Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

Demethylation of satellite I DNA during senescence of bovine adrenocortical cells in culture.

Over the finite proliferative life span of cultured bovine adrenocortical cells, satellite I DNA shows a progressive and extensive loss of methylation at CCGG sites. This was shown by Southern blotting after digestion with the methylation-sensitive enzyme HpaII alone, which provides a sensitive indicator of methylation loss, or digestion with the combination of EcoRI and HpaII, which provides a quantitative indication of loss of methylation. Bovine tissues, including adrenal cortex, all showed a much higher level of satellite methylation than cultured adrenocortical cells. After adrenocortical cells are placed in culture, some demethylation of satellite I is seen as early as 10 population doublings. By 80 population doublings, loss of satellite DNA methylation is extensive. The loss does not appear to prevent continued cell division, since an extended life span clone of bovine adrenocortical cells transfected with SV40 T antigen showed a similar pattern of extensive demethylation. Satellite demethylation has been reported in aging in vivo and the present cell culture system may provide an in vitro model for this form of genetic instability.     

Changes in gene expression during senescence of adrenocortical cells in culture

Bovine adrenocortical cells undergo a process in which expression of steroid hydroxylases is lost progressively as a function of population doubling level (PDL) in culture. Each cytochrome P450 shows a characteristic rate of loss of expression as a function of PDL (in order of rates of loss: CYP11B > CYP21 > CYP17 > CYP11A). CYP11B and CYP21 require insulin-like growth factor I as well as cyclic AMP; these are the only factors required for induction in the primary culture. Middle- and later passage cells do not express CYP11B and CYP21 under the same conditions, but will do so when cells are grown in extracellular matrix Matrigel. In late-passage cells neither CYP17, CYP21, nor CYP11B are expressed, even in the presence of Matrigel; only CYP11A is expressed in late-passage cultures. When the different environmental factors required for induction of CYP11B and CYP21 are taken into account, induction of these genes disappears with the same kinetics as previously shown for CYP17 as a function of PDL. The primary cause of the loss of expression of these genes is likely to be a phenotypic switching event similar to that previously demonstrated for CYP17 by in situ hybridization. The mechanism of phenotypic switching is unknown. However, one HpaII site at −2.3 kb of CYP17 was methylated in the bovine adrenal cortex in vivo but showed rapid and complete demethylation when adrenocortical cells were placed in culture. This indicates a unique, reproducible, environmentally determined change in methylation, with as yet undetermined consequences. However, data from reporter constructs suggest that phenotypic switching does not result from a simple loss of regulatory factors that act within 2.5 kb of the promoter. Previous data suggested that SV40 T antigen may affect phenotypic switching, and thus that SV40 may be useful for the derivation of functional adrenocortical cell lines. Adaptation of methods previously used for bovine cells to human adrenocortical cells to produce SV40 T antigen-transfected clones yielded data indicating preservation of essential aspects of the human adrenocortical cell differentiated phenotype.     

The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.     

Mythylation of human HLA-D/DR genes: derangement in chronic lymphocytic leukemia

HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.     

Marked Cortisol Production by Intracrine ACTH in GIP-Treated Cultured Adrenal Cells in Which the GIP Receptor Was Exogenously Introduced

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.     

细胞DNA低甲基化是否为化学致癌启动过程的必经途径

There seems to be a current view that the inhibition of DNA methylation may be a mechanism of initiation of carcinogenesis or one of the steps required in the carcinogenic process. However, because of the deficiencies in research works on cellular level, there is a long way to make such a general relationship between carcinogen and the inhibition of cellular DNA methylation. In this paper the effects of some chemical carcinogens on methylation of newly replicated DNA in human FL cells was analyzed by comparing the weight average length (Lw) of the DNA digests after complete digestion by restriction endonuclease Hpa II. It was observed that two non-genotoxic carcinogens (5-azacytidine and L-ethionine) and two genotoxic carcinogens (MNNG and aflatoxin B1) used in this study all caused obvious cytotoxicity on FL cells at the test concentration. Five days after the termination of 5-azacytidine treatment (2 x 10(-6) M, 24 hours), Lw (kb) of the Hpa II digests of cellular DNA was smaller than that of control (8.0 +/- 0.1 vs 10.9 +/- 1.0, P less than 0.01), the Lw change rate was -27%. When DNA was analyzed from FL cells after 9 days continuous treatment of L-ethionine (2 x 10 M), the digestibility of Hpa II was also increased as compared with that of the control, the Lw values (kb) showed a decrease of about 8% (9.8 +/- 0.3 vs 10.6 +/- 0.3, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenovirus-delivered DKK3/WNT4 and Steroidogenesis in Primary Cultures of Adrenocortical Cells.

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.     

Methylation of genomes and genes at the invertebrate-vertebrate boundary.

Patterns of DNA methylation in animal genomes are known to vary from an apparent absence of modified bases, via methylation of a minor fraction of the genome, to genome-wide methylation. Representative genomes from 10 invertebrate phyla comprise predominantly nonmethylated DNA and (usually but not always) a minor fraction of methylated DNA. In contrast, all 27 vertebrate genomes that have been examined display genome-wide methylation. Our studies of chordate genomes suggest that the transition from fractional to global methylation occurred close to the origin of vertebrates, as amphioxus has a typically invertebrate methylation pattern whereas primitive vertebrates (hagfish and lamprey) have patterns that are typical of vertebrates. Surprisingly, methylation of genes preceded this transition, as many invertebrate genes have turned out to be heavily methylated. Methylation does not preferentially affect genes whose expression is highly regulated, as several housekeeping genes are found in the heavily methylated fraction whereas several genes expressed in a tissue-specific manner are in the nonmethylated fraction.     

大鼠衰老过程中脑细胞DNA与c－Ha－ras原癌基因的甲基化

用ＭｓｐⅠ／ＨｐａⅡ酶解电泳法和高效液相色谱（ＨＰＬＣ）两种方法进行比较,研究了不同年龄大鼠的肝、脑细胞基因组ＤＮＡ的甲基化程度。从酶解电泳图谱可观察到,肝、脑细胞基因组ＤＮＡ甲基化在青年鼠和老年鼠之间没有差异。但用具有高分辨率的高效液相色谱测量ＤＮＡ中５－ｍＣ的含量时发现,老年鼠脑细胞ＤＮＡ甲基化程度较大年鼠的下降６２％,而肝细胞ＤＮＡ甲基化程度在老年鼠与青年鼠之间并没有显著差异。这些结果提示：（１）用常规的酶解电泳法所分析的ＤＮＡ甲基化结果并不能反映整个基因组ＤＮＡ甲基化的水平。（２）衰老过程中,不同组织ＤＮＡ甲基化的改变存在差异,引起这种差异的原因可能与组织的增殖和分化程度有关。进一步分析脑细胞原癌基因ｃ－Ｈａ－ｒａｓ的甲基化水平,无论ＭｓｐⅠ酶切图谱,还是ＨｐａⅡ酶切图谱均可观察到分子大小为１９ｋｂ、７．５ｋｂ、１．３ｋｂ、０．９ｋｂ的四条阳性带,说明该基因未发生甲基化,且与年龄无关。     

Mechanism of dissociation of cortisol and adrenal androgen secretion after removal of adrenocortical adenoma in patients with Cushing's syndrome

We investigated the mechanism of dissociation of cortisol and dehydroepiandrosterone sulfate (DHEA-S) secretion by the adrenal glands after the removal of an adrenal gland containing an adrenocortical adenoma in a patient with Cushing's syndrome. After removal of the adrenocortical adenoma, the serum cortisol rapidly decreased from 24.6 +/- 6.4 micrograms/dl (mean +/- SD, n = 6) to 0.7 +/- 0.5 micrograms/dl. Serum DHEA-S levels were 15 +/- 14 micrograms/dl and 6 +/- 9 micrograms/dl before and after surgery, respectively, and significantly lower than the control values. Serum cortisol levels reverted to normal levels 1.5 to 3 years after the surgery. On the other hand, DHEA-S levels reverted to normal 5 to 7 years after the serum cortisol levels had normalized. Monolayer cultures of normal human adrenal cells obtained at adrenalectomy in patients with advanced breast cancer and atrophic adrenal cells adjacent to the adrenocortical adenoma in patients with Cushing's syndrome were used to study the mechanism of the dissociation of cortisol and DHEA-S secretion. ACTH caused significant increases in the productions of pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OH-P5), 17-hydroxyprogesterone (17-OH-P4), DHEA, DHEA-S, androstenedione (delta 4-A), and cortisol. The amounts of 17-OH-P5 and 17-OH-P4 produced by ACTH in atrophic adrenal cells were significantly greater than those in normal adrenal cells. The amounts of DHEA, DHEA-S and delta 4-A produced by ACTH in atrophic adrenal cells were significantly smaller than those of normal adrenal cells. The conversion rate of 17-OH-[3H]P5 to 17-OH-[3H]P4 and 11-deoxy-[3H] cortisol was higher in atrophic adrenal cells than in normal adrenal cells, but the conversion rate to [3H]DHEA, [3H]DHEA-S and [3H]delta 4-A was significantly lower in atrophic adrenal cells than in normal adrenal cells. These results suggest that the dissociation of cortisol from DHEA-S after the removal of adrenocortical adenoma is a probably due to diminished C17,20-lyase activity in the remaining atrophic adrenal gland.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

CpG methylation regulates the Igf2/H19 insulator

The differentially methylated 5'-flank of the mouse H19 gene unidirectionally regulates the communication between enhancer elements and gene promoters and presumably represses maternal Igf2 expression in vivo [1-6]. The specific activation of the paternally inherited Igf2 allele has been proposed to involve methylation-mediated inactivation of the H19 insulator function during male germline development [1-4, 6]. Here, we addressed the role of methylation by inserting a methylated fragment of the H19-imprinting control region (ICR) into a nonmethylated episomal H19 minigene construct, followed by the transfection of ligation mixture into Hep3B cells. Individual clones were expanded and analyzed for genotype, methylation status, chromatin conformation, and insulator function. The results show that the methylated status of the H19 ICR could be propagated for several passages without spreading into the episomal vector. Moreover, the nuclease hypersensitive sites, which are typical for the maternally inherited H19 ICR allele [1], were absent on the methylated ICR, underscoring the suggestion that the methylation mark dictates parent of origin-specific chromatin conformations [1] that involve CTCF [2]. Finally, the insulator function was strongly attenuated in stably maintained episomes. Collectively, these results provide the first experimental support that the H19 insulator function is regulated by CpG methylation.     

A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA

About 1% of the mouse genome is cleaved by Hpa II to give a discrete fraction on gels. The nonmethylated fraction is present in all tested tissues, including sperm, and contains Hpa II sites at about 15 times their frequency in bulk DNA. About 80% of the fraction is composed of sequences that occur once or a few times per genome; the remainder is largely rDNA. Unlike bulk DNA, the fraction is not deficient in CpG, and this may be directly due to the lack of methylation. Genomic mapping of three nonribosomal fragments showed that they are part of islands of DNA within which nonmethylated Hpa II and Hha I sites are highly concentrated. We estimate about 30,000 islands per haploid genome and discuss evidence that many may be associated with genes.