CPG ISLAND PROMOTER REGION METHYLATION PATTERNS OF THE INACTIVE-X-CHROMOSOME HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE (HPRT) GENE

Inactive-X-chromosome genes in mammalian females have methylated CpG i slands. We have questioned whether there are variable levels of cytosi ne methylation at different CpG sites within the island that might ind icate the presence of primary sites of methylation which may be critic al for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyz ed the methylation patterns of 32 CpG sites of the X-linked hypoxanthi ne phosphoribosyltransferase (Hprt) gene oh the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprt(b-m3)) and a functional Hprt all ele were selected with 6-thioguanine. The resulting cell populations u niformly carry the intact Hprt allele on the inactive X chromosome. Th e methylation of these CpG sites was determined either by the direct s equence analysis of bisulfite-treated and amplified DNA or by the sequ ence analysis of clones derived from the amplified DNA. No CPG methyla tion was detected on the active Hprt genes from either males or the ac tive X chromosome of females. On average, 22 CpGs were methylated in t he other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in t wo cell lines showed that averages of 14 and 18 CpGs were methylated a nd that the frequency of methylation at 32 individual sites ranged fro m 3 to 100%. The highest frequency of methylation in cell lines coinci ded with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a t issue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromoso me, and it provided a quantitative estimate of the frequency of methyl ation of these sites in genomic DNA.