Banerjee S, Singh P B, Rasberry C, Cattanach B M
Centre for Reproductive Medicine, Department of Obstetrics and Gynaecology, St. George's Hospital Medical School, Cranmer Terrace, University of London, UK.
Mech Dev. 2000 Feb;90(2):217-26. doi: 10.1016/s0925-4773(99)00254-3.
Insulin-like growth factor 2 (Igf 2) and H19 genes are oppositely imprinted and as such have been most extensively studied imprinted genes both genetically and at the molecular level. Imprints of the H19 gene, being established during spermatogenesis, are epigenetically transmitted to the somatic cells of the embryo. Current hypotheses attempting to explain the allele-specific silence of the H19 gene include DNA methylation and chromatin condensation. In order to understand the molecular basis of H19 epigenesis, it is crucial to identify the markings in the chromatin organising the imprinted domain in spermatozoa. Using Micrococcal nuclease (MNase), DNase I and Methidiumpropyl-EDTA. iron II (MPE.Fe(II)) as chromatin probes, we demonstrate that in mouse epididymal spermatozoa, at least 4kb DNA upstream of the H19 'cap' site, containing the imprinted and differentially methylated domain (DMD), is heterochromatic. The cleavage sites in this domain (-2 to -4kb) exhibit approximately 425bp periodicity. This structure is maintained in the paternal allele of normal embryos and is disrupted at -2.2, -2.65 and at -3.5kb in embryos maternally disomic for the distal end of chromosome 7 (MatDp 7). The hypersensitive sites in chromatin precisely register the MPE.Fe(II) cleavage sites in chromosomal DNA. Therefore, the DNA sequences in the imprinted domain constrain the chromatin structure in a way similar to that of 1.688g/cm(3) Drosophila satellite chromatin. In addition, we find that condensation of the paternal allele correlates with methylation-dependent alteration in the structure of DNA sequences in DMD. These results suggest that CpG-methylation induces localised changes in DNA conformation and these facilitate consequent remodelling of chromatin thereby allowing the paternal and maternal H19 alleles to be distinguished.
胰岛素样生长因子2（Igf 2）和H19基因呈反向印记，因此它们是在遗传和分子水平上研究最为广泛的印记基因。H19基因的印记在精子发生过程中建立，并通过表观遗传传递给胚胎的体细胞。目前试图解释H19基因等位基因特异性沉默的假说是DNA甲基化和染色质凝聚。为了理解H19表观遗传的分子基础，识别精子中组织印记结构域的染色质标记至关重要。使用微球菌核酸酶（MNase）、脱氧核糖核酸酶I和甲基丙基-乙二胺四乙酸铁II（MPE.Fe(II)）作为染色质探针，我们证明在小鼠附睾精子中，H19“帽”位点上游至少4kb的DNA，包含印记和差异甲基化结构域（DMD），是异染色质的。该结构域中的切割位点（-2至-4kb）呈现约425bp的周期性。这种结构在正常胚胎的父本等位基因中得以维持，而在7号染色体远端母本二体的胚胎（MatDp 7）中，在-2.2、-2.65和-3.5kb处被破坏。染色质中的超敏位点与染色体DNA中的MPE.Fe(II)切割位点精确对应。因此，印记结构域中的DNA序列以类似于1.688g/cm(3)果蝇卫星染色质的方式限制染色质结构。此外，我们发现父本等位基因的凝聚与DMD中DNA序列结构的甲基化依赖性改变相关。这些结果表明，CpG甲基化诱导DNA构象的局部变化，进而促进随后染色质重塑，从而使父本和母本H19等位基因得以区分。
