Active X chromosome DNA is unmethylated at eight CCGG sites clustered in a guanine-plus-cytosine-rich island at the 5' end of the gene for phosphoglycerate kinase.

Abstract

The 5' control region and first exon for human X-chromosome-linked phosphoglycerate kinase is contained in a G + C-rich island. We measured methylation at all HpaII sites in this 5' region of leukocyte DNA. By use of a blotting procedure that allows analysis of small DNA fragments, we found that the HpaII sites are entirely methylated when from an inactive X chromosome and entirely unmethylated when from an active one. In contrast, methylation of HpaII sites in more downstream regions of the gene is essentially the same in active and inactive X chromosomes.

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.

“…To selectively reveal Xi DNA in female cells, we took advantage of the known differential methylation state of the HpaII sites in this region on the Xa and Xi in female blood cells (Keith et al 1986). For example, using a quantitative methylation assay, Steigerwald et al (1990) found that the HpaII site at position + 23 in this region is >98% unmethylated on the Xa in normal male lymphocytes and 50% methylated in the Xa/Xi mixture in normal female lymphocytes.…”

“…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}.…”

Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability.

“…Studies on X chromosome inactivation have, in fact, provided strong evidence that cytosine methylation is one of the mechanisms used by mammalian cells to aid cell memory (6) and to maintain genetic silence during development (7)(8)(9)(10)(11)(12). For X chromosome-linked genes, a strong correlation exists between the inactive state and hypermethylation of methylation-sensitive restriction sites (usually Hpa II) in the 5' region (13)(14)(15)(16)(17). Most housekeeping genes, including those on the X chromosome, have 5'-associated G+C-rich regions, termed CpG islands, which are up to 10-fold enriched for CpG dinucleotides (18).…”

“…Most housekeeping genes, including those on the X chromosome, have 5'-associated G+C-rich regions, termed CpG islands, which are up to 10-fold enriched for CpG dinucleotides (18). Autosomal CpG islands are characteristically unmethylated, but, in contrast, several X chromosome-linked CpG islands are highly methylated on the inactive X chromosome (Xi) (13)(14)(15)(16)(17). When the stabilizing effect of DNA methylation is not present (19) or is perturbed by 5-azacytidine (SzC) treatment, reactivation of genes on the inactive X is frequently observed (20,21).…”

A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

“…The 5' region of the PGKI gene is now the most highly characterized X-linked promoter region, and the relationship between methylation, transcription, and transcription factors has been studied both in normal human lymphocytes and in cultured cell lines (15)(16)(17)(18)(19). Recently, ligation-mediated genomic sequencing has been used to establish that the promoter region of the PGKI gene on the inactive X chromosome is methylated at 60 of 61 CpG sites, whereas the active promoter has no methylated sites (19).…”