Szabó P E, Pfeifer G P, Mann J R
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Mol Cell Biol. 1998 Nov;18(11):6767-76. doi: 10.1128/MCB.18.11.6767.
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.
基因组印记导致哺乳动物中少数基因呈现亲本特异性单等位基因表达。印记的本质尚不清楚，但大量证据表明DNA甲基化发挥了作用。印记基因H19的母本等位基因是活跃的且甲基化程度低；父本等位基因是不活跃的且甲基化程度高。特定位点核酸酶超敏性的等位基因特异性差异提示了其他表观遗传修饰的作用。为了进一步分析决定H19单等位基因表达的可能表观遗传机制，我们对孤雌生殖和雄核生殖胚胎干细胞编码序列上游区域进行了体内硫酸二甲酯和DNase I足迹分析。这些细胞分别只携带母本和父本来源的等位基因。我们在启动子区域观察到母本等位基因特异性的硫酸二甲酯和DNase I足迹，这表明在一个CCAAT框以及转录因子Sp1和AP-2的结合位点存在蛋白质-DNA相互作用。此外，在更上游的一个区域边界（已有研究表明该区域存在的差异甲基化构成一种印记），我们观察到一些母本等位基因特有的链特异性硫酸二甲酯反应性差异，以及一种不寻常的染色质结构，即母本来源的DNA两条链在100个核苷酸的距离内对DNase I切割都具有强烈的超敏性。因此，我们揭示了除DNA甲基化外，还存在新的亲本特异性表观遗传修饰，它们可能构成印记或维持H19的单等位基因表达。
