Chicken Ovalbumin Promoter Is Demethylated upon Expression in the Regions Specifically Involved in Estrogen-Responsiveness

Here we report the methylation status of the chicken ovalbumin promoter. Genomic DNA of oviduct from immature chickens and laying hens was analyzed through bisulfite genomic sequencing. In the ovalbumin control locus up to the 6 kb upstream region, CpG sites were methylated in immature chickens, except for several sites, and almost all CpGs residing in DNase I hypersensitive sites I, II, and III, but not IV, were selectively unmethylated in ovalbumin expressing chickens. Chromatin immunoprecipitation assays showed that the ovalbumin control region was associated with acetylated histone H3 but not with dimethylated histone H3 at Lys 27. These results demonstrate that DNA demethylation was restricted to short DNA regions of DNase I hypersensitive sites, especially to those which participated in estrogen-responsiveness, even when cells expressed extremely high levels of ovalbumin and these sites were associated with acetylated histones.     

Chicken ovalbumin promoter is demethylated upon expression in the regions specifically involved in estrogen-responsiveness

Here we report the methylation status of the chicken ovalbumin promoter. Genomic DNA of oviduct from immature chickens and laying hens was analyzed through bisulfite genomic sequencing. In the ovalbumin control locus up to the 6 kb upstream region, CpG sites were methylated in immature chickens, except for several sites, and almost all CpGs residing in DNase I hypersensitive sites I, II, and III, but not IV, were selectively unmethylated in ovalbumin expressing chickens. Chromatin immunoprecipitation assays showed that the ovalbumin control region was associated with acetylated histone H3 but not with dimethylated histone H3 at Lys 27. These results demonstrate that DNA demethylation was restricted to short DNA regions of DNase I hypersensitive sites, especially to those which participated in estrogen-responsiveness, even when cells expressed extremely high levels of ovalbumin and these sites were associated with acetylated histones.     

The vascular architecture of the developing organum vasculosum of the lamina terminalis (OVLT) in the rat

The pituitary gland of the red grouper, Epinephelus akaara, was studied by histochemical techniques, and the prolactin cells, corticotrops, somatotrops, gonadotrops, thyrotrops, pars intermedia cells and neurohypophyseal cells, were identified. Oestradiol-17 beta treatment caused PAS-positive cells in the proximal pars distalis, presumably a mixture of gonadotrops and thyrotrops, to undergo hypertrophy, vacuolation and degranulation of cytoplasmic glycoprotein granules. Disappearance of cytoplasmic granules was also evident in the PAS-positive pars intermedia cells. Oestrogen-treated fish also showed an increase in the hepatosomatic index, and hepatocytes enlarged in size, their nuclear diameter increased and large vacuoles were formed in the cytoplasm. These changes in the liver were paralleled by a secretion of vitellogenin into the serum and an increased production of mucus by the thickened skin epithelium. Testosterone injections did not affect such changes, neither in the pituitary nor liver cells, but a proliferation of skin epithelial cells was noted. Neither oestradiol-17 beta nor testosterone stimulated ovarian incorporation of vitellogenin, but treatment with high doses (5 mg/kg) of oestradiol-17 beta or testosterone brought about a slight increase in the gonadosomatic index and atresia of some of the primary oocytes. The oogonial population size decreased in response to treatment with high doses of oestradiol-17 beta.     

A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation.

Hepatoma tissue culture (HTC) cell nuclei were digested with either DNase I or micrococcal nuclease and the nucleohistone digestion products fractionated by gel electrophoresis or exclusion chromatography. Under appropriate conditions, gel electrophoresis demonstrates that for both nucleases, only cleavages within the nucleosome spacer regions and not within the nucleosome core lead to freely migrating nucleohistone particles. These particles consist of nucleosome cores, nucleosomes and nucleosome oligomers. Following DNase I digestion and fractionation by exclusion chromatography, analysis of the histones indicates a direct relationship between increased spacer region susceptibility to nuclease and increased nucleosomal histone acetylation. Evidently digestion sites outside the regions of DNA protected by core histones can reflect the degree of acetylation of core histones. Such a relationship is not found when micrococcal nuclease is used to digest the samples.     

Estrogen induces tissue specific changes in the chromatin conformation of the vitellogenin genes in Xenopus.

Nuclei from male Xenopus liver were digested extensively with DNase I and the residual amount of the four vitellogenin genes measured by hybridization with a moderate excess of vitellogenin cDNA. The saturation value was about twofold lower in chromatin isolated from liver cells of estrogen treated than from untreated males or from erythrocytes. Analyzing the disappearance of several defined restriction fragments specific for the A1 and A2 vitellogenin genes, after limited digestion with DNase I, suggested that the entire A1 and A2 vitellogenin genes are about twofold more sensitive to DNase I in chromatin of hepatocytes isolated from estrogen treated than from untreated males. Using the same assay no change in the DNase I sensitivity of the two vitellogenin genes in erythrocyte chromatin was observed. Analysis of the beta 1-globin and an albumin gene demonstrated that the DNase I sensitivity of these genes in both cell types is not altered by estrogen. All these data indicate that estrogen stimulation results in an increased DNase I sensitivity specific for the vitellogenin genes in hepatocytes.     

Condensation of chromatin into chromosomes preserves an open configuration but alters the DNase I hypersensitive cleavage sites of the transcribed gene

DNase I was used to probe the molecular organization of the chicken ovalbumin (OV) gene and glyceraldehyde 3-phosphate dehydrogenase (GPD) gene in interphase nuclei and in metaphase chromosomes of cultured chicken lymphoblastoid cells (MSB-1 line). The OV gene was not transcribed in this cell line, whereas the GPD gene was constitutively expressed. The GPD gene was more sensitive to DNase I digestion than the OV gene in both interphase nuclei and metaphase chromosomes, as determined by Southern blotting and liquid hybridization techniques. In addition, we observed DNase I hypersensitive sites around the 5' region of the GPD gene. These hypersensitive sites were not always at the same locations between the interphase nuclei and metaphase chromosomes. Our results suggest that chromatin condensation and decondensation during cell cycle alters nuclease hypersensitive cleavage sites.     

Function of the upstream hypersensitive sites of the chicken beta-globin gene cluster in mice.

We have shown previously that the chicken beta A-globin gene, with its 3' enhancer, is expressed in a copy number-dependent manner in transgenic mice. The expression level was low but increased approximately 6-fold upon inclusion of 11 kb of upstream DNA containing four DNase I hypersensitive sites. To study the effect of the individual upstream hypersensitive sites on transgene expression, we produced lines of mice in which the individual upstream sites were linked to the beta A gene and enhancer. RNA levels were measured in blood from adult animals. With each of these four constructs, the level of transgene RNA per DNA copy varied over a > 20-fold range. These data suggest that addition of a hypersensitive site to the beta A-globin/enhancer region abrogates its position independent expression. The average beta A-globin expression per copy in the lines carrying an upstream site was comparable with that in lines without an upstream site. Thus, no single upstream hypersensitive site accounts for the higher level of beta A-globin expression seen in mice containing the complete upstream region. We had shown previously that control of the chicken beta-globin cluster is distributed between at least two regions, the beta A/epsilon enhancer and the upstream region. Our current results suggest that the control mediated by the upstream DNA is itself distributed and is not due to a single hypersensitive site.     

Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain.

The distribution of core histone acetylation across the chicken beta-globin locus has been mapped in 15 day chicken embryo erythrocytes by immunoprecipitation of mononucleosomes with an antibody recognizing acetylated histones, followed by hybridization probing at several points in the locus. A continuum of acetylation was observed, covering both genes and intergenic regions. Using the same probes, the generalized sensitivity to DNase I was mapped by monitoring the disappearance of intact genomic restriction fragments from Southern transfers. Close correspondence between the 33 kb of sensitive chromatin and the extent of acetylation indicates that one role of the modification could be the generation and/or maintenance of the open conformation. The precision of acetylation mapping makes it a possible approach to the definition of chromosomal domain boundaries.     

TGGCA protein is present in erythroid nuclei and binds within the nuclease-hypersensitive sites 5' of the chicken beta H- and beta A-globin genes

The developmentally regulated 5'-flanking DNase-I-hypersensitive site of the chicken beta H-globin gene in nuclei contains a subregion which is resistant to DNase I and which disappears when nuclei are extracted with 0.3 M NaCl, suggesting that there are salt-extractable proteins bound to sequences within this region. The 0.3 M NaCl extract contains two proteins which bind in vitro to these sequences. One of the binding sequences has an inverted repeat very similar to that bound by TGGCA protein. Partially purified TGGCA protein from chicken liver binds to this sequence in vitro giving exactly the same footprint as that obtained with erythroid nuclear proteins. Similarly TGGCA protein binds to an inverted repeat with the beta A-globin 5'-hypersensitive site giving a footprint identical to that obtained with erythroid nuclear protein extracts. From competition footprinting experiments and the electrophoretic mobility of the protein-DNA complex, it is concluded that the erythroid proteins previously described as binding to the beta H- and beta A-globin inverted repeats within the 5'-flanking hypersensitive sites both belong to the TGGCA protein family.     

Steroid glucuronyltransferases of rat liver. Properties of oestrone and testosterone glucuronyltransferases and the effect of ovariectomy, castration and administration of steroids on the enzymes.

1. Microsomal preparations from rat liver, kidney and intestine were tested for UDP-glucuronyltransferase activity by using oestrone, oestradiol-17 beta, oestriol, testosterone, cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone as substrates. The microsomal preparation from the liver glucuronidated oestrone, oestradiol-17 beta and testosterone. 2. The specific activity of the enzyme was significantly higher in livers from female rats than in those from male rats. 3. Testosterone was actively glucuronidated by both sexes. Cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone were not glucuronidated by any of the three tissues. 4. The non-ionic detergent Lubrol WX activates liver microsomal UDP-glucuronyltransferase 2-3-fold with oestrone and testosterone as substrates. 5. Oestrone glucuronyltransferase was inhibited by oestradiol-17 beta, predominantly competitively and by testosterone non-competitively. Bilirubin was a non-competitive inhibitor of oestrone glucuronidation. p-Nitrophenol had no effect. 6. Oestrone glucuronyltransferase could not be stimulated by either acute or prolonged treatment of animals with phenobarbital, whereas a single dose of 3-methylcholanthrene led to a moderate stimulation. 7. Ovariectomy leads to a 56% decrease in oestrone glucuronyltransferase activity; administration of oestradiol-17 beta induces the enzyme to normal activity after 12 days, and after 15 days the activity is twice the control value. Actinomycin D and cycloheximide block the oestradiol-17 beta-induced increase in enzyme activity. 8. Castration has no effect on the activity of testosterone glucuronyltransferase, nor does administration of testosterone influence enzyme activity. The results provide strong evidence for the existence of multiple steroid glucuronyltransferases in the liver of the rat.     

Oestradiol-17 alpha dehydrogenase from chicken liver.

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.     

Histone acetylation and the deoxyribonuclease I sensitivity of the Tetrahymena ribosomal gene

Under appropriate conditions, up to 8.5% of the total acetate can be removed from the histones of isolated Tetrahymena macronuclei by an endogenous histone deacetylase activity. After in vitro deacetylation, the ribosomal genes are still preferentially digested by DNase I. These observations suggested that either the majority of histone-bound acetate is unnecessary to maintain the DNase I sensitive state or ribosomal chromatin (rChromatin) histones remain acetylated under these conditions. The characteristics of histones acetylation were studied in Tetrahymena rChromatin, which can be isolated in a relatively pure form. Histones associated with the presumably active, DNase I sensitive ribosomal genes have a high steady-state level of histone acetylation which, surprisingly, is maintained by very low acetate turnover rates.     

Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene

Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats. Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure. By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene. An S1 nuclease sensitive site is located close to a DNase I site. Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites. Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene. This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes. The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.