Non-expressing Tissues Research Articles

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.

DNA methylation and the regulation of aldolase B gene expression.

DNA methylation was studied as a potential factor for the regulation of tissue-specific and developmentally specific expression of the rat aldolase B gene. We examined cytosine methylation in the HpaII and HhaI recognition sequences in the aldolase B gene in aldolase expressing and nonexpressing tissues and cells. Out of the 15 methyl-sensitive restriction sites examined, the sites in the 3'-half and 3'-flanking regions were found to be heavily methylated in all the tissues or cells, regardless of the level of aldolase B gene expression. However, the methylation pattern in the region immediately upstream and in the 5'-half of the gene exhibited tissue-specificity: the site located about 0.13 kb upstream of the cap site (just next to the CCAAT box), and the sites in the first intron (intron 1) were heavily methylated in nonexpressing cells and tissues (ascites hepatoma AH130 and brain), whereas those in an expressing tissue (liver) were considerably less methylated. These results suggest that cytosine methylation at the specific sites in the 5'-flanking and 5'-half regions of the gene is associated with repression of the gene activity. However, the gene is still substantially methylated in the fetal liver on day 16 of gestation, when it is in a committed state for rapid activation in the period immediately afterwards (Numazaki et al. (1984) Eur. J. Biochem. 152, 165-170). This suggests that demethylation of the methylated cytosine residues in the specific gene region is not necessarily required before activation of the gene during development, but it may occur along with or after the activation.

Sequential changes in DNA methylation patterns of the rat phosphoenolpyruvate carboxykinase gene during development.

The cytosolic phosphenolpyruvate carboxykinase [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] gene was isolated from a rat genomic library, and a map of the methylatable sites C-C-G-G and G-C-G-C has been constructed. The extent of methylation of 18 sites in the PEPCK gene in adult liver, kidney, spleen, and heart muscle and in fetal liver has been analyzed using the 5-methylcytosine sensitive enzymes Hpa II and Hha I. This analysis revealed extensive undermethylation of the PEPCK gene in the adult liver and kidney (PEPCK-expressing tissue), whereas the gene in adult spleen and heart muscle as well as in fetal liver (PEPCK-nonexpressing tissues) was heavily methylated. However, unlike the gene in the adult nonexpressing tissues, a region in the middle of the gene was found to be partially hypomethylated in fetal liver. This hypomethylation correlates with the competence of the fetal liver gene to be expressed. Treatment of fetuses by in utero injection of 5-azacytidine causes a hypomethylation-associated activation of the PEPCK gene. Taken together, the present findings suggest a sequential loss of methyl groups during development. When related to PEPCK gene expression, the sequential loss of methyl groups demonstrates an early stage prior to transcription characterized by hypomethylation of discrete sites and a later developmental hypomethylation of all sites associated with the mature active PEPCK gene around the time of birth.

The cardiac myosin light chain (MLC-2A) gene in chicken is methylated in both expressing and nonexpressing tissues.

Certain specific methylation sites of the chicken cardiac myosin light chain (MLC-2A) gene in DNA from embryonic heart tissue as well as from embryonic livers and brains were studied. MLC-2A specific mRNA was present in the heart only, indicating that the gene is transcriptionally active in this tissue and inactive in liver and brain. Using the methylation-sensitive restriction enzymes Hpa II and Hha I, the gene was found to be identically methylated in DNA from all three examined tissues, irrespective of its expression. Moreover, the gene was extensively methylated throughout, including the 5' end and 3' flanking regions, with the exception of 3 Hpa II sites, one located in the middle part of the gene, one in the last intron and one in the 3' flanking sequence. These results suggest that specific genes can be actively expressed even when they are highly modified, and that changes in the methylation pattern from one tissue to another are not necessarily associated with the gene activation.

Developmental regulation of albumin and alpha-fetoprotein gene expression in the rat.

Albumin and AFP comprise a developmentally regulated gene family expressed predominantly in liver and yolk sac. In this study, we have analyzed the developmental changes in the levels of transcripts in liver and yolk sac, and the methylation of the genes in yolk sac. We have previously analyzed the gene methylation in developing liver and non-expressing tissues (Kunnath and Locker, 1983, EMBO J., 2, 317-324). From these analyses we can distinguish 3 active modes of coordinated expression. The adult liver synthesizes albumin, the yolk sac synthesizes AFP, and the fetal liver synthesizes both. In the yolk sac, the relatively inactive albumin gene is hypermethylated, while the active AFP gene is undermethylated. Our data suggest that the genes are activated simultaneously but that subsequent enhancement is specific for tissue and developmental stage.

Tissue- and cell-specific casein gene expression. II. Relationship to site-specific DNA methylation.

The relationship between DNA methylation and the expression of the gamma- and beta-casein genes was investigated in both expressing and nonexpressing tissues and in isolated tumor cell subpopulations displaying differential casein gene expression. MspI/HpaII digestions of DNA isolated from liver, a totally nonexpressing tissue, indicated that specific sites of hypermethylation existed in these genes as compared to the DNA isolated from casein-producing lactating mammary gland. The positions of these sites were mapped in the gamma-casein gene by comparing total genomic DNA Southern blots to the restriction digests of several overlapping phage clones constituting the gamma-casein gene. In contrast, the methylation status of the HhaI sites in the gamma-casein gene was found to be invariant regardless of the expression status of the gene. The inverse correlation between the hypermethylation of certain MspI/HpaII restriction sites in the casein genes and their potential expressibility was further substantiated by studies in 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced mammary carcinomas, which have an attenuated casein gene expression, and in cell subpopulations isolated from the 7,12-dimethylbenz(a)-anthracene tumor which were either depleted or enriched in casein-producing cells. Analysis of total tumor DNAs indicated that the casein genes were hypermethylated at the same sites observed in liver. However, a very faint hybridization signal was observed in the HpaII digests, suggesting cell-specific methylation differences. We have confirmed the hypomethylation of at least two of these MspI/HpaII sites within the subpopulation containing the casein-producing cells at a level consistent with the relative enrichment in that fraction. These results demonstrate differential site-specific casein gene methylation not only between tissues but also between cell subpopulations within a single tissue.

Comparison of the methylation patterns of the two rat insulin genes.

We have investigated whether DNA methylation is involved in regulating the expression of the rat insulin genes. We studied cytosine methylation in and near the two insulin genes to examine the correlation of site-specific methylation with expression in different tissues and to compare the demethylation patterns of the two genes. For both genes, we found certain sites undermethylated only in insulin-producing tissues. However, the overall patterns of the two genes in expressing tissues are quite different. The insulin I gene is significantly less methylated than the insulin II gene in a tumor that makes equal amounts of the two insulin RNAs. In another tumor and cell line that make 5-fold higher levels of insulin I RNA than insulin II RNA, the changes in the methylation levels of the two genes do not correlate with the increased expression of insulin I. In nonexpressing tissues, the insulin I gene is completely methylated, while the insulin II gene is demethylated at a number of sites. Thus, the general level of methylation of the insulin genes does not correlate with their differential expression. Furthermore, the methylation at specific sites does not correlate either; some CG dinucleotides that appear to be important for one gene are not present in the other. Our results suggest that there is no specific control by methylation of the expression of the insulin genes in the rat.

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.