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Inactive X chromosome DNA does not function in DNA-mediated cell transformation for the hypoxanthine phosphoribosyl transferase gene
Abstract
The molecular nature of the X chromosome inactivation process has been investigated by utilizing the techniques of DNA-mediated cell transformation of the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus. The findings indicate that purified DNA from the inactive X chromosome of a near-euploid mouse cell line is not functional in transformation for HPRT, but the DNA from its "homologous" active X readily elicits transformation for HPRT in the same hamster cell recipient. These findings suggest that there is a difference between the DNA, per se, of the active and inactive X at (or near) the HPRT locus and that this difference could account, at least in part, for its inactivation.
... This effect is also not limited to in vitro-methylated templates. Both the inactive X-linked Hprt gene (46) and a modified muscle determination gene in 1OT1/2 cells (45) have been shown to be inactive in the transfection assay, and the same can be shown for the transcriptionally silent mouse endogenous viral sequences (74). ...
A large body of evidence demonstrates that DNA methylation plays a role in gene regulation in animal cells. Not only is there a correlation between gene transcription and undermethylation, but also transfection experiments clearly show that the presence of methyl moieties inhibits gene expression in vivo. Furthermore, gene activation can be induced by treatment of cells with 5-azacytidine, a potent demethylating agent. Methylation appears to influence gene expression by affecting the interactions with DNA of both chromatin proteins and specific transcription factors. Although methylation patterns are very stable in somatic cells, the early embryo is characterized by large alterations in DNA modification. New methodologies are now becoming available for studying methylation at this stage and in the germ line. During development, tissue-specific genes undergo demethylation in their tissue of expression. In tissue culture cells this process is highly specific and appears to involve an active mechanism which takes place in the absence of DNA replication. The X chromosome undergoes inactivation during development; this is accompanied by de novo methylation, which appears necessary to stably maintain its silent state. As opposed to the programmed changes in DNA methylation which occur in vivo, immortalized tissue culture cells demonstrate alterations in DNA modification which take place over a long time scale and which appear to be the result of selective pressures present during the growth of these cells in culture.
... Transfection experiments which tested the ability of purified DBA to confer the HPRT-*' phenotype on HPRT~ recipient cells showed that the HPRT gene on the somatic inactive X is unable to express. However, DBA from the active X chromosomes can transform cells to HPRT-*" (Liskay & Evans 1980;Chapman et 2a. 1983;Venolia & Gartler 1983), as can DBA from an inactive X chromosome that has been re-activiated by the inhibitor of DBA methylation, 5-azacytidine (Venolia et al.1982). ...
Genetic analysis of the human Y chromosome has been slow despite its central role in sex determination. At present genetic analysis has identified only about ten genes on the Y chromosome, and only three genes (one coding for a cell surface antigen, a second coding for a regulatory zinc finger protein and a third coding for a testis specific transcript) have been cloned. This study has attempted to identify further genes on the Y chromosome using techniques which also facilitate cloning. This thesis therefore describes an investigation into the GC rich regions (HTF - Hpall Tiny Fragment Islands) of the Y chromosome as an approach to cloning Y-1inked genes. Using restriction analysis, 150 Y-specific cosmid clones have been screened. Four putative HTF island containing clones were selected and studied in more detail using Southern mapping, northern analysis, library screening, DNA sequencing and methylation analysis. Although data indicate that three of the clones are not associated with transcribed sequences, the fourth clone has been shown through sequence analysis, to contain a possible open reading frame. A direct approach to cloning Hpall tiny fragments from a Y chromosome specific library, obtained from the American Type Culture Collection, is also described. Following the isolation and purification of the human inserts away from the phage vector within this library, the inserts were further restricted using the enzyme Hpall and the resulting tiny fragments re-cloned and analysed for their possible association with transcribed sequences. Although a clone was identified which hybridizes to human genomic sequences, data indicates this to be of autosomal origin. Evidence is also discussed that the human Y chromosome appears to be relatively deficient of HTF islands when compared with the remainder of the genome, and that this deficiency could also reflect the small number of genes which are carried on the human Y chromosome.
... The maintenance of inactivity of genes on the inactive X-chromosome in adult somatic tissues appears to involve DNA methylation (reviewed in Monk, 1986). Early studies showed that DNA from the inactive X-chromosome would only transform recipient cells defective for HPRT function if the DNA was isolated from cells treated with the demethylating agent, 5-azacytidine (Liskay & Evans, 1980;Venolia et al, 1982). In addition, differential patterns of DNA methylation between the active and inactive Xchromosome have been directly demonstrated (Wolf et al, 1984;Yen et al, 1984;Toniolo et al, 1984;Lindsay et al, 1985). ...
Maternal (oocyte) and embryonic programmes of hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT) gene expression have been investigated in mouse oocytes and during preimplantation development. The onset of the embryonic HPRT gene occurs following fertilization, or parthenogenetic activation, before the 4-cell stage. The oocyte-determined increase in HPRT activity due to preformed mRNA continues in aging oocytes, as well as in fertilized or activated eggs, i.e. irrespective of the initiation of embryonic development. Attempts were made to determine whether there is a detectable time difference in the onset of maternal and paternal genomes by assaying the onset of embryo- coded HPRT activity in embryos of different maternal and paternal X-chromosome constitution, but due to difficulties in comparable staging of embryos, no definite conclusion could be drawn. The expression of an exogenously introduced HPRT minigene has been monitored throughout preimplantation development. The embryos injected with supercoiled HPRT minigene showed an approximately twofold increase in HPRT activity at the 2-cell stage compared with control uninjected embryos. Linear minigene DNA was less efficient in giving active enzyme. The efficacy of three different promoters were studied in 2-cell mouse embryos using the expression of the HPRT minigene as a reporter function. The mouse HPRT promoter and the uninduced mouse metallothionein-1 (MT-1) promoter functioned equally well whereas the viral SV40 promoter did not allow HPRT expression. The mouse MT-1 promoter linked to the HPRT minigene allowed induction of HPRT gene expression in mouse embryos cultured in the presence of cadmium. The inhibition of enzyme expression from injected minigene DNA is mediated by simultaneous injection of a fivefold molar excess of HPRT antisense DNA. The same negation of exogenous HPRT activity was observed with simultaneous injection of HPRT exon-1 antisense DNA. The use of an inducible HPRT antisense construct achieved repression of gene activity with equivalent molar amounts of antisense to the sense molecules. Transgenic mice were produced with an antisense HPRT minigene construct attached to the inducible mouse MT-l promoter. The prospect of "cancelling" the endogenous "sense" gene activity in these mice at a specific stage of development by applying the induction stimulus is discussed.
... DNA methylation at CpG islands of X-linked genes (Riggs 1975;Gartler and Riggs 1983). Particularly telling were experiments in which a methylated DNA plasmid containing the X-linked gene HPRT was shown to remain silent after transfection into HPRT-deficient cells, but became competent after removal of DNA methylation by 5-azacytidine (Liskay and Evans 1980;Venolia et al. 1982;Venolia and Gartler 1983). Maintenance of XCI is also ensured by the histone modifications that are progressively added throughout early development (see spreading). ...
X-chromosome inactivation, which was discovered by Mary Lyon in 1961 results in random silencing of one X chromosome in female mammals. This review is dedicated to Mary Lyon, who passed away last year. She predicted many of the features of X inactivation, for e.g., the existence of an X inactivation center, the role of L1 elements in spreading of silencing and the existence of genes that escape X inactivation. Starting from her published work here we summarize advances in the field.
... This effect is also not limited to in vitro-methylated templates. Both the inactive X-linked Hprt gene (46) and a modified muscle determination gene in 1OT1/2 cells (45) have been shown to be inactive in the transfection assay, and the same can be shown for the transcriptionally silent mouse endogenous viral sequences (74). ...
... The resulting animals will carry a single copy of the human WT-YFG, or YFRV-YFG BAC, on the Yfg mouse null background (Yfg 2/2 , Hprt WT-YFG /Y or Yfg 2/2 , Hprt YFRV-YFG /Y). Animals studied on the null background will be males, thus avoiding X inactivation [56,75]. Using this HuGX strategy, the phenotype of the Yfg 2/2 , Hprt YFRV-YFG /Y animals can be directly compared to that of the Yfg 2/2 , Hprt WT-YFG /Y animals. ...
An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant-harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation.
... Also, treatment of cells with 5-azacytidine, which causes heritable hypomethylation of cytosine in the DNA, can cause reactivation of genes on X i (Lock et al., 1986). Finally, DNAs isolated from X i versus X a show different transformation efficiencies (Liskay and Evans, 1980; Chapman et al., 1982 ), indicating that X i and X a DNA fragments are differentially mod- ified. Although most X-linked genes are believed to be subject to X inactivation, the number of genes is growing that have been demonstrated to escape the inactivation process. ...
Transgenic mice carrying one complete copy of the human alpha 1(I) collagen gene on the X chromosome (HucII mice) were used to study the effect of X inactivation on transgene expression. By chromosomal in situ hybridization, the transgene was mapped to the D/E region close to the Xce locus, which is the controlling element. Quantitative RNA analyses indicated that transgene expression in homozygous and heterozygous females was about 125% and 62%, respectively, of the level found in hemizygous males. Also, females with Searle's translocation carrying the transgene on the inactive X chromosome (Xi) expressed about 18% transgene RNA when compared to hemizygous males. These results were consistent with the transgene being subject to but partially escaping from X inactivation. Two lines of evidence indicated that the transgene escaped X inactivation or was reactivated in a small subset of cells rather than being expressed at a lower level from the Xi in all cells, (i) None of nine single cell clones carrying the transgene on the Xi transcribed transgene RNA. In these clones the transgene was highly methylated in contrast to clones carrying the transgene on the Xa. (ii) In situ hybridization to RNA of cultured cells revealed that about 3% of uncloned cells with the transgene on the Xi expressed transgene RNA at a level comparable to that on the Xa. Our results indicate that the autosomal human collagen gene integrated on the mouse X chromosome is susceptible to X inactivation. Inactivation is, however, not complete as a subset of cells carrying the transgene on Xi expresses the transgene at a level comparable to that when carried on Xa.
... The molecular mechanisms responsible for initiating, spreading, and maintaining X chromosome inactivation are unknown. However, DNA-protein interactions (8,28), chromatin structure (20,34,36), DNA replication (7,47), and DNA methylation (19,24,25,31,38,55,57) have all been postulated to be involved. Though X inactivation is a chromosome-wide phenomenon and process, some degree of regulation at the level of individual X-linked genes must also be involved, as indicated by the ability to independently reactivate individual genes on the inactive X chromosome by 5-azacytidine (5-azaC) (12,13,31,49,50). ...
Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.
... It is our speculation that the lack of large muscle colonies following loT1/2 DNA transfection reflects the methylation status of a myogenie determination locus in these cells. Genes that are fully methylated in vitro or that are present on the hypermethylated, inactive X-chromosome are similarly inactive following DNA transfection (Liskay and Evans, 1980;5usslinger et al., 1963;Venolia and Gartler, 1983). Definitive proof that the methylation status of this gene modulates its expression, and therefore IOTlB cell determination, awaits manipulation of the cloned locus. ...
Stable myoblast cell lines were isolated after a brief exposure of mouse fibroblasts (10T1/2 cells) to 5-azacytidine. We show that transfection of 10T1/2 cells with DNA from these azacytidine-induced myoblasts (or from mouse C2C12 myoblasts) results in myogenic conversion of approximately 1 in 15,000 transfected colonies. In contrast, transfection of 10T1/2 cells with DNA from nonmyogenic cells (parental 10T1/2 cell DNA) does not give rise to myoblast colonies. These results indicate that an azacytidine-induced structural modification (presumably demethylation) in the DNA of a single locus is sufficient to convert 10T1/2 cells into determined myoblasts.
... 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. ...
We have obtained a mouse transformant cell line containing two herpes viral thymidine kinase (tk) genes integrated in pericentromeric
heterochromatin. Restriction analysis of tk- revertant and tk+ rerevertant derivatives suggest that one of the two tk genes
is repressed in tk- cells, but is reactivated in tk+ rerevertants. The results of Northern analysis indicated that repression-activation
is probably controlled at the transcriptional level. To examine the molecular basis for this repression, we cloned the tk
gene from a tk- revertant cell line. Then, using the cloned tk gene as donor DNA to select for tk+ transformants, we found
that it has a transfection efficiency indistinguishable from the viral tk gene. This indicates that repression is probably
not mediated via any DNA sequence changes within the tk gene. The results of further studies by restriction analysis, azacytidine
treatments, and secondary DNA transfection assays demonstrated that tk repression is associated with changes in DNA methylation.
Surprisingly, derepression of the tk gene was accompanied by rearrangements in the flanking DNA. The latter result suggests
that the flanking DNA may exert cis effects on tk gene expression. Additional studies with this system may provide insights
into the molecular basis underlying position effects in heterochromatin.
Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.
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 142 CpG 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.
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 cell 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.
HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).
The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.
We have obtained a mouse transformant cell line containing two herpes viral thymidine kinase (tk) genes integrated in pericentromeric heterochromatin. Restriction analysis of tk- revertant and tk+ rerevertant derivatives suggest that one of the two tk genes is repressed in tk- cells, but is reactivated in tk+ rerevertants. The results of Northern analysis indicated that repression-activation is probably controlled at the transcriptional level. To examine the molecular basis for this repression, we cloned the tk gene from a tk- revertant cell line. Then, using the cloned tk gene as donor DNA to select for tk+ transformants, we found that it has a transfection efficiency indistinguishable from the viral tk gene. This indicates that repression is probably not mediated via any DNA sequence changes within the tk gene. The results of further studies by restriction analysis, azacytidine treatments, and secondary DNA transfection assays demonstrated that tk repression is associated with changes in DNA methylation. Surprisingly, derepression of the tk gene was accompanied by rearrangements in the flanking DNA. The latter result suggests that the flanking DNA may exert cis effects on tk gene expression. Additional studies with this system may provide insights into the molecular basis underlying position effects in heterochromatin.
X8/6T2, a hamster-human hybrid cell line which contains an inactive human X chromosome, was treated with 5-azacytidine and selected for derepression of hypoxanthine-guanine phosphoribosyltransferase. Clones were examined for coreactivation of the phosphoglycerate kinase gene (Pgk). Of 68 of these hybrids, approximately 20% expressed measurable human phosphoglycerate kinase (PGK) activity. A 600-base-pair region of the Pgk 5' CpG cluster was examined for the methylation status of eight CCGG sites (site 1 being 5'-most) in a number of PGK-negative and PGK-positive cell lines. The inactive X chromosome is normally methylated at all eight sites, and this was also true for the majority of X8/6T2 cells. However, several PGK-negative hybrids were demethylated in the site 3 to site 6 region. PGK activity correlated with demethylation at both sites 6 and 7. The data for PGK-positive and -negative hybrids indicate that demethylation at or near site 7 was necessary for reactivation of Pgk. Chromatin sensitivity to MspI digestion in the nuclei of male lymphoblastoid cells and several PGK-positive and PGK-negative hybrids was examined. PGK-positive cell lines were hypersensitive to digestion, while PGK-negative hybrids were resistant. Cleavage at sites 6 and 7 was observed in all PGK-positive cell lines at each MspI concentration examined. Sites 7 and 8 were less accessible to digestion than site 6. Cleavage in the site 2 to site 5 region was observable at the lowest MspI concentration. In most PGK-positive hybrids, a nonspecific endogenous nuclease detected the presence of a hypersensitive region spanning at least 450 base pairs, bounded at the 3' end near HpaII site 6. Nuclease hypersensitivity appears to be related to promoter activity, because sites 7 and 8 are in transcribed regions of the gene. These data indicate that specific sites within the CpG cluster have a dominant controlling influence over the Pgk promoter conformation and the transcriptional activation of Pgk.
The X chromosome in placental mammals is subject to a unique system of developmental regulation which involves the coordinate activation and inactivation of all or most of an entire chromosome in a cis-limited fashion. Females begin life with two active X chromosomes but, in the course of their development, genes on one of these X chromosomes become transcriptionally silent and late replicating. X-inactivation results in dosage compensation whereby female (XX) somatic cells become equivalent to male (XY) cells in terms of their X-linked gene products. The inactivation event in the embryonic lineages is random with respect to the parental origin of X chromosomes such that the developing embryo and resulting adult is a mosaic of cells with one or the other X chromosome active. However, X-inactivation is non-random in female marsupials and in the extraembryonic tissues of developing female rodents; in these cases, the paternally-inherited X chromosome is preferentially inactivated. Paternal X-inactivation is one of the best known examples of imprinting, i.e., the differential expression of the genetic material dependent on its gamete of origin. It has been suggested that imprinting of the X chromosome and of autosomal regions are manifestations of the same phenomenon and that evidence from X-inactivation studies may help to elucidate factors responsible for the imprinting of autosomal genes. Although the molecular mechanisms which underly the phenomena of X-inactivation and imprinting have remained elusive, there is growing evidence that DNA modification, in the form of cytosine methylation, may be involved. There is considerable evidence that DNA methylation is associated with changes in chromatin structure and potential for gene expression and critical methylation changes in the promoter regions and other sites correlate with X-linked gene silencing on the inactive X chromosome. The main subject of this thesis is the investigation of the changes in methylation of specific CpG sequences associated with X-linked gene inactivation and an imprinted transgene at different stages of embryonic development. The approach used is PCR amplification of sequences containing informative CCGG sites, 5’ to the X-linked Pgk-1, Hprt and G6pd genes and within the CAT 17 transgene. The DNA is cut with HpaII before amplification; if the site is methylated, amplification will be resistant to HpaII digestion. Primordial germ cells, oocytes, sperm, individual preimplantation embryos, dissected regions of postimplantation embryos and embryonic stem cells have been analysed. Consistent with the activity of both X chromosomes in female embryos as determined by biochemical criteria, it has been shown that oocytes, individual preimplantation embryos and embryonic stem cells are unmethylated. Methylation of the Pgk-1 gene on the inactive X chromosome occurs at the time of X-inactivation in the blastocyst, whereas methylation of the G6pd gene occurs later, but within two days of the initiation of X-inactivation (by 5.5 days’ gestation). The methylation process may be progressive in that it occurs earlier for the Pgk-1 gene which is located close to the inactivation centre. Methylation also occurs in female extraembryonic tissues (paternal X-inactivation) although the sites are unmethylated on the inactive X chromosome in sperm. Hence, X-linked gene methylation is not part of the gamete imprinting mechanism distinguishing the paternal X chromosome. In the female germ cell lineage, which is derived from the epiblast after X-inactivation has occurred, these sites do not become methylated on the inactive X chromosome. Thus, it appears that the germ cell lineage remains separate and undifferentiated with respect to méthylation. HpaII-sensitive PCR analysis of specific CpG sites failed to elucidate the role of methylation in the phenomenon of imprinting associated with this transgene. It is still unclear whether or not the parent-of-origin-dependent differential methylation observed in some transgenes is a parallel system to the genomic imprinting of endogenous genes. These studies have advanced our knowledge of the role of methylation as a molecular mechanism regulating the differential activation and silencing of specific genes in different lineages in early development. The sensitive techniques devised will undoubtedly be informative when applied to an analysis of specific CpG sites associated with the X-inactivation centre gene, Xist, and to endogenous imprinted genes in mouse and human.
It has been clear for a number of years that the functional analysis of biological systems is highly dependent upon the availability of a large number of mutants of broad phenotypic classes. It was with this understanding that our laboratory undertook about ten years ago to develop methods for selection of mutants in somatic cells. Over this decade the field has developed extremely rapidly both in terms of methodological expertise and in the wide spectrum of mutants which can now be used for studies on gene function and regulation.
The origin of uric acid in the body is reviewed, both from exogenous sources, degradation of purine precursors, and de novo purine synthesis. The pathways of the biosynthesis of purines in the body are reviewed, and regulatory enzyme abnormalities which may lead to overproduction of uric acid are described.
We have known for the greater part of the 20th century that mammalian females and males differ in both number and kind of sex chromosomes. While females have two X-chromosomes (XX), males have one X- and one Y-chromosome (XY). Both the X and Y carry genes that enhance female and male reproductive function, respectively. Because the Y-chromosome is relatively small and carries only a few genes, this sex chromosome difference means that females essentially have double the number of sex-chromosome genes compared to males. Yet, biochemical evidence indicates that this difference in chromosome number does not result in sex-specific differences in the total amount of X-encoded RNAs or proteins. In other words, female cells with two copies of every X-linked gene synthesized the same amount of protein products as males cells with half the number of genes. For years, this equation puzzled biologists. Clearly, some form of dosage compensation akin to those found in other sexual organisms must exist in mammals.
Recent approaches towards an understanding of the molecular basis of X-chromosome inactivation in mammals suggest that regulation is due to multiple events including DNA methylation.
In 1975, two papers suggested a role for DNA methylation in X chromosome inactivation. In one paper (Riggs, 1975), I argued that: 1) DNA methylation should affect protein-DNA interactions; 2) methylation patterns and a maintenance methylase should exist; and 3) DNA methylation should be involved in mammalian cellular differentiative processes. Holliday and Pugh (1975) argued similarly, although less weight was given to X inactivation and more weight was given to the possibility that 5-methylcytosine (5-meCyt) might be deaminated to thymidine; thus a specific mutational change would be generated, as suggested by Scarano (1971). Recently, several studies of X chromosome inactivation have contributed to the emerging body of evidence supporting a role for DNA methylation in mammalian gene regulation; it is these studies that will be reviewed in this chapter. More comprehensive reviews of X chromosome inactivation have been published recently (Gartler and Riggs, 1983; Graves, 1983).
Changing DNA methylation patterns during embryonic development are discussed in relation to differential gene expression, changes in X-chromosome activity and genomic imprinting. Sperm DNA is more methylated than oocyte DNA, both overall and for specific sequences. The methylation difference between the gametes could be one of the mechanisms (along with chromatin structure) regulating initial differences in expression of parental alleles in early development. There is a loss of methylation during development from the morula to the blastocyst and a marked decrease in methylase activity. De novo methylation becomes apparent around the time of implantation and occurs to a lesser extent in extra-embryonic tissue DNA. In embryonic DNA, de novo methylation begins at the time of random X-chromosome inactivation but it continues to occur after X-chromosome inactivation and may be a mechanism that irreversibly fixes specific patterns of gene expression and X-chromosome inactivity in the female. The germ line is probably delineated before extensive de novo methylation and hence escapes this process. The marked undermethylation of the germ line DNA may be a prerequisite for X-chromosome reactivation. The process underlying reactivation and removal of parent-specific patterns of gene expression may be changes in chromatin configuration associated with meiosis and a general reprogramming of the germ line to developmental totipotency.
It is estimated that at least 10% of ill health in man is a direct consequence of the inheritance of defective or disease predisposing genes (UNSCEAR 1977), and this estimate does not include diseases, such as certain forms of cancer, which may be associated with acquired somatic genetic changes as opposed to inherited defects. Approximately l% of the live newborn inherit major single gene defects and a proportion of these are a consequence of new mutations. The mutation rate for these changes differs enormously for different genes with at one extreme a gene responsible for a form of X-linked non-specific mental retardation having a mutation rate of around 1 per 104 gametes per generation, whereas other genes may undergo mutation at rates which are at least a thousand fold lower. In addition to the inherited major single gene defects, around 1 in every 150 live newborn babies inherit a chromosomal abnormality which may take the form of an alteration in chromosome number or in chromosome structure, and the majority of these abnormalities are new mutations arising in parental germ lines (Evans 1977a). The incidence of chromosomal mutations in still births and in spontaneous abortuses is very much higher and indeed more than 50% of early abortions are associated with a chromosomal mutation (Boue et al. 1975) and the overall numbers would imply that almost 1 in 10 human gametes carries a chromosomal mutation. Chromosomal and, in some cases, gene mutations can also be readily demonstrated in human somatic cells and chromosomal abnormalities in blood lymphocytes from healthy individuals occur with a frequency of between 1 in 102 to 103 cells. Mutation therefore is by no means a rare event in human somatic and germ cells and the human genome may be a rather less stable entity than is sometimes assumed.
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 nuclease-sensitive 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) hemi-demethylation 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.
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Treatment with 5-azacytidine, a potent inhibitor of DNA methylation, was used to induce activation of the selectable hprt gene on the inactive X chromosome in a diploid female Chinese hamster cell line. The transformed, stably diploid cell line F3B was selected in media containing the lethal purine analogue 6-thioguanine, to generate a phenotypically HPRT- mutant, F3BT1, of presumed genotype hprt-/hprt(+), where (+) represents the presumably wild-type allele on the inactive X chromosome. Treatment of F3BT1 with 5-azacytidine resulted in phenotypic reversion to HPRT+ at a frequency greater than 10^-3. Similar treatment of 6-thioguanine-resistant control lines derived from male cells, or from CHO (which has no inactive X chromosome), had no effect on the frequency of phenotypic reversion, indicating that activation of the hprt(+) allele, rather than reversion of the hprt- is responsible. This conclusion is substantiated by documentation of the low mutagenic capacity of 5-azacytidine in this system. Proof that the hprt(+) allele can be activated by 5-azacytidine treatment was obtained in somatic cell hybrids in which hprt gene products from the active and inactive X chromosomes could be distinguished by isoelectric focusing. Our results demonstrate that X-linked gene activation associated with generalized DNA demethylation occurs with high frequency in transformed diploid Chinese hamster cells.
We have investigated several of the experimental factors that affect calcium phosphate-DNA-mediated gene transfer of thymidine kinase (tk) into mouse LM tk– Cl 1D cells using unfractionated DNA from both Chinese hamster ovary cells and L6 rat myoblasts. Increases in the length of exposure to DNA (24 h) and the expression time (48 h) before selection result in a 20-fold enhancement in the efficiency of transformation. These modifications yield frequencies up to 35 HATR colonies/20 g tk+ DNA/106 recipient cells. Exposure to dimethyl sulfoxide enhances transformation efficiencies slightly for short DNA exposure times, but has no effect when optimal DNA exposure times are used. Several other variations in our standard transformation protocol were also examined: these include the concentration and size of the DNA and exposure to low concentrations of the nonionic detergent, Tween-80. We have also isolated and characterized a subclone of Cl 1D that is a high-efficiency recipient for the tk+ marker. Segregation analysis reveals that the majority of the Tk+ transformants derived from this subclone are stable, in contrast to those derived from the Cl 1D parent. The combination of improved methodology and the highefficiency recipient subclone permits DNA-mediated transformation for tk at frequencies on the order of 10–4 transformants per recipient cell.
The methylation status of the nuclear DNA from a mealybug, aPlanococcus species, has been studied. Analysis of this DNA by High Performance Liquid Chromatography and Thin Layer Chromatography revealed
the presence of significant amounts of 5-—methylcytosine. Since analysis of DNA methylation using the Msp I/Hpa II system
showed only minor differences in susceptibility of the DNA to the two enzymes, it seemed possible that 5-methylcytosine (5mC)
occurred adjacent to other nucleotides in addition to its usual position, next to guanosine. This was verified by dinucleotide
analysis of DNA labelledin vitro by nick translation. These data show that the total amount of 5-methylcytosine in this DNA is slightly over 2.3 mol %, of
which 0.61% occurs as the dinucleotide 5mCpG, 0.68% as 5mCpA, 0.59% as 5mCpT and 0.45% as 5mCpC. 5mCpG represents approximately
3.3% of all CpG dinucleotides. The experimental procedure would not have permitted the detection of 5mCp5mC, if it occurs
in this system. Unusually high amounts of 6-methyladenine (approximately 4 mol %) and 7-methylguanine (approximately 2 mol
%) were also detected, 6-methyladenine and 7-methylguanine occurred adjacent to all four nucleotides. The total G+C content
was 33.7% as calculated from dinucleotide data and 32.9% as determined from melting profiles.
Inactivation of the X chromosome during mammalian spermatogenesis has been postulated to occur by the same mechanism that controls female somatic X chromosome inactivation. We have used DNA-mediated transformation of HPRT– cells to test this idea, because it has been shown previously that inactive X chromosome DNA from somatic cells will not transform HPRT– cells. Isolated DNA from the mature sperm of five mammals (human, mouse, horse, bull, rabbit) were all capable of HPRT transformation, and transformants were confirmed electrophoretically. Measures were taken to ensure that the transformation frequencies observed could not be due to somatic contamination. The positive HPRT transformation result indicates that mature sperm X chromosomal DNA is not modified in the same manner as that of female inactive X chromosomal DNA. Since there is evidence for methylation of the somatic inactive X chromosome, it is possible that methylation, at least for the genes studied, is not involved in sperm X chromosome inactivation.
Nucleoids prepared by gentle lysis of non-complementing diploid cells resulting from Bacillus subtilis protoplast fusion have been used to transform competent cultures of appropriate recipient strains. The yields of transformants were regularly much larger when the transforming allele was expressed in vivo than when it was unexpressed. Ribonuclease treatment of the lysates prior to their use as donors in transformation did not change the yields of transformants. Proteinase treatment had no effect when the selected trait was expressed in vivo, but it restored transforming activity of unexpressed markers to the level of expressed markers.
Proteins bound to the nucleoids of non-complementing diploids are thus responsible for their inability in vitro to transform for unexpressed markers. Whether these proteins are also responsible in vivo for the chromosomal extinctions observed remains unknown.
DNA methylation plays an important role in the regulation of gene expression during development. Methyl moieties at CpG residues suppress transcription by affecting DNA—protein interactions, thus altering the accessibility of genes to trans-acting factors in the cell. Because it works in cis, this mechanism is important in the control of X inactivation and genomic imprinting.
Nr2e1 encodes a stem cell fate determinant of the mouse forebrain and retina. Abnormal regulation of this gene results in retinal,
brain, and behavioral abnormalities in mice. However, little is known about the functionality of human NR2E1. We investigated this functionality using a novel knock-in humanized-mouse strain carrying a single-copy bacterial artificial
chromosome (BAC). We also documented, for the first time, the expression pattern of the human BAC, using an NR2E1-lacZ reporter strain. Unexpectedly, cerebrum and olfactory bulb hypoplasia, hallmarks of the Nr2e1-null phenotype, were not fully corrected in animals harboring one functional copy of human NR2E1. These results correlated with an absence of NR2E1-lacZ reporter expression in the dorsal pallium of embryos and proliferative cells of adult brains. Surprisingly, retinal histology
and electroretinograms demonstrated complete correction of the retina-null phenotype. These results correlated with appropriate
expression of the NR2E1-lacZ reporter in developing and adult retina. We conclude that the human BAC contained all the elements allowing correction of
the mouse-null phenotype in the retina, while missing key regulatory regions important for proper spatiotemporal brain expression.
This is the first time a separation of regulatory mechanisms governing NR2E1 has been demonstrated. Furthermore, candidate genomic regions controlling expression in proliferating cells during neurogenesis
were identified.
Three cytidine analogs containing modifications in the 5-position of the cytosine ring (5-azacytidine, 5-aza-2'-deoxycytidine and pseudoisocytidine) induced the expression of human hypoxanthine/guanine phosphoribosyltransferase (IMP; pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene (HPRT) from a structurally normal inactive human X chromosome retained in a mouse-human somatic cell hybrid. Between 0.1% and 8% of the cells surviving treatment with these analogs were able to form colonies in selective medium (hypoxanthine/aminopterin/thymidine/glycine medium), but two other analogs, 5-fluoro-2'-deoxycrytidine and 5.6-dihydro-5-azacytidine, did not induce HPRT expression. The inactive X chromosome present in the hybrid, was found to be late replicating, and experiments with synchronized cells showed that the induction of HPRT expression by 5-aza-2'-deoxycytidine occurred maximally in cells treated in the latter half of the S phase. Two division cycles were required after analog treatment for the highest frequency of expression of the induced gene. Because these analogs are powerful inhibitors of the methylation of cytosine residues in DNA, the results imply that demethylation of specific DNA sequences may be required for the reexpression of human HPRT.
In mammals, dosage compensation for X-linked genes between males and females is achieved by the inactivation of one of the X chromosomes in females. The inactivation event occurs early in development in all cells of the female mouse embryo and is stable and heritable in somatic cells. However, in the primordial germ cells, reactivation occurs around the time of meiosis. Owing to random inactivation in somatic cells, all female mice and humans are mosaic for X-linked gene function. Variable mosaicism can result in expression of disease in human females heterozygous for an X-linked gene defect.
In the extra-embryonic lineages of female mouse embryos, and in the somatic cells of female marsupials, the paternally inherited X chromosome is preferentially inactivated. The X chromosomes in the egg and sperm must be differentially marked or imprinted, so that they are distinguished by the inactivation mechanism in these tissues.
Initiation of inactivation of an entire X chromosome appears to spread from a single X-inactivation centre and may involve the recently discovered gene,XIST, which is expressed only from the inactive X chromosome. The maintenance of inactivation of certain household genes on the inactive X chromosome involves methylation of CpG islands in their 5' regions. Critical CpG sites are methylated at, or very close to, the time of inactivation in development.
The mouse and the human X chromosomes carry the same genes but their arrangement is different and there are some genes in the pairing segment and elsewhere on the human X chromosome which can escape inactivation. Regions of homology between the mouse and human X chromosomes allow prediction of the map positions of homologous genes and provide mouse models of genetic disease in the human.
A common mutation causing thalassemia in Mediterranean populations is an amber (UAG) nonsense mutation at the 39th codon of the human beta-globin gene, the beta-39 mutation. Studies of mRNA metabolism in erythroblasts from patients with beta-39 thalassemia and studies using heterologous transfection systems have suggested the possibility that this mutation not only affects protein synthesis but also alters mRNA metabolism. The effects of this mutation on several steps in the metabolism of mRNA have been investigated by transfection of the gene into permanent cell lines bearing a temperature-sensitive RNA polymerase II. Several RNA expression studies were performed, including analysis of transcription, mRNA stability, mRNA splicing accuracy, and mRNA polyadenylation. The results suggest that the defect in expression of the beta-39 mRNA occurs at a step prior to the accumulation of mRNA in the cytoplasm.
A large body of evidence demonstrates that DNA methylation plays a role in gene regulation in animal cells. Not only is there a correlation between gene transcription and undermethylation, but also transfection experiments clearly show that the presence of methyl moieties inhibits gene expression in vivo. Furthermore, gene activation can be induced by treatment of cells with 5-azacytidine, a potent demethylating agent. Methylation appears to influence gene expression by affecting the interactions with DNA of both chromatin proteins and specific transcription factors. Although methylation patterns are very stable in somatic cells, the early embryo is characterized by large alterations in DNA modification. New methodologies are now becoming available for studying methylation at this stage and in the germ line. During development, tissue-specific genes undergo demethylation in their tissue of expression. In tissue culture cells this process is highly specific and appears to involve an active mechanism which takes place in the absence of DNA replication. The X chromosome undergoes inactivation during development; this is accompanied by de novo methylation, which appears necessary to stably maintain its silent state. As opposed to the programmed changes in DNA methylation which occur in vivo, immortalized tissue culture cells demonstrate alterations in DNA modification which take place over a long time scale and which appear to be the result of selective pressures present during the growth of these cells in culture.
Tissue-specific animal cell genes are usually fully methylated in the germ line and become demethylated in those cell types in which they are expressed. To investigate this process, we inserted a methylated IgG kappa gene into fibroblasts and lymphocytes at various stages of development. The results show that this gene undergoes demethylation only in the mature lymphocytes and therefore suggest that the ability to demethylate a gene is developmentally regulated. These studies were supported by similar experiments using the rat Insulin I gene, and in this case it appears that the cis-acting elements that control demethylation may be different from those responsible for gene activation. The ability to demethylate the housekeeping gene APRT is also under developmental control, because this occurs only in embryonic cells, both in tissue culture and in transgenic mice.
The extent of methylation of DNA sequences upstream and within the two X-linked genes, Pgk-1 and Hprt, was analyzed in male and female somatic cells and in female embryonal carcinoma cells carrying either two active X chromosomes (Xa) or one active and one inactive X chromosome (Xi). Sites upstream and within the first intron of both Pgk-1 and Hprt were heavily methylated on the Xi in somatic cells and in embryonal carcinoma cells with an Xi. Reactivation of this Xi was accompanied by extensive demethylation of these sites. In female embryonal carcinoma cells with two active X chromosomes, one X inactivates during differentiation in culture; however, methylation did not occur during differentiation, consistent with the idea that DNA methylation does not play a role in the initiation of X inactivation but may be involved in maintaining inactivation of those genes on the Xi.
I am indebted to Mary Lyon as her X-inactivation hypothesis stimulated my mentor, Barton Childs, and in turn, myself, to think about the consequences of X-inactivation in heterozygous females. I often reread her original papers setting forth the single active X hypothesis, and still marvel at the concise and compelling exposition of the hypothesis and the logical predictions which seemed prophetic at my first reading, and have survived the test of time.
My contribution to this Festschrift reviews evidence derived from studies of DNA methylation, species variation and DNA replication that reveals an important role for methylated CpG islands and suggests a role for late DNA replication in propagating X inactivation from one cell to its progeny. These studies also show that X inactivation is a powerful research tool for identifying the factors which program and maintain developmental processes.
During development of the female mouse embryo, one of the two X chromosomes is inactivated in a random manner in most cell lineages. However, in the extraembryonic trophectoderm and primary endoderm lineages there is preferential inactivation of the paternally derived X chromosome. The inactivated X chromosomes of the extraembryonic and somatic tissues appear equally inactive at the level of the expression of X-linked genes. However, there are differences in the timing of their replication and the extent of DNA modification as determined by gene transfer. The identification of transgenic animals carrying X-linked modified alpha-fetoprotein (AFP) genes allowed us to examine whether the inactivation process extends to an autosomal gene which is normally expressed at high levels in specific extraembryonic and somatic cells, and if so, whether the inactivation process is different in these two tissues. Our results demonstrate that the X-linked AFP genes were expressed on the inactive X chromosome in the visceral endoderm of the yolk sac but not in fetal liver. Thus, the transcriptional activity of the AFP minigene on the inactive X chromosome is dependent on the tissue in which it resides, and most probably reflects differences in the nature of the maintenance of the inactive state of the extraembryonic and embryonic X chromosomes.
It is likely that most vertebrate genes are associated with 'HTF islands'--DNA sequences in which CpG is abundant and non-methylated. Highly tissue-specific genes, though, usually lack islands. The contrast between islands and the remainder of the genome may identify sequences that are to be constantly available in the nucleus. DNA methylation appears to be involved in this function, rather than with activation of tissue specific genes.
The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell
cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine
phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted
in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked
genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT
had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond
to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and
perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were
differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as
a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.
HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).
Three genes on the human inactive X chromosome retained in the Chinese hamster X human hybrid cell line X8/6T2 have been reactivated using the demethylating agent, 5-azacytidine (5-aza-CR). Pulse-labeling and histochemical methods permitted detection and measurement of reactivation rates of the hypoxanthine phosphoribosyltransferase (Hpt) and glucose-6-phosphate dehydrogenase (G6pd) genes within 48 h of treatment. About 50% of the cells became active for these genes, which represents a reactivation rate some 30-fold greater than previously reported in similar systems. The phosphoglycerate kinase (Pgk) gene was not reactivated as frequently as the Hpt or G6pd genes. Segregation analysis of progeny of treated cells showed that enzyme-positive and enzyme-negative cells were produced in proportions supporting the notion that 5-aza-CR causes demethylation by replicative loss and that demethylation leads to reactivation.
Transformation of human cells from a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75)-negative to a thymidine kinase-positive phenotype has been achieved by using purified DNA from herpes simplex virus type 2. The specific activity of the DNA was in the range 0.5 to 2.0 transformants per microng and the efficiency of gene transfer was up to 1 transformant per 10(5) recipient cells. Several transformed lines able to grow continuously in medium selective for thymidine kinase-positive cells have been established. All of these lines express a thymidine kinase activity of viral origin but they differ from each other in the stability of enzyme expression. Subclones derived from a given transformed line inherited the degree of stability of the parental line.
A new technique for assaying infectivity of adenovirus 5 DNA has been developed. Viral DNA was diluted in isotonic saline containing phosphate at a low concentration, and calcium chloride was added, resulting in the formation of a calcium phosphate precipitate. DNA coprecipitated with the calcium phosphate and, when the resulting suspension was added to human KB cell monolayers, became adsorbed to the cells. Following adsorption, uptake of DNA into the cells occurred during an incubation in liquid medium at 37 ° in the continued presence of extra calcium chloride.For adenovirus 5 DNA the assay resulted in up to 100-fold more plaques than could be obtained using DEAE-dextran. Furthermore a reproducible relationship between amounts of DNA inoculated per culture and numbers of plaques produced was demonstrated. The assay was most efficient at high DNA concentrations (10–30 μg/ml); below this range the addition of carrier DNA was necessary for optimum results.In addition to adenovirus 5 DNA, the technique has been used successfully to assay infectivity of DNA from adenovirus 1 and simian virus 40.
Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 510–8 colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK– deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK– cells, 310–6. Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10–4/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.
We have constructed ideograms of human prometaphase chromosomes from synchronized and from standard 72-h lymphocyte cultures. G banding was achieved by a trypsin-Giemsa (or Wright's stain) method.
In addition to light (white) and dark (black) bands, we have distinguished three different shades of grey. This distinction is essential for proper identification of the increasing number of bands displayed by high-resolution chromosomes. The relative amount of chromatin in each category of staining intensity has been calculated and expressed as ‘light value.’
The ideograms represent the maximal number of bands discernible with some consistency on prometaphase chromosomes, i.e., 721 euchromatic and 62 ‘variable’ heterochromatic or heteromorphic bands.
The ideograms are based on measurements. On selected printed copies of each chromosome derived from different cells and different individuals, the relative width of each band was measured in relation to the length of the respective chromosome arm. The measurements per chromosome were averaged and used for construction of the ideograms. The distance of each border between bands or sub-bands from the centromere has been calculated on a relative scale, with positions 0 at the centromere and 1.0 at the p terminus or q terminus.
The numbering system for bands and sub-bands follows the Paris Conference (1971) recommendations.
The late-replicating X of the mouse can be clearly identified in a large proportion of metaphase spreads as an unlabelled chromosome when labelling of cells is done early in the S period. The Y chromosome follows this same pattern. Studies on Cattanach’s translocation are also reported, and it has been shown that the Xt can be distinguished from the normal X in early labelled material.Copyright © 1970 S. Karger AG, Basel
Clones of fibroblasts from a G6PD A heterozygote transformed with SV-40 did not express the G6PD silent allele in the transformed heteroploid cultures. In addition, transformed fibroblasts from a woman heterozygous for both G6PD A and HGPRT deficiency, subjected to selective pressure, did not reveal a single cell expressing either silent allele. Since the incidence of sex chromatin was significantly lower in these cells after transformation, it is likely that the loss of sex chromatin reflects the loss of the inactive X-chromosome at an early stage following transformation.
Mouse L cells lacking the enzyme thymidine kinase (LMTK-) have been converted to a TK+ phenotype by infection with fragmented HSV2 strain 333 DNA. The DNA fragments used were either unique, produced by cleavage with the restriction endonucleases Eco RI and Hild III, or randomly produced by mechanical shearing. Survival in HAT medium was used initially to establish the TK+ phenotype; clones possessing the ability to grow in selective medium were picked on the basis of differing morphology and growth rates. Cytosol extracts of these clones possessed virus-specified TK activity identical to that present in cells lytically infected with HSV2, as indicated by thermolability and mobility on polyacrylamide gel electrophoresis. The transformed cells also exhibit HSV-specific immunofluorescence. Based on these transformation studies, it is possible to assign a map location to the TK gene on the HSV genome.
Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.
Five embryonic mouse cultures and one human fibroblast culture were transformed with SV40. The cultures were studied cytologically to see if the normal pattern of sex chromosome replication was maintained in SV40 transformed cells. Characteristic late replication patterns were observed for both the X and Y chromosomes, and there was no evidence for loss of the inactive X chromosome, even in cells with 4 or more X chromosomes. The human line was heterozygous at two X-linked loci and a clonal analysis showed that the expression of X-linked genes was not affected by SV40 transformation.
Cells of the Chinese hamster line V79-8 multiply without a G1 period (i.e., they are G1(-)) and have an average generation time of 9.5 hr. After mutagenesis and selection we have derived five stable mutants (or variants) of this line that have longer generation times. In each case the increase in generation time is due solely to the introduction of a G1 period into the cell cycle, with no measurable effect on S, G2, or M. Fusions among these five G1(+) mutant lines and another presumably nonmutant G1(+) line (V79-743) produce hybrid cells lacking a G1 period in all but one case. These complementation tests define five complementation groups among these six G1(+) cell lines. The six G1(+) lines represent five different causes or bases for the presence of a G1 period. The two G1(+) mutants belonging to complementation group V are temperature sensitive for expression of the G1(+) phenotype (G1 congruent with 0, 4, and 6 hr at 33 degrees , 37 degrees , and 39 degrees , respectively). In all cases the G1(-) state is dominant over the G1(+) state, suggesting that the presence of G1 represents a "deficient" condition. Mutants of this type may be useful in the analysis of the switch from G1(-) to G1(+) that occurs normally in cleaving embryos and in elucidation of the genetic mechanism(s) responsible for the presence of a measurable G1 in most cells.
Under selective growth conditions a revertant of mouse cells, defective in hypoxanthine phosphoribosyltransferase activity (HPRT, EC-No. 2.4.2.8), was isolated, which contained an electrophoretically abnormal form of HPRT activity. The specific HPRT activity in crude extracts of the revertant cells is about 30% of the level determined in normal wild type cells. The variant HPRT reacts with antiserum against normal mouse HPRT but the rate of heat inactivation of the variant activity is different from the wild type form. By isozyme and karyotype analyses of somatic cell hybrids between the revertant mouse cells and Chinese hamster cells we found that the abnormal HPRT activity is coded for by the mouse X-chromosome as expected for a mutation in the structural HPRT gene.
DNA has been purified from the abnormal HPRT revertant cells and incubated with mouse A9 cells (HPRT-). After growth in selective medium one clone was isolated which expressed the electrophoretically abnormal form of HPRT. Six clones showed the normal form of HPRT due to reversion of the defective HRRT locus in A9 cells. This result indicates DNA-mediated transfer of the mouse HPRT gene at a frequency of about 0.5×10-7. A similar frequency has been found for transfer of the variant HPRT locus via isolated metaphase chromosomes to A9 recipient cells. When placed in non-selective media the DNA-mediated transferent cells gradually lost their ability to express the HPRT transgenome at a rate of about 6% per average cell generation.
As a means of obtaining insights into the mechanisms for maintaining X-chromosome inactivation, we have carried out a series of experiments in an attempt to reverse the process. To identify cells in which the silent X has been derepressed, we have developed a model based on the one described by Comings (1966) using human fibroblasts heterozygous for the common A electrophoretic variant of glucose-6-phosphate dehydrogenase (G6PDA). In our model, however, the cells are also heterozygous for the Lesch-Nyhan mutation specifying deficiency of hypoxanthine guanine phosphoribosyl transferase (HGPRT-), so that we can select for rare cells in which reactivation has occurred (Migeon 1972). Clonal populations of skin fibroblasts from females heterozygous for both G6PDA and HGPRT-, but expressing only the alleles on the active X, are subjected to a variety of treatments, and the phenotype with regard to both loci is ascertained following treatment. The resultant phenotype is interpreted according to Table 1. The G6PD heteropolymer, because it is never found in mixtures of the two cells under conditions used for these studies, is a sensitive indicator of two functional X chromosomes within the same cell, while the presence of variants at two X-linked loci helps distinguish reactivation from other events such as reversion, somatic crossing over, or contamination with cells of other phenotype.
Several authors have put forward theories explaining the mechanism of inactivation of one X chromosome in mammals that underlies the Lyon hypothesis. In these theories, more attention is given to the question of which of the two X chromosomes is chosen for inactivation than to the problem of how, once the decision has been made, inactivation of the same chromosome is maintained in somatic cells.
The initial step in mammalian sexual differentiation is based on the XX: XY chromosomal system. In order to function properly, this chromosomal mechanism must be regulated to eliminate the aneuploidy effects in somatic tissues and still insure normal sexual differentiation and development. In mammalian forms, an X-chromosome regulatory mechanism has evolved to carry out these developmental functions. The two X chromosomes in the female germ line remain active through most of their ontogeny to bring about normal ovarian function; a single X chromosome is active in the female soma so as to eliminate gross aneuploidy effects between males and females; and in the male germ line the single X chromosome is inactivated or eliminated at an apparently critical stage in spermiogenesis. This is the broad outline of mammalian X-chromosome regulation. The specifics vary in different forms: random X-chromosome inactivation in most eutherian mammals, a possible nonrandom mechanism in marsupials, and a chromosomal elimination system in the creeping vole, Micron’s oregoni.
THE X linkage of loci coding for glucose-6-phosphate de-hydrogenase (G6PD), hypoxanthine-phosphoribosyl transferase (HPRT), and phosphoglycerate kinase (PGK) has been determined for the mouse species Mus musculus and M. caroli. Evidence was obtained from a somatic cell genetic analysis of cultured embryonic cells of an interspecific hybrid foetus. The X-chromosomal assignment of these loci has been determined already for several other mammalian species. This evidence was obtained from genetic analyses of electrophoretic variants1 and enzyme deficiencies2,3; from the expression of these genes in viable interspecific hybrids4-6 from somatic cell genetic analyses of heterozygotes7-9, and from the expression of these loci in interspecific somatic cell hybrids10.
Evidence for derepression of the gene for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) on the human inactive X chromosome was obtained in hybrids of mouse and human cells. The mouse cells lacked HPRT and were also deficient in adenine phosphoribosyltransferase (APRT; AMP: pyrophosphate phosphoribosyltransferase; EC2.4.2.7). The human female fibroblasts were HPRT-deficient as a consequence of a mutation on the active X but contained a normal HPRT gene on the inactive X. The two human X chromosomes were further distinguished by differences in morphology: the inactive X was morphologically normal while the active X included most of the long arm of autosome no. 1 translocated to the distal end of the X long arm. Forty-one hybrid clones were first isolated by selection for the presence of APRT; when these clones were selected for HPRT, six of them yielded derivatives having human HPRT with incidences of about 1 in 10-6 APRT-selected hybrid cells. The HPRT-positive derivatives contained a normal-appearing X chromosome indistinguishable from the inactive X of the parental human fibroblasts. The active X with the translocation was not found in any of the HPRT-positive hybrid cells. Human phosphoglycerokinase (ATP:3-phospho-D-glycerate 1-phosphotransferase. EC 2.7.2.3) and glucose-6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP 1-oxidoreductase, EC 1.1.1.49), which are specified by X-chromosomal loci, were not detected in the hybrids expressing HPRT even though they contained an apparently intact X chromosome. The observations are most simply explained by the infrequent, stable derepression of inactive X chromosome segments that include the HPRT locus but not the phosphoglycerokinase and glucose-6-phosphate dehydrogenase loci.
A model based on DNA methylation is proposed to explain the initiation and maintenance of mammalian X inactivation and certain aspects of other permanent events in eukaryotic cell differentiation. A key feature of the model is the proposal of sequence-specific DNA methylases that methylate unmethylated sites with great difficulty but easily methylate half-methylated sites. Although such enzymes have not yet been detected in eukaryotes, they are known in bacteria. An argument is presented, based on recent data on DNA-binding proteins, that DNA methylation should affect the binding of regulatory proteins. In support of the model, short reviews are included covering both mammalian X inactivation and bacterial restriction and modification enzymes.
Most earthquake losses result from damage to structures, but much can be done to mitigate urban earthquake risk. Because basic data and analysis are lacking for how buildings and other structures perform under extreme loads, this [Policy Forum][1] describes two U.S. government programs for expanding placement of recording instruments and sensors in seismic regions and for developing tools for modeling and simulation to predict building performance during earthquakes. These combined efforts could lead to significant progress toward building resiliency in the urban environment.
[1]: http://www.sciencemag.org/cgi/content/full/304/5677/1604
A near-diploid mouse ceil line has been produced from C57BL/6J X AKR/J F1-hybrid embryonic cells by spontaneous transformation. The modal chromosome number of this line is 41 (2n = 40), with low numerical and structural variation relative to other mouse lines which have been reported. Giemsa-banded karyotypes on a number of clones and subclones from this line have been analyzed. The modal karyotypes deviate very little from that of a normal diploid female. Most of the cells have two X chromosomes, one of which is unlabeled early in the S phase.Copyright © 1974 S. Karger AG, Basel
Using human somatic cell/mouse cell hybrids, no evidence for reactivation of the silent X chromosome in human somatic cells has been obtained.
It is suggested that cellular differentiation is established during
development by the development of higher order structural polymorphism
of the chromosomes. The differentiated superstructure, once adopted,
would be maintained during replication.
In the normal XX female, one of the two X chromosomes is inactivated at an early stage in development. This article discusses the current theories proposed to account for X inactivation.
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