Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression.
X8/6T2, a hamster-human hybrid cell line which contains an inactive human X chromosome, was treated with 5-azacytidine and selected for derepression of hypoxanthine-guanine phosphoribosyltransferase. Clones were examined for coreactivation of the phosphoglycerate kinase gene (Pgk). Of 68 of these hybrids, approximately 20% expressed measurable human phosphoglycerate kinase (PGK) activity. A 600-base-pair region of the Pgk 5' CpG cluster was examined for the methylation status of eight CCGG sites (site 1 being 5'-most) in a number of PGK-negative and PGK-positive cell lines. The inactive X chromosome is normally methylated at all eight sites, and this was also true for the majority of X8/6T2 cells. However, several PGK-negative hybrids were demethylated in the site 3 to site 6 region. PGK activity correlated with demethylation at both sites 6 and 7. The data for PGK-positive and -negative hybrids indicate that demethylation at or near site 7 was necessary for reactivation of Pgk. Chromatin sensitivity to MspI digestion in the nuclei of male lymphoblastoid cells and several PGK-positive and PGK-negative hybrids was examined. PGK-positive cell lines were hypersensitive to digestion, while PGK-negative hybrids were resistant. Cleavage at sites 6 and 7 was observed in all PGK-positive cell lines at each MspI concentration examined. Sites 7 and 8 were less accessible to digestion than site 6. Cleavage in the site 2 to site 5 region was observable at the lowest MspI concentration. In most PGK-positive hybrids, a nonspecific endogenous nuclease detected the presence of a hypersensitive region spanning at least 450 base pairs, bounded at the 3' end near HpaII site 6. Nuclease hypersensitivity appears to be related to promoter activity, because sites 7 and 8 are in transcribed regions of the gene. These data indicate that specific sites within the CpG cluster have a dominant controlling influence over the Pgk promoter conformation and the transcriptional activation of Pgk.
Hansen
R S
RS
Department of Medicine, University of Washington, Seattle 98195.
Ellis
N A
NA
Gartler
S M
SM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004720:Endonucleases; D005786:Gene Expression Regulation; D008040:Genetic Linkage; D006801:Humans; D008745:Methylation; D008968:Molecular Conformation; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D014960:X Chromosome
D002843:Chromatin; D004247:DNA; D010735:Phosphoglycerate Kinase; D004720:Endonucleases