High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors.

Hornstra, Ian; Yang, Thomas P.

doi:10.1128/mcb.14.2.1419

Cited by 56 publications

(50 citation statements)

References 59 publications

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“…Recent studies of high-resolution methylation patterns in the human HPRT gene and the human and mouse PGK-1 gene 5Ј regions suggest that direct, methylation-induced alteration of sequence-specific DNA-protein interactions in the promoter region is unlikely to be the primary mechanism by which X-linked genes are silenced by DNA methylation. In these studies methylation patterns in the 5Ј region of the human and mouse PGK-1 genes (15, 16, 34) and human and mouse HPRT genes (14,40) reveal no strict correlation between methylated CpG dinucleotides and binding sites for transcriptional activators and no discernible conserved pattern of CpG methylation among the inactive alleles of these genes (other than a generally higher level of methylation compared with the active allele). In particular, the methylation pattern seen in the mouse PGK-1 gene makes it unlikely that DNA methylation functions by directly modifying individual interactions between transcriptional activators and their DNA binding sites in the immediate promoter because only a single CpG dinucleotide is fully methylated on the inactive X chromosome (16).…”

Section: Discussionmentioning

confidence: 99%

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

Litt¹,

Hansen²,

Hornstra³

et al. 1997

Journal of Biological Chemistry

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During the process of 5-aza-2-deoxycytidine (5aCdr)-induced reactivation of the X-linked human hypoxanthine phosphoribosyltransferase (HPRT) gene on the inactive X chromosome, acquisition of a nucleasesensitive chromatin conformation in the 5 region occurs before the appearance of HPRT mRNA. In vivo footprinting experiments reported here show that the 5aCdr-induced change in HPRT chromatin structure precedes the appearance of three footprints in the immediate 5 flanking region that are characteristic of the active HPRT allele. These and other data suggest the following sequence of events that lead to the reactivation of the HPRT gene after 5aCdr treatment: (a) hemidemethylation of the promoter, (b) an "opening" of chromatin structure detectable as increased nuclease sensitivity, (c) transcription factor binding to the promoter, (d) assembly of the transcription complex, and (e) synthesis of HPRT RNA. This sequence of events supports the view that inactive X-linked genes are silenced by a repressive chromatin structure that prevents the binding of transcriptional activators to the promoter.A unique system of differential gene expression in mammals is established during female embryogenesis by X chromosome inactivation (1, 2). The inactivation of one X chromosome within each female somatic nucleus generates a transcriptionally active and inactive allele of most X-linked genes and results in dosage compensation for X-linked genes between males and females. A variety of molecular mechanisms have been implicated in regulating the initiation, spreading, and maintenance of X inactivation (1-7). The involvement of DNA methylation in this process has been established by studies using methyl-sensitive restriction enzymes (8 -10), DNA-mediated transformation (11-13), genomic sequencing (14 -16), and the DNA demethylating agent 5-azacytidine (6,13,(17)(18)(19). All of these studies support the notion that hypermethylation of the 5Ј CpG island associated with many X-linked housekeeping genes is involved in the transcriptional silencing of these genes on the inactive X chromosome.The ability to demethylate and reactivate individual genes on the human inactive X chromosome in rodent-human somatic cell hybrids by treatment with 5-azacytidine or 5-aza-2Ј-deoxycytidine (5aCdr) 1 (6, 20) suggests that transcriptional regulation of X-linked genes by X chromosome inactivation involves some measure of local control either at the level of individual genes or at the level of chromatin domains. Reactivation of inactive X-linked genes such as the hypoxanthine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase (PGK-1) genes after 5-azacytidine or 5aCdr treatment is associated with both a change in chromatin structure from a nuclease-inaccessible to a nuclease-accessible conformation and a reduction in DNA methylation levels in the 5Ј CpG island (17,21).In previous studies, Sasaki et al. (22) assayed four parameters during 5aCdr reactivation of the human HPRT gene in a hamster-human somatic cell hybrid cell line (X8 -6T2) conta...

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Section: Discussionmentioning

confidence: 99%

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

Litt¹,

Hansen²,

Hornstra³

et al. 1997

Journal of Biological Chemistry

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“…For many, but not all, MDBP sites, including the ~-GAL A site, binding is much better when the site is methylated at its CPG dinucleotides. Much evidence indicates that DNA methylation is involved in negatively controlling expression of many genes in mammals, including X-linked genes subject to lyonization [24,25]. MDBP sites have been implicated in the negative control of gene expression from certain sites as well as in positive control from others [26][27][28].…”

Section: Discussionmentioning

confidence: 99%

A mutation in the 5′ untranslated region of the human α‐galactosidase A gene in high‐activity variants inhibits specific protein binding

1995

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Recently, normal individuals were identified who had high levels of plasma ¢x-galactosidase A activity and a G to A transition in the 5' untranslated (5' UT) region of the ol-galactosidase A gene. Electrophoretic mobility shift assays revealed that the wild-type sequence at the site of this mutation complexed with specific nuclear proteins. A standard NF-KB site competed with the 5' UT site for formation of these DNA-protein complexes. Complex formation was inhibited by the transition mutation. Therefore, the wild-type site might down-modulate expression of the o~-galactosidase A gene from this 5' untranslated region, which includes a previously described protein-binding site for another family of sequence-specific DNA-binding proteins, methylated DNA-binding protein.

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“…This region was chosen for analysis because previous studies suggest the region surrounding transcription initiation sites is crucial for transcriptional repression by DNA methylation (13)(14)(15)(16)(17). In the parental 8121 cell line, this region contains 20 symmetrically methylated CpG sites (where paired CpGs on the upper and lower strand count as a single site) and 12 unmethylated sites (18). Purified genomic DNA from each clone was subjected to the MaxamGilbert cytosine-specific sequencing reaction followed by ligation-mediated PCR (LMPCR).…”

Section: Identification Of Critical Cpgmentioning

confidence: 99%

“…High resolution methylation analysis by ligation-mediated PCR (LMPCR)-assisted genomic sequencing indicates that the promoter on the active X chromosome is unmethylated, whereas the promoter on the inactive X chromosome is methylated at most, but not all, CpG dinucleotides (18). The promoter methylation of the inactive HPRT allele, like that of other X-linked housekeeping genes, is unusual in that it occurs in a CpG island, which is typically unmethylated in autosomes.…”

mentioning

confidence: 99%

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

Chen¹,

Yang²,

Yang³

2001

Journal of Biological Chemistry

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The strong correlation between promoter hypermethylation and gene silencing suggests that promoter methylation represses transcription. To identify methylation sites that may be critical for maintaining repression of the human HPRT gene, we treated human/hamster hybrid cells containing an inactive human X chromosome with the DNA demethylating agent 5-azadeoxycytidine (5aCdr), and we then examined the high resolution methylation pattern of the HPRT promoter in single cell-derived lines. Reactivation of HPRT correlated with complete promoter demethylation. In contrast, the 61 5aCdr-treated clones that failed to reactivate HPRT exhibited sporadic promoter demethylation. However, three specific CpG sites remained methylated in all unreactivated clones, suggesting these sites may be critical for maintaining transcriptional silencing of the HPRT gene. Re-treatment of partially demethylated (and unreactivated) clones with a second round of 5aCdr did not increase the frequency of HPRT reactivation. This is consistent with mechanisms of methylation-mediated repression requiring methylation at specific critical sites and argues against models invoking overall levels or a threshold of promoter methylation. Treatment of cells with the histone deacetylase inhibitor, trichostatin A, failed to reactivate HPRT on the inactive X chromosome, even when the promoter was partially demethylated by 5aCdr treatment, suggesting that transcriptional repression by DNA methylation is unlikely to depend upon a trichostatin A-sensitive histone deacetylase.In mammals, DNA methylation at CpG dinucleotides in the 5Ј region of genes is frequently associated with transcriptional silencing (1), particularly in housekeeping genes on the inactive X chromosome. Numerous studies suggest that this association between promoter hypermethylation and transcriptional repression has a functional basis. For instance, individual loci on the inactive human X chromosome in human/ hamster hybrid cell lines may be reactivated using DNA-demethylating agents such as 5-azacytidine (2, 3) and 5-azadeoxycytidine, which inhibit the maintenance methyltransferase and incorporate into newly synthesized DNA (4), resulting in a failure to maintain methylation patterns during growth. Likewise, in vitro methylation of various promoter constructs results in inhibition of transcription in transient expression assays (5-9). However, despite significant evidence that DNA methylation represses transcription, specific mechanisms of this repression are only now becoming apparent.Recent reports suggest that DNA methylation mediates transcriptional repression indirectly, via binding of the methylated DNA-binding protein MeCP2, which in turn recruits histone deacetylases that modify the local chromatin structure (10, 11). However, additional mechanisms may also act to repress transcription by methylation, such as direct inhibition of transcription factor binding to its cognate site in DNA. Indeed, methylation of the binding sites of several transcription factors has been shown to alter ...

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High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors.

Cited by 56 publications

References 59 publications

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

A mutation in the 5′ untranslated region of the human α‐galactosidase A gene in high‐activity variants inhibits specific protein binding

Evidence That Silencing of the HPRT Promoter by DNA Methylation Is Mediated by Critical CpG Sites

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