Comparison of transformation efficiency of human active and inactive X-chromosomal DNA

Venolia, Lee; Gartler, Stanley M.

doi:10.1038/302082a0

Cited by 64 publications

(27 citation statements)

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“…3. RT-PCR analysis of human HPRT mRNA in X8 -6T2 cells after treatment with 5aCdr. Levels of human HPRT mRNA were assayed by 12,24,32, 48, and 60 h after initiating 5aCdr treatment of X8 -6T2 cells (lanes marked by numbered brackets). Numbers above each lane denote micrograms of RNA used in RT-PCR reaction; RNA from each 5aCdr-treated sample was assayed by RT-PCR in duplicate.…”

Section: Binding Of Transcription Factors During 5acdr-induced Reactimentioning

confidence: 99%

“…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)(12)(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.…”

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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: Binding Of Transcription Factors During 5acdr-induced Reactimentioning

confidence: 99%

mentioning

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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“…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). These experimental approaches include methylation analysis by methyl-sensitive restriction enzymes in conjunction with Southern blotting (28,41,48,53,55), DNA-mediated transformation studies using DNA from the active or inactive X chromosomes (27,49), and analysis of the reactivation of genes from the inactive X chromosome using the DNA-demethylating agent 5-azacytidine (5-azaC) (16,34,48,50). All support the view that the 5' CpG island of housekeeping genes on the inactive X chromosome is hypermethylated in comparison to its corresponding alleles on the active X chromosome.…”

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confidence: 99%

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.

Hornstra¹,

Yang²

1994

Mol. Cell. Biol.

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DNA methylation within GC-rich promoters of constitutively expressed X-linked genes is correlated with transcriptional silencing on the inactive X chromosome in female mammals. For most X-linked genes, X chromosome inactivation results in transcriptionally active and inactive alleles occupying each female nucleus.To examine mechanisms responsible for maintaining this unique system of differential gene expression, we have analyzed the methylation of individual cytosine residues in the 5' CpG island of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Methylation analysis of 142CpG dinucleotides by genomic sequencing was carried out on purified DNA using the cytosine-specific Maxam and Gilbert DNA sequencing reaction in conjunction with ligation-mediated PCR. These studies demonstrate the 5' CpG islands of active and 5-azacytidine-reactivated alleles are essentially unmethylated while the inactive allele is hypermethylated. The inactive allele is completely methylated at nearly all CpG dinucleotides except in a 68-bp region containing four adjacent GC boxes where most CpG dinucleotides are either unmethylated or partially methylated. Curiously, these GC boxes exhibit in vivo footprints only on the active X chromosome, not on the inactive X. The methylation pattern of the inactive HPRT gene is strikingly different from that reported for the inactive X-linked human phosphoglycerate kinase gene which exhibits methylation at all CpG sites in the 5' CpG island. These results suggest that the position of methylated CpG dinucleotides, the density of methylated CpGs, the length of methylated regions, and/or chromatin structure associated with methylated DNA may have a role in repressing the activity of housekeeping promoters on the inactive X chromosome. The pattern of DNA methylation on the inactive human HPRT gene may also provide insight into the process of inactivating the gene early in female embryogenesis.During early mammalian female embryogenesis, one of the two transcriptionally active X chromosomes is randomly inactivated in each cell of the embryo. The inactivation of one X chromosome in each female somatic cell creates a unique system of differential gene expression where a transcriptionally active X chromosome and a transcriptionally inactive X chromosome occupy the same nucleus. The inactivation of genes on one of the two X chromosome in females compensates for the dosage imbalance of X-linked genes between males and females. 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 methylation has been widely implicated in the regulation of gene expression in mammalian cells (3,42 produce 5-methylcytosine (26). CpG dinucleotides are gener...

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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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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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Comparison of transformation efficiency of human active and inactive X-chromosomal DNA

Cited by 64 publications

References 16 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

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.

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

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