The Babraham Institute
In many imprinted genes, regulatory sequence elements have been identified that are methylated on one of the parental chromosomes only. For some of these "differentially methylated regions" (DMRs), such as the region upstream of H19, the 5'-portion of Snrpn and the 5'-UTR of U2af1-rs1, the allelic methylation originates from the germ line and is maintained throughout development. What ensures that at these DMRs, DNA methylation is established in only tile male or the female germ line is unknown. It is also unclear, which mechanisms are involved in maintaining their allele-specific methylation status in the developing embryo. As a step towards addressing these questions, we analyzed the in vivo chromatin organization of DMRs in the mouse H19 and U2af1-rs1 genes.
Genetic studies have established that the DMR upstream of the H19 gene functions as a chromatin boundary on the unmethylated maternal chromosome (1). At this differentially methylated imprinting-control region, we detected constitutive DNase-I hypersensitive sites (HS) and a non-nucleosomal chromatin organization on the unmethylated maternal chromosome (see contribution of S. Khosla). A similar situation was apparent at the 5'-UTR of the U2af1-rs1 gene where constitutive HS were detected on the unmethylated paternal chromosome only. From our data it appears that DNase-1 hypersensitivity and methylation are mutually exclusive at the H19 and U2af1-rs1 DMRs, and represent alternative epigenotypes. That they are inter-dependent, is supported by our finding that alterations in the one affect the other. In the H19 DMR, for example, in vitro-induced loss of maternal DNase-I hypersensitivity was consistently associated with gain of maternal methylation. Similarly, at the U2af1-rs1 locus, gain of maternal hypersensitivity was associated with loss of maternal DNA methylation.
Our studies establish that the H19 and U2af1-rs1 DMRs have a specialized organization of chromatin characterized by nuclease hypersensitivity at the unmethylated chromosome. Such an allelic chromatin organization might be present at other DMRs as well, and a recent study by Schweizer et al. (2) shows that the imprinting-control region in the 5% portion of the SNRPN gene has strong hypersensitive sites on the unmethylated maternal chromosome only. Parental allele-specific hypersensitive sites at DMRs are likely to indicate binding of non-histone proteins to the unmethylated chromosome. A model will be discussed which proposes that such protein factors and associated chromatin features regulate the allele specificity of DNA methylation at differentially methylated imprinting-control regions.
Thorvaldsen et al., Genes Dev. 12: 3693, 1998.
Schweizer et al., Hum. Mol. Genet. 8: 555, 1999.