics; D011034:Podophyllotoxin / Q000031:analogs & derivatives; D011401:Promoter Regions, Genetic / Q000235:genetics; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013234:Stem Cells; D013463:Sulfuric Acid Esters / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics
D002843:Chromatin; D004268:DNA-Binding Proteins; C094335:H19 long non-coding RNA; D009124:Muscle Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D013463:Sulfuric Acid Esters; D050656:Transcription Factor AP-2; D014157:Transcription Factors; C003170:mitopodozide; D011033:Podophyllin; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate; D011034:Podophyllotoxin