Avian Vitellogenin Research Articles

Cloning and characterization of vitellogenin cDNA from the common Japanese conger ( Conger myriaster) and vitellogenin gene expression during ovarian development

The major yolk protein precursor, vitellogenin (VTG) was detected in plasma from vitellogenic females and estradiol-17β (E 2)-treated immature females, but not in males and immature females by Western blotting in common Japanese conger Conger myriaster. Its molecular mass was approximately 180 kDa under denaturing and reducing conditions. The common Japanese conger VTG cDNA was cloned from the liver of vitellogenic female. It contains 5110 nucleotides including an open reading frame that encodes 1663 amino acids. The deduced amino acid sequence of the common Japanese conger VTG shares 80% identity with that of eel Anguilla japonica VTG-1, and 45–55%, 32–34% and 27–29% identity with the deduced amino acid sequences of other fish, amphibian and avian VTG with polyserin domain, respectively. In female common Japanese conger, VTG gene was highly expressed in the liver of this species similar with other oviparous vertebrates. The expression levels of VTG gene in the liver increased from the oil droplet stage to the tertiary yolk globule stage and were maintained until the migratory nucleus stage.

Avian vitellogenin: In vitro mRNA induction and comparison of nucleotide sequences

Avian vitellogenin: In vitro mRNA induction and comparison of nucleotide sequences

Structure of a fish ( Oncorhynchus mykiss) vitellogenin gene and its evolutionary implication

In this paper we describe the first complete structure of a fish vitellogenin gene. A 22 kb genomic region from rainbow trout ( Oncorhynchus mykiss) was cloned and analysed. This region was shown to contain two tandemly arranged vitellogenin genes. Both genes are 98.7% similar, indicating that they result from a recent local duplication. The complete sequence encoding one of the two genes was determined and the gene organization was established. The gene is 10.3 kb long and has 34 exons, it lacks one exon compared to amphibian and avian vitellogenin genes. Exons 22 and 23 of the Xenopus and chicken genes were shown to be merged into a single exon in the trout genome. Other splicing sites appeared highly conserved between the three vertebrate genes. In contrast, little similarity between invertebrate and vertebrate vitellogenin genes was observed with respect to the number and organization of introns. The comparison of 17 independent invertebrate splicing sites with the 34 vertebrate sites indicated that a few sites are probably ancient. However, most of the splicing junctions compared appeared unrelated. Results suggest that vitellogenin genes have been reshaped through multiple insertions and deletions of intervening sequences during evolution.

Phosphorylation/dephosphorylation of the repressor MDBP-2-H1 selectively affects the level of transcription from a methylated promoter in vitro

We have previously shown that in vivo estradiol-dependent dephosphorylation of MDBP-2-H1 (a member of the histone H1 family) correlates with the loss of in vitro preferential binding to methylated DNA. To study the effects of the phosphorylation/dephosphorylation of MDBP-2-H1 on the expression of the avian vitellogenin II gene, we optimised an in vitro transcription system using HeLa nuclear extracts. We show that in the absence of the phosphorylated form of MDBP-2-H1 from rooster, methylation of the vitellogenin II promoter does not affect the transcription. Addition of purified MDBP-2-H1 from rooster to the in vitro transcription system inhibits transcription more efficiently from a methylated than an unmethylated DNA template. Dephosphorylation of rooster MDBP-2-H1 by phosphatase treatment or estradiol treatment of rooster lead to the loss of inhibitory activity of the protein when added to the in vitro transcription assays. These findings indicate that the phosphorylation of MDBP-2-H1 is essential for the repression of the transcription. Taken together these results establish the relationship between the dephosphorylation of MDBP-2-H1 caused by estradiol, the down regulation of its binding activity to methylated DNA and the derepression of vitellogenin II transcription.

Non-histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes

We have previously purified and characterized a ubiquitous non-histone protein (NHP1) which has a high affinity ( K d 10 −11 M) for different avian vitellogenin gene sequences containing CpGs (Hughes et al. (1989) Biochemistry 28, 9137–9142; Hughes and Jost (1989) Nucleic Acids Res. 17, 8511–8520). Here we show by microsequencing that the peptides derived from the purified p75 and p85 subunits of NHP1 from HeLa cells have between 64 and 100% identity with the human Ku autoantigen. During the differentiation of human HL-60 promyelocytes there is an increase in the amount of p85 subunit protein whereas the level of the p75 subunit is unchanged. In differentiating mouse G8 myoblasts there is, however, an upregulation of both the p75 and p85 subunits and of the p85 mRNA. An inhibition of mouse myoblast differentiation by either cAMP, 3-aminobenzamide or sodium butyrate abolishes the upregulation of the p85 subunit. In G8 myoblasts chemical, or physical stress by UV light or X-rays does not upregulate the level of the p85 subunit. The possible involvement of NHP1 in the active demethylation of bifilarly methylated DNA will be discussed.

The VBP and a1/EBP leucine zipper factors bind overlapping subsets of avian retroviral long terminal repeat CCAAT/enhancer elements

Two long terminal repeat (LTR) enhancer-binding proteins which may regulate high rates of avian leukosis virus (ALV) LTR-enhanced c-myc transcription during bursal lymphomagenesis have been identified (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 9:5660-5668, 1989). The genes encoding the a1/EBP and a3/EBP binding factors were cloned by expression screening of a lambda gt11 cDNA library from chicken bursal lymphoma cells. The a1/EBP cDNA encodes a novel leucine zipper transcription factor (W. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). The partial a3/EBP cDNA clone encodes amino acids 84 to 313 of vitellogenin gene-binding protein (VBP), a leucine zipper factor that binds the avian vitellogenin II gene promoter (S. Iyer, D. Davis, and J. Burch, Mol. Cell. Biol. 11:4863-4875, 1991). Multiple VBP mRNAs are expressed in B cells in a pattern identical to that previously observed for VBP in other cell types. The LTR-binding activities of VBP, a1/EBP, and B-cell nuclear extract protein were compared and mapped by gel shift, DNase I footprinting, and methylation interference assays. The purified VBP and a1/EBP bacterial fusion proteins bind overlapping but distinct subsets of CCAAT/enhancer elements in the closely related ALV and Rous sarcoma virus (RSV) LTR enhancers. Protein binding to these CCAAT/enhancer elements accounts for most of the labile LTR enhancer-binding activity observed in B-cell nuclear extracts. VBP and a1/EBP could mediate the high rates of ALV and RSV LTR-enhanced transcription in bursal lymphoma cells and many other cell types.

An avian 40 KDa nucleoprotein binds preferentially to a promoter sequence containing one single pair of methylated CpG

In vitro transcription competition with oligonucleotides has shown that a down regulating factor can be displaced by a methylated oligonucleotide covering a specific region of the avian vitellogenin II gene promoter (Proc. Natl. Acad. Sci USA, (1990) 87, 3047-3051). Gel mobility shift and competition assays show that a protein binding preferentially to methylated DNA (MDBP-2) is present in fractionated hen and rooster nuclear extracts. The protein(s) bind to the methylated sequence 5' TTCACCTTmCGCTATG-AGGGGGATCATACTGG' 3' (nucleotide positions +2 to +32) of the vitellogenin II promoter and not to other methylated DNA sequences. Contact points of the MDBP-2 with DNA were studied by DNA binding interference experiments with partially depurinated and depyrimidinated oligonucleotides. The protein has an approximate molecular weight of 40 KDa and is mainly found in the liver and oviduct. Proteolytic clipping bandshift assays of the MDBP-2 from rooster and hen liver nuclear extracts indicate that the protein from the two sources are different. In vitro transcription experiments show that the addition of a purified nuclear fraction containing the addition of a purified nuclear dependent manner the transcription of vitellogenin II gene.

Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site

A negative regulating protein (MDBP-2) from rooster liver nuclear extracts binds preferentially to a methylated promoter region 5'TTCACCTTmCGCTATGAGGGGGATCATACTGG3' of the avian vitellogenin II gene (Nucleic Acids Res. 19, 1029-1034, 1991). Treatment of adult and immature roosters with estradiol results in a 90% decrease in the binding activity of MDBP-2 within three days. This corresponds to the level found in egg laying hens. The decrease in the binding activity of MDBP-2 precedes the onset of vitellogenin gene transcription. At the same time, there is a two-fold increase in the binding activity of NHP-1 (tested with the same oligonucleotide as for MDBP-2), a protein thought to be involved in the active demethylation of DNA. The methylated oligonucleotide binds either MDBP-2 or NHP-1 and there is no complex formation between the two proteins and DNA. Estradiol treatment does not change the equilibrium binding constant of MDBP-2 which is about 10(-9)M for the methylated oligonucleotide. The early kinetics of demethylation of the mCpG pair in the binding site of MDBP-2 was studied by means of genomic sequencing. A low level of demethylation of mCpG starts gradually on both DNA strands already 4 hours after estradiol treatment during the lag phase of vitellogenin mRNA synthesis. It is concluded that the lowering of the binding activity of MDBP-2 may have a stronger effect on the derepression of the gene than the slow demethylation of MDBP-2 DNA binding site. The role of the methylated CpG is to assure a high binding affinity of the repressor to DNA.

Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions

The avian vitellogenin gene is expressed only in the liver of egg-laying hens. It can, however, be activated in immature chicks or roosters by oestradiol. Parallel to the onset of transcription, there is a demethylation of specific mCpGs in the promoter region and in the oestrogen response element (ERE). The methylation pattern in the promoter region is hormone and expression specific, whereas in the ERE it is only hormone and not organ specific. The demethylation occurring in the promoter region is correlated with the appearance of DNase I hypersensitivity sites and changes in the specific protein-DNA interactions. In vivo genomic footprinting of the ERE with varying concentrations of dimethylsulphate revealed, upon gene activation, only minor changes in the protein-DNA interaction. We present evidence that there is another protein that binds with high affinity to the ERE, besides the oestrogen receptor.

Genomic sequencing and in vivo footprinting of an expression-specific DNase I-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA.

Genomic sequencing was used to study the in vivo methylation pattern of two CpG sites in the promoter region of the avian vitellogenin gene. The CpG at position +10 was fully methylated in DNA isolated from tissues that do not express the gene but was unmethylated in the liver of mature hens and estradiol-treated roosters. In the latter tissue, this site became demethylated and DNase I hypersensitive after estradiol treatment. A second CpG (position -52) was unmethylated in all tissues examined. In vivo genomic footprinting with dimethyl sulfate revealed different patterns of DNA protection in silent and expressed genes. In rooster liver cells, at least 10 base pairs of DNA, including the methylated CpG, were protected by protein(s). Gel-shift assays indicated that a protein factor, present in rooster liver nuclear extract, bound at this site only when it was methylated. In hen liver cells, the same unmethylated CpG lies within a protected region of approximately equal to 20 base pairs. In vitro DNase I protection and gel-shift assays indicate that this sequence is bound by a protein, which binds both double- and single-stranded DNA. For the latter substrate, this factor was shown to bind solely the noncoding (i.e., mRNA-like) strand.

Interaction of two nonhistone proteins with the estradiol response element of the avian vitellogenin gene modulates the binding of estradiol-receptor complex.

The DNA sequence corresponding to the estradiol response element has been synthesized and tested in vitro for the binding of specific proteins. Gel retardation experiments combined with dimethyl sulfate protection experiments revealed that this region binds two nonhistone proteins (NHPs). One of them, NHP-1, has a molecular weight of 70,000 and binds specifically to the dyad symmetry sequence GGTCAGCGTGACC. The NHP-1 can be separated from the estradiol receptor chromatographically; it does not bind estradiol and does not cross-react with an antibody directed against the estradiol receptor. A series of synthetic "mutant" oligonucleotides were tested in a protein-DNA binding competition assay. Deletion of the GCG in the center of the dyad symmetry sequence suppressed the binding of NHP-1 by 90%, and the conversion of any GC pair to an AT pair decreased the affinity of the binding site for NHP-1. Methylation of the two CpGs on both strands of the dyad symmetry sequence decreased the affinity of the binding site for NHP-1 by 60%, whereas hemimethylation of the same structure did not inhibit the binding of NHP-1. NHP-1 and NHP-2, the NHP binding to the DNA next to the dyad symmetry sequence, bind exclusively to double-stranded DNA. NHP-2 has a molecular weight of 60,000. NHP-1 and NHP-2 are neither tissue nor species specific. In vitro reconstitution experiments show that NHP-1 and NHP-2 increase the binding efficiency of the estradiol-receptor complex to the estradiol response element.

Estradiol-dependent trans-acting factor binds preferentially to a dyad-symmetry structure within the third intron of the avian vitellogenin gene.

The secondary activation of the avian vitellogenin II gene in isolated liver nuclei by cytoplasmatic liver extracts of estradiol-treated chicks is accompanied by the binding of a protein from the extract to the structural part of the cloned gene. Both the DNA-binding and gene-stimulatory activities, which cochromatograph on heparin-Sepharose, are apparently present only in the cytoplasmatic liver extracts of estradiol-treated roosters and in the oviduct extracts of egg-laying hens. DNA-binding competition assays combined with exonuclease III footprinting showed that the factor binds to the imperfect dyad-symmetry structure 5'GTCTTGTTCCAAAC3' within the third intron of the gene. The factor is sequence specific and binds equally well to both single-and double-stranded DNA with an estimated dissociation constant of 3.5 X 10(-10) M.

Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis.

Genomic sequencing was used to study the extent of cytosine methylation of four CpG sites within the regulatory region of the estradiol-inducible avian vitellogenin II gene. Three of these sites, two of which lie within the estradiol-receptor binding site and one in a short stretch of alternating purines and pyrimidines, were initially fully methylated. Analysis of DNA isolated from liver nuclei revealed that hormone treatment of immature White Leghorn roosters resulted in a demethylation of these sites, which occurred initially in only one DNA strand. This demethylation correlated well with the induction of vitellogenin mRNA synthesis. The demethylation of the complementary DNA strand lagged approximately equal to 24 hr behind. The fourth CpG, located within an overlapping glucocorticoid-receptor binding site, was already hemimethylated at the onset of the experiment. The demethylation of this site also occurred with kinetics similar to the rate of vitellogenin mRNA synthesis. All four CpGs remained demethylated even after cessation of gene transcription. A comparison of the methylation state of these four sites in DNA from different tissues demonstrated a clear dependence of the demethylation on estradiol. Our results suggest that this hormone-dependent event occurs via an active pathway through excision repair and/or enzymatic demethylation.

Optimized genomic sequencing as a tool for the study of cytosine methylation in the regulatory region of the chicken vitellogenin II gene

Some critical parameters of genomic sequencing are described. As an example we show a 200-nucleotide (nt) sequence of the estradiol-regulated avian vitellogenin gene II upstream region containing four CpG nt pairs. The two CpG's at positions −612 and −618 are in the sequence binding estradiol-receptor complex. While all four CpG's are methylated in erythrocytes, they are hypomethylated in the DNA of estradiol-responsive organs of egg-laying hens. A simple electroblot DNA transfer system which gives no distortion of DNA bands and quantitative transfer of denatured DNA from the gel to the filter membrane is described. Using Gene Screen membranes, a maximal hybridization signal was obtained when 30–50% of the input DNA was stably bound to the filters. Hybridization background signal and exposure time could be largely reduced by using highly purified fractionated DNA. Using a 90–120 nt long homogeneous single-stranded DNA probe of high specific activity it was possible to read a genomic sequence of up to 200 nt. The resolution was further improved by reducing the extent of chemical modifications of the DNA during the Maxam-Gilbert sequencing reactions.

In vitro secondary activation (memory effect) of avian vitellogenin II gene in isolated liver nuclei.

The vitellogenin II gene is specifically reactivated in vitro (secondary stimulation, memory effect) in purified liver nuclei that had ceased to express the gene in vivo a month after the roosters had received a single injection of estradiol (primary stimulation). The in vitro reactivation depends on the addition to the nuclei of nuclear and cytoplasmic extracts from estradiol-stimulated livers, polyamines (0.1-1.0 mM), and calmodulin (0.1 mM). Under identical incubation conditions the vitellogenin gene could not be reactivated in oviduct, embryonic, and immature chicken liver nuclei. Two other genes, those for ovalbumin and lysozyme, which are regulated by estradiol in the oviduct, could not be activated in the liver nuclei. The correct initiation of vitellogenin gene transcription in the liver nuclei was tested by primer extension studies. Addition of the antiestrogen tamoxifen (0.1 microM) to the system decreased vitellogenin mRNA synthesis by about 45% without affecting total RNA synthesis. Addition of quercetin (0.1 mM) and trans-flupenthixol (0.2 mM), inhibitors of nuclear protein kinase II and calmodulin-dependent kinase, respectively, inhibited the synthesis of vitellogenin mRNA by about 55% without affecting total RNA synthesis. The inhibitory effects of the antiestrogen and the kinase inhibitors were not additive, suggesting that both classes of inhibitor act on the same target or related targets. Depleting the estradiol receptors from the cell and nuclear extracts by means of estradiol-receptor antibodies covalently bound to Matrex beads reduced the stimulation of the vitellogenin gene by 40%. We conclude that in addition to the estradiol receptor and phosphorylation of nuclear protein(s) there are additional factors responsible for the in vitro secondary activation of the avian vitellogenin II gene.

Purification of avian vitellogenin III: comparison with vitellogenins I and II

Purification of avian vitellogenin III: comparison with vitellogenins I and II

Differential responsiveness of avian vitellogenin I and vitellogenin II during primary and secondary stimulation with estrogen

Avian vitellogenin consists of two major species, VTG I and VTG II, which show major differences in structure and immunological properties suggesting that VTG I and VTG II are distinct gene products. During primary stimulation with estrogen, VTG I was found to accumulate in plasma much more slowly than VTG II. At 1 day after hormone treatment VTG I was only 1–3% of VTG II, but by day 5 VTG I increased to approximately 25% of VTG II. Measurements of hepatic vitellogenin synthesis confirmed the slower induction and reduced expression of VTG I. A further difference was noted in the amnestic or memory response to secondary estrogen treatment. Measurements of VTG I and VTG II accumulation and synthesis after primary and secondary estrogen treatment showed that the memory response occurs to a much greater extent for VTG I than VTG II. These differences indicate that the inductions of VTG I and VTG II are not tightly coupled.

Biosynthesis of the vitellogenins. Identification and characterization of nonphosphorylated precursors to avian vitellogenin I and vitellogenin II.

Avian vitellogenin consists of two major species designated VTG I and VTG II. Rooster hepatocytes were employed to identify intracellular forms of the vitellogenins and to characterize biosynthetic intermediates of VTG I and VTG II. After labeling with [3H]serine, intracellular vitellogenin radioactivity was seen in mature VTG I and VTG II but was primarily found in two species, pVTG I and pVTG II, which showed greater mobilities in sodium dodecyl sulfate-polyacrylamide gels. The pVTG species were identified as vitellogenins by reaction with antibodies against plasma VTG II and against the mixture of VTG I and VTG II. Immunological and peptide mapping procedures were used to relate pVTG I and pVTG II to secreted VTG I and VTG II, respectively. Pulse-labeling and pulse-chase experiments showed that the pVTG species are precursors to the secreted vitellogenins and are thus discrete intermediates in the biosynthesis of the vitellogenins. Additional labeling experiments showed that the pVTG species are glycosylated but not phosphorylated. The stages of vitellogenin biosynthesis may be ordered as follows: polypeptide synthesis leads to glycosylation leads to phosphorylation leads to secretion. The presence of only small quantities of the phosphorylated vitellogenins intracellularly indicates that when phosphorylation is completed, the vitellogenins are rapidly secreted from the hepatocyte. The differences in the electrophoretic mobilities of the pVTG and VTG species suggested that sodium dodecyl sulfate-polyacrylamide gel electrophoresis does not accurately estimate the molecular weights of the heavily phosphorylated vitellogenins. This was confirmed directly by showing that the mobility of plasma vitellogenin increased upon dephosphorylation. An independent estimate of vitellogenin molecular weight was made by gel chromatography in 7 M guanidine-HCl. With this method, the molecular weights of the pVTG and VTG species were indistinguishable and in agreement with the molecular weight of the pVTG species as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These analyses indicate that the vitellogenin polypeptide has Mr approximately equal to 180,000. This value is 60,000-70,000 less than commonly estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The implications of this lower molecular weight are discussed in relation to vitellogenin structure and the egg yolk polypeptides which may derive from each vitellogenin.

Isolation and characterization of a cDNA clone specific for avian vitellogenin II.

A clone for vitellogenin, a major avian, estrogen responsive egg yolk protein, was isolated from the cDNA library of estrogen-induced rooster liver. Two forms of plasma vitellogenin, vitellogenin I (VTG I) and vitellogenin II (VTG II), distinguishable on the basis of their unique partial proteolysis maps, have been characterized and their corresponding hepatic precursor forms identified. We have used this criterion to specifically characterize which vitellogenin protein had been cloned. Partial proteolysis maps of BTG I and VTG II standards, synthesized in vivo, were compared to maps of protein synthesized in vitro using RNA hybrid-selected by the vitellogenin plasmid. Eight major digest fragments were found common to the in vitro synthesized vitellogenin and the VTG II standard while no fragments were observed to correspond to the VTG I map. A restriction map of the VTG II cDNA clone permits comparison to previously described cDNA and genomic vitellogenin clones.

Isolation and fine structure organisation of an avian vitellogenin gene coding for the major estrogen-inducible mRNA

Two phage λ recombinant DNA clones covering the entire sequence of an avian vitellogenin gene, plus flanking regions, have been isolated from an erythrocyte DNA gene library and characterized by R-loop and restriction mapping. The total length of this avian vitellogenin gene is 23 kb. The cloned sequences flanking the gene at the 5' and 3' end are 7 and 3 kb, respectively. The total length of exons in the two clones is 6.7 kb (vitellogenin mRNA is 6.6 kb). The gene is interrupted by at least 25 introns with a mean intron length of 940 bp. Some 6–10 additional very small introns may also be present but they were not observed reproducibly. The mean exon length is 250 bp. Restriction endonuclease digests of total liver genomic DNA and λ recombinant DNA were also analyzed by electrophoresis, Southern blotting and hybridization with cloned vitellogenin cDNA. The results show an identity of organisation of this vitellogenin in the DNA from the two sources, thus ruling out a possible cloning artifact. In contrast to Xenopus vitellogenin we have found no evidence to suggest that avian vitellogenin is encoded by a small family of related genes.