Differential methylation of hypoxanthine phosphoribosyltransferase genes on active and inactive human X chromosomes.

Abstract

Previous theoretical considerations and some experimental data have suggested a role for DNA methylation in the maintenance of mammalian X chromosome inactivation.The isolation of specific X-encoded sequences makes it possible to investigate this hypothesis directly. We have used cloned fragments of the human hypoxanthine phosphoribosyltransferase (HPRT) gene and methylation-sensitive restriction enzymes to study methylation patterns in genomic DNA of individuals with different numbers of X chromosomes and in somatic cell hybrid lines containing human X chromosomes that are either active or inactive or have been reactivated by treatment with 5-azacytidine. The results of these analyses show that there is hypomethylation of active X chromosomes relative to inactive X chromosomes in the 5' region of this gene. In the middle region of the gene, however, a site that is consistently undermethylated on inactive X chromosomes was identified. Taken together, the data suggest that the overall pattern of methylation, rather than methylation of specific sites, plays a role in the maintenance of X chromosome inactivation.

Sequence and Comparative Analysis of the Mouse 1-Megabase Region Orthologous to the Human 11p15 Imprinted Domain

“…This also appears to be true for imprinted genes on other chromosomes (Yen et al 1984;Brandeis et al 1993;Shemer et al 1997;Wutz et al 1997), although, to our knowledge, this has not been commented on in the literature, likely because interspecies global sequence comparisons have not been possible. We suggest that there may be a two-island rule for imprinting, that is, in most cases more than one CpG island is required to maintain normal imprinting.…”

“…Feinberg, unpubl. ), including the CpG islands, which are normally unmethylated except for the inactive X-chromosome and imprinted genes (Yen et al 1984;Brandeis et al 1993;Shemer et al 1997;Wutz et al 1997). We are currently determining which of these sequences might show allele-specific methylation.…”

“…CpG islands are normally unmethylated, but allele-specific methylation of CpG islands appears to mark both the inactive X chromosome (Yen et al 1984) and many imprinted genes, for example, H19, Snrpn, and Igf2r (Brandeis et al 1993;Shemer et al 1997;Wutz et al 1997). In addition, GC-rich sequences that are not CpG islands (i.e., they meet the first, but not the second criterion above) may also be differentially methylated (termed a differentially methylated region) in the vicinity of imprinted genes, for example Igf2 (Sullivan et al 1999) and a second site 2-4 kb upstream of the H19 CpG island (Thorvaldsen et al 1998).…”

DNA hypomethylation can activate Xist expression and silence X-linked genes.

“…This conclusion was based upon the patchy reactivation of some X-linked genes such as hypoxanthine phosphoribosyl transferase (HPRT) but not of other genes following drug treatment of somatic cell hybrids containing a human Xi (Mohandas et al 1981). Reactivation of HPRT correlated with demethylation of the CpG island associated with the HPRT gene (Yen et al 1984;Wolf et al 1984b). It is important to point out here that treatment of cells with 5-aza-dC achieves a moderate level of genomic DNA hypomethylation (Flatau et al 1984;Taylor et al 1984;Michalowsky and Jones 1989) that may be insufficient for the activation of Xist.…”

In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1.

“…In contrast to autosomal CpG islands, several on the inactive X chromosome were found to be highly methylated at HpaII sites {Wolf et al 1984;Yen et al 1984;Keith et al 1986;Toniolo et al 1988}. X chromosome inactivation is a gene-silencing mechanism, unique to mammals, that affects almost all genes of a whole chromosome (for review, see Gartler and Riggs 1983;Grant and Chapman 1988}.…”