Methylation patterns in the gene for the alpha subunit of chorionic gonadotropin are inherited with variable fidelity in clonal lineages of human fibroblasts.

Cytosine methylation in DNA of an endogenous single-copy gene locus, the alpha-subunit of human chorionic gonadotropin (alpha-hCG), was assessed in a mass culture and individual clonal lineages of human diploid fibroblasts. Progressive hypomethylation at -CCGG- sites in this gene occurred during the replicative lifespan of the mass culture and was shown to arise randomly during clonal expansion. Thus, some clones and subclones lost -CCGG- methylation in the alpha-hCG gene region while others maintained essentially complete methylation. These data indicate significant interclonal variability in the fidelity with which DNA methylation is transmitted in an endogenous gene.     

Clonal variation in gene methylation: c-H-ras and alpha-hCG regions vary independently in human fibroblast lineages

The stability of DNA methylation has been followed in clonal lineages of human diploid fibroblasts, for the gene regions encoding the c-H-ras proto-oncogene and the alpha subunit of human chorionic gonadotropin (alpha-hCG). Although methylation losses predominated, both de novo gains and losses of cytosine methylation were observed in subclones and sub-subclones, at frequencies which differed between individual clonal lineages, and between the 2 gene regions compared. Methylation of these loci varied independently among clones; e.g., a lineage which showed frequent methylation loss in the c-H-ras gene region remained highly methylated for alpha-hCG, and vice versa. Thus, the fidelity with which DNA methylation is inherited in specific endogenous gene regions must be governed by a clone-specific property affecting local chromatin structure, but apparently not by gene expression per se. Late in the replicative life-span of diploid fibroblasts, as cell replication slowed, restriction patterns for methylation-sensitive enzymes became simpler and more discrete, while those for other enzymes did not change. This is interpreted as a consequence of 'clonal succession', in which the fastest-replicating or longest-lived clones/subclones eventually predominate in a cell population; it could also reflect a decreased rate or a non-random selection of methylation changes in late-passage cells.     

Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3'' enhancer in the stage-specific expression of globin genes.

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

A novel type of secondary modification of two CCGG residues in the human gamma delta beta-globin gene locus

The majority of the CCGG residues in the human gamma delta beta-globin gene locus are cleaved by Msp I, irrespective of the tissue of origin of the DNA, although these sites show differential sensitivity to Hap II as a result of methylation of the internal C residue of the cleavage site (ref 6). Two CCGG sites, at homologous positions 54 nucleotides in front of the G gamma- and A gamma-globin genes respectively, are not cleaved by Msp I in DNA from several human tissues, although DNA from placenta, foetal liver and from some established cell lines is cut at these sites. We have cloned the A gamma-globin gene from foetal blood DNA where the relevant CCGG site is not cut by Msp I. After cloning, the CCGG site can be cut by Msp I. The failure to cleave at this CCGG site in foetal blood DNA therefore, is not the result of a change in the DNA sequence of the cleavage site. Most likely the external C residue and perhaps both C residues are blocked by methylation at these two specific sites.     

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

DNA modification, differentiation, and transformation

Substantial evidence has accumulated over the last 5 years that the methylation of cytosine residues in vertebrate DNA is implicated in the control of gene expression. We have used analogs of cytidine, modified in the 5 position, as specific inhibitors of DNA methylation to probe the relationship between this process and cellular differentiation. 5-Azacytidine effected marked changes in the differentiated state of cultured cells and induced the formation of biochemically differentiated muscle, fat, and chondrocytes from mouse fibroblast cell lines. Since the analog is a powerful inhibitor of DNA methylation, we suggest that this inhibition is causally related to the mechanism of phenotypic conversion. DNA extracted from cells treated with 5-azacytidine was hemimethylated and was used as an efficient acceptor of methyl groups in an in vitro reaction in the presence of eukaryotic methylases. In vitro methylation was inhibited if the substrate DNA was preincubated with a diverse range of chemical carcinogens including benzo(a)pyrene diolepoxide. Thus, chemical carcinogens may induce changes in gene expression by alteration of cellular methylation patterns. Recent experiments have also demonstrated that freshly explanted diploid fibroblasts from mice, hamsters, and humans lose substantial quantities of 5-methylcytosine during cell division and aging in culture. Taken together, these experiments suggest that the genomic distribution of 5-methylcytosine might have importance in normal differentiation and also in the aberrant gene expression found in cancer and senescence in culture.     

DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.     

Mitochondrial DNA in mortal and immortal human cells. Genome number, integrity, and methylation

Mitochondrial DNA was quantitated in total DNA of various normal and mutant strains of human diploid fibroblasts (finite replicative lifespan) and permanent cell lines, using Southern-transfer hybridization to 32P-labeled pure mtDNA probe and saturation hybridization to 3H-labeled cRNA copied from mtDNA. In six normal fibroblast strains, mtDNA copy number increased during serial passage roughly in proportion to cell volume or protein content, whereas normalized mtDNA content per pg of protein depended upon in vivo donor age but not passage level ("in vitro" age). Copy numbers for mtDNA varied much more widely in individual fibroblast clones than in mass cultures, but were not well correlated with longevity or growth rate. Five mutant fibroblast strains associated with reduced replicative lifespan, and four permanent cell lines, were also examined; in each group, mtDNA values were observed both lower and higher than any obtained for normal fibroblasts. No evidence was found of petite-type deletions from human mtDNA, either at late passage or in individual clones of fibroblasts. Methylation of mtDNA genomes was strikingly non-random and apparently decreased with culture age.     

Humans and old world monkeys have similar patterns of fetal globin expression

The expression of epsilon- and gamma-globin mRNA and protein has been determined in three Old World monkey species (Macaca mulatta, Macaca nemestrina, and Cercopithecus aethiops). Using RT-PCR with primers for epsilon- and gamma-globin, both mRNAs were detected in early fetal stages, whereas at 128 days (85% of full term), only gamma was expressed. High-performance liquid chromatography was used for separation and quantitation, and matrix-assisted laser desorption/ionization mass spectrometry was used for identification of globin polypeptides. An alpha-globin polymorphism was observed in all of the species examined. During fetal life, gamma-globin was the predominant expressed beta-type globin. The red blood cells of infants still contained substantial amounts of gamma-globin, which declined to negligible levels in 14 weeks as beta-globin expression reached adult values. The ratio of gamma1- to gamma2-globins (equivalent to Ggamma/Agamma in humans) was approximately 2.5, similar to the Ggamma/Agamma ratio observed in humans. Thus, gamma-globin gene expression in these Old World monkeys species has three features in common with human expression: expression of both duplicated gamma genes, the relative preponderance of gamma1 over gamma2 expression, and the delay of the switch from gamma- to beta-globin until the perinatal period. Thus, the catarrhines seem to share a common pattern of developmental switching in the beta-globin gene cluster, which is distinct from the timing of expression in either prosimians or the New World monkeys. Our results indicate that an Old World monkey, such as Rhesus, could serve as a model organism (resembling humans) for experimentally investigating globin gene expression patterns during the embryonic, fetal, and postnatal stages.     

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.     

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.