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.

Abstract

The 5' region of the gene encoding human X chromosome-linked phosphoglycerate kinase 1 (PGKI) is a promoter-containing CpG island known to be methylated at 119 of 121 CpG dinucleotides in a 450-base-pair region on the inactive human X chromosome in the hamster-human cell line X8-6T2. Here we report the use of polymerase chain reactionaided genomic sequencing to determine the complete methylation pattern of this region in clones derived from X8-6T2 cells after treatment with the methylation inhibitor 5-azacytidine. We find (i) a clone showing full expression of human phosphoglycerate kinase is fully unmethylated in this region; (ii) clones not expressing human phosphoglycerate kinase remain methylated at =50% ofCpG sites, with a pattern ofinterspersed methylated (M) and unmethylated (U) sites different for each clone; (iN) singles, defined as M-U-M or U-M-U, are common; and (iv) a few CpG sites are partially methylated. The data are interpreted according to a model of multiple, autonomous CpG sites, and estimates are made for two key parameters, maintenance efficiency (Em 99.9% per site per generation) and de novo methylation efficiency (Ed 5%). These parameter values and the hypothesis that several independent sites must be unmethylated for transcription can explain the stable maintenance of X chromosome inactivation. We also consider how the active region is kept free of methylation and suggest that transcription inhibits methylation by decreasing Em so that methylation cannot be maintained. Thus, multiple CpG sites, independent with respect to a dynamic methylation system, can stabilize two alternative states of methylation and transcription.Inheritance of DNA methylation patterns is generally observed in tissue culture, consistent with the maintenance methylase concept (1, 2) and the in vitro observed preference of DNA methyltransferase for hemimethylated sites (3-5). However, little is known about the in vivo ratio of maintenance to de novo methylation. Methylation maintenance probably is a key part of the maintenance of X chromosome inactivation, a phenomenon where one has extremely stable, clonally heritable differentiation of identical DNA sequences. 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 (7-12). 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 (13-17). 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). Autosomal CpG islands are characteristically unmethylated, but, in contrast, several X chromosome-linked CpG islands are highly methylated on the inactive X chromosome (Xi) (13-17). When the stabilizing effect of DNA methylation is not present (19) or i...

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