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.

Hansen, R. Scott; Ellis, Nathan A.; Gartler, Stanley M.

doi:10.1128/mcb.8.11.4692

Cited by 87 publications

(77 citation statements)

References 56 publications

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“…MeCP binding at methylated CCGG sites inhibits the action of MspI, a methylationinsensitive restriction endonuclease. Studies on isolated nuclei by Hansen et al (1988)have shown that the methylated human PGK-1 promoter on the Xi is > 100-fold less sensitive to MspI digestion than the same unmethylated region is on the Xa. Antequera et al {1989) obtained similar results for mouse HPRT.…”

Section: Heterochromatin Structure and The XImentioning

confidence: 99%

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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.

Pfeifer

Tanguay²,

Steigerwald³

et al. 1990

Genes Dev.

240

182

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The promoter region of the X-linked human phosphoglycerate kinase-I [PGK-I) gene is a CpG island, similar to those often found near autosomal genes. We used ligation-mediated polymerase chain reaction (PCR) for a genomie 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 lymphoeytes 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 lymphoeytes is also highly methylated, but some GCG or CGC trinueleotides 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 Spl. 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 hyperreaetive 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 ehromatin structure of faeultative heteroehromatin are discussed.

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Section: Heterochromatin Structure and The XImentioning

confidence: 99%

“…Chinese hamster hybrid cells containing either a human Xa {Y162-11C) or Xi (X86T2) (Hansen et al 1988} were kindly provided by R.S. Hansen and S.M.…”

Section: Cells and Cell Linesmentioning

confidence: 99%

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.

Pfeifer

Tanguay²,

Steigerwald³

et al. 1990

Genes Dev.

240

182

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show abstract

“…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).…”

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confidence: 99%

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.

Pfeifer

Steigerwald

Hansen

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

243

191

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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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“…It is thought to play a prominent role in the transcriptional regulation of gene cxpresvion in various organisms. For instance, the inactivation of X-chromosomal manimalian genes is linked to inethylation (Hansen et al, 1988).…”

mentioning

confidence: 99%

“…These critical sites for niethylation are often situated in the 5'-noncoding region of the gene (Hansen et al, 1988). Apart from affecting gene regulation in hcalthy organisms, methylation can havc an effect on virus infection and cancer cell proliferation (Ben-Hattar and Jiricny, 1988;Prcndergast el at., 1992).…”

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confidence: 99%

A Tobacco Nuclear Protein that Preferentially Binds to Unmethylated CpG‐rich DNA

Fisscher

Weisbeek

Smeekens

1996

European Journal of Biochemistry

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(Rcceivcd X Aug~i~tI30 Octoher 1995) -E3R 95 1321)/2 Thc methylatinn of cytosine rcsidues in CpG dinucleotides of cukaryolic DNA is an important inechunistn for the regulation of gene expression. Higher plants havc a high content of inethylated cytosine residues in CpG as well as CpNpC sites, and experimental evidence suggests a role in gene expression for DNA methylation. In this article, we dcscribe ii tobacco nuclear protcin whose binding to various DNA sequences is positively correlated with the CpG density of thc probes. This protein, CpG-binding protein 1 (CGBP-1). has reduced affinity for DNA when the CpC; sites are methylated. Ribonucleasc treatment also reduces thc formation of the CGRP-I complex. The hinding characteristics of' CGBP-1 make it an interesting protein with respect to methylation-meJiated gene expression in pl tits.

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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.

Cited by 87 publications

References 56 publications

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.

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.

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.

A Tobacco Nuclear Protein that Preferentially Binds to Unmethylated CpG‐rich DNA

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