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

Abstract

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...

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.

“…CpG islands are often located in the 5' region of constitutively expressed housekeeping genes and are frequently unmethylated in mammalian DNA (2, 26). However, CpG islands associated with the 5' region of housekeeping genes on the inactive X chromosome are characteristically hypermethylated (28,(53)(54)(55).Numerous studies have examined the role of DNA methylation in the process of X chromosome inactivation. Using a variety of experimental approaches, these studies have investigated a correlation between DNA methylation and maintenance of the transcriptionally silent state of genes on the inactive X chromosome (13,14).…”

“…CpG islands are often located in the 5' region of constitutively expressed housekeeping genes and are frequently unmethylated in mammalian DNA (2, 26). However, CpG islands associated with the 5' region of housekeeping genes on the inactive X chromosome are characteristically hypermethylated (28,(53)(54)(55).…”

“…The molecular mechanisms that initiate inactivation, propagate the inactivation signal, and maintain this novel system of differential gene expression through subsequent cell divisions are unknown. DNA methylation (21,27,28), chromatin structure (22,38,41), DNA-protein interactions (13,31), and DNA replication (12,13) have all been proposed to have roles in this process.…”

DNA hypomethylation can activate Xist expression and silence X-linked genes.

“…Inactivation is initiated at the X-inactivation center (Xic), a cis-acting sequence from which the process of inactivation spreads into adjacent chromatin (Russel 1963;Cattanach 1975;Mattei et al 1981). Propagation and maintenance of X-inactivation are thought to involve heterochromatinization, because the inactive X chromosome (Xi) shows many characteristics of constitutive heterochromatin, including delayed replication timing (Takagi 1974), maintenance of the condensed chromatin throughout interphase (Barr and Cart 1962), lack of acetylation of histone H4 (Jeppesen and Turner 1993), and methylation of CpG islands (Wolf et al 1984a, b;Lock et al 1986). Dem3Corresponding author.…”

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

“…The methylation of cytosines occurring in CpG dinucleotides has also been shown to be correlated with lack of expression of certain X-linked genes (20,21), and it has been suggested that some nutritional auxotrophic mutants of mammalian cells may have arisen from DNA hypermethylation (14)(15)(16)(17)30). Several cell lines that require asparagine for growth because of a lack of asparagine synthetase (AS) activity have been isolated (24,34).…”