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.

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Abstract
The promoter region of the X-linked human phosphoglycerate kinase-1 (PGK-1) gene is a CpG island, similar to those often found near autosomal genes. We used ligation-mediated polymerase chain reaction (PCR) for a genomic sequencing study in which 450 bp of the human PGK-1 promoter region was analyzed for the presence of in vivo protein footprints and cytosine methylation at all CpG sites. A technique was devised to selectively visualize the DNA of the inactive X chromosome (Xi), even in the presence of the active X chromosome (Xa). We found that the human Xa in both normal male lymphocytes and hamster-human hybrids is completely unmethylated at all 120 CpG sites. In contrast, 118 of the CpG sites are methylated on the human Xi in hamster-human hybrids. The Xi in normal female lymphocytes is also highly methylated, but some GCG or CGC trinucleotides partially escape methylation; all other CpGs are fully methylated. In vivo footprinting studies with dimethylsulfate (DMS) revealed eight regions of apparent protein-DNA contacts on the Xa. Four of the footprints contained the consensus sequence of the binding site for transcription factor Sp1. The other regions include potential binding sites for transcription factors ATF, NF1, and a CCAAT-binding protein. The Xi did not show any specifically protected sequences, and with the exception of four hyperreactive sites, the in vivo DMS reactivity profile of Xi DNA was very similar to that of purified, linear Xi DNA. The implications of these findings with regard to the maintenance of methylation-free islands, X chromosome inactivation, and the chromatin structure of facultative heterochromatin are discussed.