Ovalbumin Gene Transcription Research Articles

Gene gun-mediated in vivo analysis of tissue-specific repression of gene transcription driven by the chicken ovalbumin promoter in the liver and oviduct of laying hens.

In order to search tissue-specific elements in the 5'-upstream promoter region, gene gun was used to transfect in vivo plasmid DNAs with varying lengths of truncated ovalbumin promoter fused to the CAT reporter gene to the oviduct and liver of laying hens. The results indicated that in the oviduct, consistently high reporter gene expression was observed irrespective of the length of the truncated ovalbumin gene promoters, whereas in the liver the ovalbumin promoter extending from -3200 to +8 bp suppressed substantially the reporter gene expression compared with consistently high gene expression obtained by the ovalbumin promoters from -2800 to +8 bp or shorter length. It was concluded, therefore, that a tissue-specific silencer-like element might reside most likely in the ovalbumin gene promoter region between -3200 and -2800 bp which represses the ovalbumin gene transcription in the liver, but not in the oviduct of laying hens.

Rapid down-regulation of c-jun protooncogene transcription by progesterone in the avian oviduct.

Previous work in this and other laboratories has shown that steroids rapidly regulate the expression of nuclear protooncogenes. In this present study, we have investigated the effect of progesterone (Pg) on the expression of c-jun in the avian oviduct system and its promoter activity in avian liver cells. Pg treatment of estrogen-withdrawn chickens brings about a decrease in the steady state mRNA level of the protooncogene c-jun within 30 min. This decrease is steroid dose dependent and gene specific. Using nuclear run-off transcription analyses, this rapid regulation was shown to occur at the level of gene transcription, as the rate of c-jun transcription decreases by more than 80% within 15 min after progesterone treatment. As expected, ovalbumin gene transcription is increased only after a lag period of 4 h following Pg treatment. In other studies, we have linked the c-jun promoter sequences between -1000 and +192 to chloramphenicol acetyltransferase reporter gene and cotransfected them into transformed avian liver cells along with the expression vector for the Pg receptor. Pg treatment of these cells causes a decrease in chloramphenicol acetyltransferase gene expression, albeit to a lesser extent than Pg inhibition of c-jun gene transcription. These results suggest that the 5'-domain of the chicken c-jun gene contains sequence elements that negatively regulate c-jun promoter activity in response to Pg.

Members of the steroid hormone receptor superfamily interact with TFIIB (S300-II).

The S300-II factor was discovered as an activator of ovalbumin gene transcription with the chicken ovalbumin upstream promoter-transcription factor (COUP-TF, Sagami, I., Tsai, S. Y., Wang, H., Tsai, M.-J., and O'Malley, B. W. (1986) Mol. Cell. Biol. 6, 4259-4267). Although S300-II does not bind DNA selectively, it stabilizes the binding of COUP-TF to its ciselement (Tsai, S. Y., Sagami, I., Wang, H., Tsai, M.-J., and O'Malley, B. W. (1987) Cell 50, 701-709). Purified S300-II is also required for steroid receptor-activated transcription. Cloning and sequencing of S300-II showed that it is the general transcription factor TFIIB. Specific protein-protein interactions between recombinant S300-II/TFIIB and three members of the steroid hormone receptor superfamily, COUP-TF, estrogen receptor, and progesterone receptor, indicate that S300-II/TFIIB is one of the targets of these transactivators. Interestingly, a truncated estrogen receptor construct containing only the N-terminal transcription activation function 1 did not interact with S300-II/TFIIB in our assay, revealing that individual transcription activation functions of a single steroid hormone receptor may contact different targets. Demonstration of a direct association of S300-II/TFIIB and COUP-TF, independent of additional "adaptor" proteins, suggests that members of the steroid hromone receptor superfamily facilitate the transcription of activated genes at least in part via protein-protein interactions with the general transcription factor TFIIB.

Active beta-globin gene transcription occurs in methylated, DNase I-resistant chromatin of nonerythroid chicken cells.

We report active, inappropriate transcription of the chicken beta A-globin gene in normal fibroblasts, cultured MSB cells, and brain. We were unable to detect ovalbumin gene transcription in these same tissues. Most of the globin gene transcripts were found to be truncated near the beginning of the gene, suggesting the existence of a premature termination process that is preferentially active under conditions of inappropriate transcription. The inappropriately transcribed beta A-globin gene chromatin remained DNase I resistant and highly methylated. Thus, the DNase I-sensitive conformation of erythrocyte beta A chromatin was correlated not with beta A transcription per se but with beta A expression. Although both transcribed and nontranscribed genes within the globin domain exhibited the same DNase I sensitivity in erythrocyte nuclei, a housekeeping gene active in erythrocytes exhibited a different level of DNase I sensitivity. However, this gene exhibited the same level of DNase I sensitivity in both erythrocytes and a cultured cell line. These observations are consistent with the proposal (G. Blobel, Proc. Natl. Acad. Sci. USA 82:8527-8529, 1985) that the DNase I sensitivity of a gene may reflect properties of chromatin related to cotranscriptional and posttranscriptional aspects of mRNA production rather than to transcription per se.

Active β-Globin Gene Transcription Occurs in Methylated, DNase I-Resistant Chromatin of Nonerythroid Chicken Cells

We report active, inappropriate transcription of the chicken beta A-globin gene in normal fibroblasts, cultured MSB cells, and brain. We were unable to detect ovalbumin gene transcription in these same tissues. Most of the globin gene transcripts were found to be truncated near the beginning of the gene, suggesting the existence of a premature termination process that is preferentially active under conditions of inappropriate transcription. The inappropriately transcribed beta A-globin gene chromatin remained DNase I resistant and highly methylated. Thus, the DNase I-sensitive conformation of erythrocyte beta A chromatin was correlated not with beta A transcription per se but with beta A expression. Although both transcribed and nontranscribed genes within the globin domain exhibited the same DNase I sensitivity in erythrocyte nuclei, a housekeeping gene active in erythrocytes exhibited a different level of DNase I sensitivity. However, this gene exhibited the same level of DNase I sensitivity in both erythrocytes and a cultured cell line. These observations are consistent with the proposal (G. Blobel, Proc. Natl. Acad. Sci. USA 82:8527-8529, 1985) that the DNase I sensitivity of a gene may reflect properties of chromatin related to cotranscriptional and posttranscriptional aspects of mRNA production rather than to transcription per se.

A far upstream ovalbumin enhancer binds nuclear factor-1-like factor.

Band-shifting and DNase I footprinting analyses detected a specific DNA binding protein extracted from oviduct nuclei that binds to the ovalbumin gene 5' sequence between -1094 and -1125. This "-1100" fragment, when inserted upstream of the SV40 or ovalbumin promoters fused to a chloramphenicol acetyltransferase reporter gene, enhances chloramphenicol acetyltransferase activity 5-10-fold following transfection into CV1 cells. The sequence to which the oviduct factor binds contains a nuclear factor-1 (NF-1) half-site (GCCAA). An oligonucleotide matching the sequence of the adenovirus NF-1 binding site competed for binding to the -1100 footprinted region with a higher affinity than an oligonucleotide for the -1100 region itself. Similarly, the -1100 region oligonucleotide also competes for binding of the factor to the NF-1 oligonucleotide. These data suggest that the oviduct factor which binds to the -1100 region is an NF-1-like protein that serves as a steroid hormone-independent enhancer of the ovalbumin gene transcription.

Receptor interconversion model of hormone action. I. ATP-mediated conversion of estrogen receptors from a high to lower affinity state and its relationship to antiestrogen action.

We have previously reported that the estrogen receptor exists in three distinct states in the oviduct of the estrogen-treated chick. Since two of these forms bind estrogen and possess a number of similar properties, the intrinsic relationship between these two receptor forms has been investigated. We now show that a quantitative conversion of the high affinity (Rx) to the lower affinity (Ry) state can be induced by mild heating (30 degrees C) in the presence of estradiol and ATP, or the synthetic analogue alpha,beta-methylene adenosine triphosphate and the divalent cation Mg2+. Other nucleotides, including ADP, GTP, CTP, cAMP, and cGMP, as well as the nonhydrolyzable analogues beta,gamma-methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate are ineffective. The conversion occurs only partially in the absence of estradiol but completely in its presence. The process is not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride and alpha 2-macroglobulin, and when conversion is induced by low concentrations of ATP (1 mM), a time dependent reequilibration back to Rx occurs. These observations and the fact that the pure hormone-binding peptides Rx and Ry have similar molecular weights on sodium dodecyl sulfate-polyacrylamide gels (66,000) confirm that proteolysis is not involved in the conversion. Moreover their physical properties suggest that Rx and Ry exist in alternate conformations, with Ry being favored as a result of an ATP-mediated event involving the gamma-phosphoryl moiety. The biological relevance of the receptor conversion is suggested by studies with the antiestrogen hydroxytamoxifen. This antiestrogen binds to Rx with higher affinity than either estradiol or diethylstilbestrol but with low affinity to Ry. Hydroxytamoxifen also inhibits the conversion of Rx to Ry. Since this antiestrogen is a complete antagonist in the chick oviduct and prevents estradiol-induced stimulation of ovalbumin gene transcription, it is speculated that Rx to Ry conversion is crucial for ovalbumin gene activation and that Rx may act as a transcriptional repressor. Furthermore, since Rx and Ry both bind to nuclei and DNA, it is proposed that the presence of Ry is a better predictor of ovalbumin gene activation than DNA binding alone.

Effects of progesterone and tamoxifen on glucocorticosteroid-induced egg-white protein synthesis in the chick oviduct.

A single injection of either natural (cortisol, corticosterone) or synthetic [dexamethasone (DEX), triamcinolone acetonide] glucocorticosteroids to estradiol-primed, withdrawn chicks, resulted in a dose-dependent increase in the relative rates of ovalbumin and conalbumin synthesis. The simultaneous injection of equal doses of DEX and progesterone resulted in an additive effect on the relative rate of ovalbumin synthesis at all doses tested (range: 0.05-15 mg/chick), even when the induction of ovalbumin synthesis was maximal at 6 h, for either hormone injected alone. Moreover, the simultaneous injection of DEX and progesterone yielded an additive effect on the relative rates of ovalbumin and conalbumin gene transcription. The nonsteroidal antiestrogen tamoxifen does not increase ovalbumin synthesis and only slightly increases conalbumin synthesis. The simultaneous injection of tamoxifen and DEX potentiated the effect of DEX on the relative rates of ovalbumin and conalbumin synthesis, and amplified the DEX-induced increase in the relative rates of ovalbumin and conalbumin gene transcription. These results were supported by morphological studies carried out after 4 days of stimulation, which showed an increased accumulation of secretory granules in the magnum cells of the oviducts of chickens treated by tamoxifen plus DEX, as compared to that observed in chickens injected with DEX alone. In conclusion, these results suggest that glucocorticosteroids likely act through a mechanism distinct from that of sex steroids, and may modulate the effects of the latter on egg-white protein synthesis.

Reversible activation of non-steroid binding oestrogen receptor.

Two high-affinity oestrogen receptors have been identified in the chick oviduct with equilibrium dissociation constants (Kd) of 0.1 and 1 nM, differing in their binding kinetics, role in ovalbumin synthesis and independent regulation in vivo. The higher-affinity receptor (X) increases RNA polymerase II activity directly, whereas the low-affinity receptor (Y) seems to be necessary to confer specificity to transcription of oestrogen-dependent genes. Acute administration of progesterone to oestrogen-stimulated chicks results in preferential destruction of the nuclear Y receptor accompanied by interruption of ovalbumin gene transcription. Here we demonstrate that receptor Y exists in a non-oestradiol binding form (Ynb) which can be activated to the binding form in vitro by treatment with either ATP or ADP. Furthermore, dialysis of oviduct cytosol, which has no effect on the high-affinity receptor X, converts receptor Y to Ynb; receptor Y can then be recovered by treatment with ATP in the presence of Mg2+ and independently of Ca2+. This is the first report of the controlled interconversion between a non-steroid binding form of oestrogen receptor and active receptor in a tissue that contains two independently regulated oestrogen receptor types.

Correlation in isolated nuclei of template-engaged RNA polymerase II, ovalbumin mRNA synthesis, and estrogen receptor concentrations.

Template-engaged and total RNA polymerase II molecules were quantitated in isolated nuclei at various stages of estrogen withdrawal and secondary stimulation by using [3H]amanitin titration assays. Estrogen receptors, RNA transcriptional activity, and ovalbumin mRNA were also measured, and comparisons were made between these parameters to determine whether any significant correlations exist. In isolated nuclei, the highest positive correlations existed between template-engaged RNA polymerase II, ovalbumin mRNA synthesis in vitro, and estrogen receptor concentration. Interestingly, restimulation of estrogen-withdrawn chicks results in replenishment of RNA polymerase II activity to prewithdrawal levels within 4 h; however, the recovery of the numbers of template-engaged polymerase II molecules, ovalbumin gene transcription, and nuclear receptor binding lags behind. These findings suggest that the estrogen effect on RNA polymerase activity is more rapid than the increase in template-engaged RNA polymerase II and ovalbumin-specific gene transcription. The excellent correlation that exists between nuclear estrogen receptor concentrations, template-engaged RNA polymerase II, and ovalbumin gene transcription strongly supports the hypothesis that estrogen receptors mediate RNA polymerase II binding to sequences associated with preferential transcription of the ovalbumin gene.

The association of transcriptionally active genes with the nuclear matrix of the chicken oviduct.

Eucaryotic DNA is organized into a series of supercoiled loops that are anchored to the nuclear matrix. When these DNA loops are cleaved by endonucleases, the DNA sequences which remain associated with the nuclear matrix can be recovered and analyzed for their content of specific genes. Using restriction endonucleases to cleave the loops, we demonstrate that ovalbumin and conalbumin gene sequences are preferentially associated with the nuclear matrix of hen oviduct cells but not with the nuclear matrix of hen brain cells. Furthermore, we determined that several regions of the ovalbumin gene were independently attached to the nuclear matrix of hen oviduct cells. This included sequences located 3.8 kb downstream from the 3' end of the ovalbumin gene transcription unit. To determine whether the nuclear matrix association of the ovalbumin gene was regulated by hormones, we examined the oviduct cells of chicks that underwent primary estrogen stimulation, estrogen withdrawal and secondary estrogen stimulation. Ovalbumin gene sequences selectively dissociated from the chick oviduct nuclear matrix during estrogen withdrawal and reassociated with the nuclear matrix following restimulation.

Estrogen receptors as mediators of gene transcription

Three high affinity estrogen binding proteins which bind estradiol with equilibrium dissociation constants ( K d ) of 0.1 nM, 4nM and 30 nM have been quantitated in chick oviduct cytosol. The two highest affinity binding proteins with K d s of 0.1 nM and 4 nM have the characteristics of extrogen receptors. The concentration of these receptors in isolated nuclei is highly correlated with ovalbumin mRNA sequences synthesized in vitro in these same nuclei. The two receptors have been separated using a low capacity estrogen affinity resin and the steroid binding specificities of each was determined. When the higher affinity receptor ( K d 0.1 nM) is highly purified and added to nuclei from estrogen withdrawn oviducts RNA polymerase II activity is increased 2–3-fold, but selective transcription of the ovalbumin gene is only observed when nuclei from short term estrogen withdrawn oviducts (24 hr) are utilized. The administration ofDanazol (17α-pregn-4-en-20-yno-[2,3- disoxazol-17-ol) to estrogen stimulated chicks blocks the formation of the lower affinity receptor ( K d , 4 nM). Upon stimulation with estrogen the nuclei from the Oanazol treated oviducts have the same RNA polymerase activity as control nuclei but ovalbumin mRNA synthesis is inhibited. It would appear that both receptors are required for estrogen mediation of ovalbumin gene transcription.

Steroid hormone regulation of ovalbumin and conalbumin gene transcription. A model based upon multiple regulatory sites and intermediary proteins.

Changes in rates of ovalbumin and conalbumin gene transcription and mRNA levels were monitored during an entire cycle of estrogen withdrawal and restimulation. Correlations of transcription rates with nuclear estrogen receptor levels in this experiment and in dose-response experiments reveal that conalbumin gene transcription is directly proportional to nuclear receptor levels, whereas ovalbumin gene transcription is related to receptor levels in a way that suggests cooperative interactions among receptors. Conalbumin mRNA accumulation and transcription are transiently inhibited by administration of progesterone to estrogen-stimulated chicks, whereas ovalbumin gene transcription is stimulated by this regimen. This puzzling observation can be rationalized if a single receptor binding site is involved in conalbumin gene regulation and multiple sites are involved in ovalbumin gene regulation. These ideas are combined with our recent observations that protein synthesis inhibitors and butyrate selectively, but reversibly, inhibit ovalbumin and conalbumin gene transcription. We describe a new hypothesis of how steroid hormones regulate egg white gene transcription in the chick oviduct.

A somatomedin-like peptide hormone is required during the estrogen-mediated induction of ovalbumin gene transcription

Expression of the ovalbumin gene in chicken oviduct explant cultures requires the presence of a somatomedin-like peptide hormone in addition to estrogen. Insulin, proinsulin and multiplication-stimulating activity (MSA) are equally active substitutes for this peptide hormone, and maximal induction requires about 0.5μg/ml; fetal calf serum can partially substitute for these factors. The equipotency of insulin and proinsulin indicates that insulin receptors are not involved, and the activity of MSA suggests that the active receptor is specific for somatomedins. The permissive effect of the peptide factor occurs within 1–2 hr and is required for the initiation of estrogen-mediated transcription on the ovalbumin gene. In contrast, transcription from the conalbumin gene is fully induced by estrogen in the presence or absence of peptide factors or serum, despite the fact that these two egg white genes are both transcribed in the same cells in response to the same steroid hormones. We suggest that the interaction of a somatomedin with its membrane-bound receptor generates an intracellular signal that interacts specifically with the ovalbumin gene.

Androgens regulate ovomucoid and ovalbumin gene expression independently of estrogen.

Administration of dihydrotestosterone to estrogen-stimulated chicks induces a synergistic increase in ovomucoid mRNA synthesis and accumulation in the oviduct without affecting the synthesis of ovalbumin mRNA. This synergism is accompanied by an increase in nuclear estrogen receptors which could not be produced by simply increasing the maintenance dose of estrogen. Synergistic interactions between estrogen and dihydrotestosterone are also observed when they are added to oviduct explants in vitro. In addition to these synergistic effects, dihydrotestosterone is capable of maintaining the synthesis of ovalbumin and ovomucoid mRNAs after estrogen withdrawal in vivo. Analysis of total biologically active estrogen in the serum indicates that dihydrotestosterone does not interfere with estrogen elimination. Dihydrotestosterone also reinduces ovalbumin and ovomucoid gene transcription when injected into birds withdrawn from estrogen for less than 5 days; however, after several weeks of estrogen withdrawal, dihydrotestosterone is ineffective. Dihydrotestosterone alone acts directly on cultured oviduct explants to restimulate transcription of the ovalbumin and ovomucoid genes. Moreover, dihydrotestosterone effectively induces ovalbumin mRNA in the presence of the anti-estrogen, tamoxifen. These results indicate that androgen receptors can act either in concert with or independently of estrogen receptors to mediate specific gene transcription.

Butyrate and related inhibitors of histone deacetylation block the induction of egg white genes by steroid hormones

The short chain aliphatic acid salts, butyrate and propionate, are effective inhibitors of histone deacetylation in chick oviduct at 2–5 mM; they also prevent the hormonal induction of the ovalbumin and transferrin genes. The less potent deacetylase inhibitor isobutyrate is correspondingly less effective in blocking egg white mRNA induction; acetate has little effect at concentrations up to 15 mM. Butyrate does not appear to alter estrogen receptor binding in the nucleus, total RNA synthesis, or protein synthesis during the early hours of treatment when its specific effects on deacetylation and egg white gene transcription are observed. In addition to preventing the induction, butyrate also causes a rapid deinduction when added to preinduced cultures; ovalbumin and transferrin gene transcription decline with a half-life of 15–30 min. The effects of butyrate on egg white mRNA induction and deacetylation are completely reversible, and mRNA induction resumes within 1 hr after removal of butyrate from the medium. These results suggest that the modification of either histones or other unidentified regulatory proteins by acetylation may play a role in the mechanism of estrogen-mediated gene induction.

Effect of estrogen on gene expression in chicken oviduct: Evidence for transcriptional control of ovalbumin gene

The transcription of structural and intervening sequences of the chicken ovalbumin gene was studied in nuclei isolated from the oviduct, liver, and spleen of chickens in different states of estrogen simulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [(3)H]RNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [(3)H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription.