Evidence for DNA modification in the maintenance of X-chromosome inactivation of adult mouse tissues.

Chapman, Verne M.; Kratzer, Paul G.; Siracusa, Linda D.; Quarantillo, Barbara A.; Evans, Ronald J.; Liskay, R. Michael

doi:10.1073/pnas.79.17.5357

Cited by 46 publications

(16 citation statements)

References 28 publications

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“…It has been proposed that DNA methylation has a role in the process of X inactivation (5,7,26,28 phosphoribosyltransferase (HPRT)-deficient cells to an HPRT-positive phenotype, whereas the hprt allele carried on the active X can transform these cells efficiently (2,13,14,35). This fact suggests that DNAs of the inactive and active X chromosomes are differentially modified.…”

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confidence: 99%

“…Several studies of the expression of X-linked genes, including the -hprt gene, provide indirect evidence that DNA methylation plays a role in at least the maintenance of inactivation. The hprt allele carried on the inactive X in fibroblasts is inefficient in transformation of hypoxanthine phosphoribosyltransferase (HPRT)-deficient cells to an HPRT-positive phenotype, whereas the hprt allele carried on the active X can transform these cells efficiently (2,13,14,35). This fact suggests that DNAs of the inactive and active X chromosomes are differentially modified.…”

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confidence: 99%

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Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

Lock¹,

Melton²,

Caskey³

et al. 1986

Mol. Cell. Biol.

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It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cel lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene.In mammals, female and male cells express equivalent levels of most X-linked gene products despite the fact that females have twice the number of X-linked genes per diploid complement as do ma-les. This "dosage compensation" of X-linked genes in mammals results from the inactivation of one of the two X chromosomes in each female cell (16) concomitant with cellular differentiation in the early embryo (24,25,32). Once it is established, inactivity of the X chromosome is stably inherited through successive mitotic divisions. In germ cells, however, the inactive X is reactivated just prior to meiosis (4,8,12). X chromosome inactivation and reactivation thus represent stable changes in gene expression that are developmentally regulated.The mechanisms by which X inactivation and reactivation occur are unknown, but it has been suggested that X inactivation occurs in several phases, i.e., the primary inactivation event, spreading of inactivation from the site(s) of the initial event, and a process that results in maintenance of the inactive state. It has be...

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confidence: 99%

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confidence: 99%

Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

Lock¹,

Melton²,

Caskey³

et al. 1986

Mol. Cell. Biol.

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“…Riggs (5) suggested that such DNA methylation could theoretically account for many of the biological observations regarding X chromosome inactivation, but no direct evidence was available to support this concept. Recently, however, the technique of DNA-mediated gene transfer has been used to show that a hypoxanthine phosphoribosyltransferase (HPRT) gene on an active X chromosome, but not one on an inactive X, is competent in transferring the HPRT+ phenotype to HPRT-recipient cells (6,7). This functional distinction between active and inactive X chromosomes is apparently true for cultured cells and somatic tissues but not for yolk sac endoderm derived from mouse embryos (8).…”

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confidence: 99%

Differential methylation of hypoxanthine phosphoribosyltransferase genes on active and inactive human X chromosomes.

Yen¹,

Patel²,

Chinault³

et al. 1984

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

195

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Previous theoretical considerations and some experimental data have suggested a role for DNA methylation in the maintenance of mammalian X chromosome inactivation.The isolation of specific X-encoded sequences makes it possible to investigate this hypothesis directly. We have used cloned fragments of the human hypoxanthine phosphoribosyltransferase (HPRT) gene and methylation-sensitive restriction enzymes to study methylation patterns in genomic DNA of individuals with different numbers of X chromosomes and in somatic cell hybrid lines containing human X chromosomes that are either active or inactive or have been reactivated by treatment with 5-azacytidine. The results of these analyses show that there is hypomethylation of active X chromosomes relative to inactive X chromosomes in the 5' region of this gene. In the middle region of the gene, however, a site that is consistently undermethylated on inactive X chromosomes was identified. Taken together, the data suggest that the overall pattern of methylation, rather than methylation of specific sites, plays a role in the maintenance of X chromosome inactivation.

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“…But we note that in D. melanogaster, DNA methylation is not observed (53) and probably plays no role in position effect variegation. However, in the mammalian genome, DNA methylation is associated with X-chromosome inactivation (7,27,30,54,55, and 60), a facultative heterochromatization process (18,19,33) which also results in gene inactivation and is associated with chromosome condensation. Thus, the mechanisms of tk repression in our cell line may be analogous to mammalian processes where X-linked genes become repressed during the process of X-chromosome inactivation.…”

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confidence: 99%

Modulation of tk expression in mouse pericentromeric heterochromatin.

Butner¹,

Lo²

1986

Mol. Cell. Biol.

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Constitutive heterochromatin is that part of the chromosome which does not decondense or unravel during interphase (for a review, see reference 2). It is commonly associated with centromeres and contains large amounts of satellite DNA, a class of highly repetitive DNA composed of tandem arrays of simple sequence repeats (for a review, see references 3, 26, and 47). Constitutive heterochromatin is generally associated with transcriptional inactivity. One indication of this is the low density of major gene loci in constitutive heterochromatin (25). Furthermore, genes translocated to the vicinity of heterochromatin are frequently repressed or variegated. In Drosophila melanogaster, this repression has been correlated with the spread of the condensed state from the heterochromatic region to the translocated euchromatin (29), and the probability of repression has been observed to increase with increasing proximity to the heterochromatin. This type of cis effect is classically referred to as position effect or position effect variegation. A similar phenomenon occurs in mice in which autosomal loci translocated to a heterochromatic X chromosome also are observed to be inactivated (6).The molecular mechanism underlying classical position effects remains obscure. A detailed molecular analysis of genes modulated within heterochromatin may provide insights into this intriguing phenomenon. In the long term, such studies may allow the identification of cis-acting elements which play a role in exerting position effects. In this study, we describe a cell line, LC2-3, which can serve as a valuable model system for such studies. LC2-3 is a cell line derived from thymidine kinase-deficient (tk-) mouse L cells (LMTK-cells) satellite DNA was found to immediately flank these tk inserts and, using the tk DNA as a marker, the adjacent satellite DNA was shown to undergo a high frequency of DNA rearrangements (5), a process which is reminiscent of the known fluidity of satellite DNA. In the current study, we characterized three pairs of tk-revertant and tk+ rerevertant derivatives of LC2-3. Our results indicate that one of the tk genes in LC2-3 is probably not expressed but can be reactivated in association with DNA rearrangements and changes in DNA methylation. Through these and other studies in the future, we hope to obtain insights into the molecular mechanism mediating tk repression and, furthermore, to determine whether and how the flanking centromeric heterochromatin may participate in this proc- 5-AzaC treatments. Treatments with 5-azacytidine (5-azaC) were performed by plating 106 cells into a 100-mm dish containing medium supplemented with 5-azaC at a concentration of 0, 1, 3, or 10 ,uM. Cells were exposed to the nucleotide analog for 2 days and subsequently grown for 2 days in medium without 5-azaC. Cells were then harvested from each dish and replated at a density of 5 x 106 cells per 100-mm dish in HAT medium and 200 cells per 100-mm dish in BrdU medium. After 7 to 10 days of growth, colonies were fixed in methanol-acetic ...

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Evidence for DNA modification in the maintenance of X-chromosome inactivation of adult mouse tissues.

Cited by 46 publications

References 28 publications

Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

Methylation of the mouse hprt gene differs on the active and inactive X chromosomes.

Differential methylation of hypoxanthine phosphoribosyltransferase genes on active and inactive human X chromosomes.

Modulation of tk expression in mouse pericentromeric heterochromatin.

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