Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae.

The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms. The sequences responsible for this activity have been mapped to within the -1339 region of the gene. We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor. When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity. This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor. This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors.

Retinoid X receptor isotype identity directs human vitamin D receptor heterodimer transactivation from the 24-hydroxylase vitamin D response elements in yeast.

Unlike estrogen and progesterone receptors that operate as homodimers on response elements, retinoid X receptors (RXRs) and vitamin D receptors (VDRs) can function as heterodimers. Studies concerning the significance of heterodimeric partnerships are usually performed utilizing mammalian or insect cells. These cells express endogenous nuclear receptors, making it impossible to assign a role for one receptor subtype over another while studying the function of transfected receptor(s). Yeast lacks endogenous VDRs and RXRs and their ligands and provides a unique cellular context to study nuclear receptor function. We examined the interaction between human VDR and human RXR alpha, mouse RXR beta 2, and mouse RXR gamma to identify physiologically important receptor interactions. DNA binding studies on consensus, osteocalcin, or the rat 24-hydroxylase vitamin D response elements (VDREs) indicated that although RXR complexes can form on the consensus DNA elements, RXR:VDR heterodimers preferentially interact with the natural VDREs. The interaction is RXR isotype-specific and affected by ligands. Transactivation studies using the rat 24-hydroxylase VDREs indicated that VDR preferentially associated with RXR alpha or RXR gamma to stimulate transcription, and the activity was potentiated by ligand. Although RXR beta 2:VDR bound tightly to DNA, the resulting heterodimer transactivated poorly. The regulation of the 24-hydroxylase promoter observed in yeast is similar with respect to transactivation potential of specific VDRE and fold activation observed in osteosarcoma cells. Ligand binding to both receptors in a RXR:VDR complex is required for maximal transcriptional activity, indicating that the isotype-specific RXR partner significantly contributes to the ability of RXR:VDR heterodimers to transactivate from target response elements in yeast.

GCN5 and ADA adaptor proteins regulate triiodothyronine/GRIP1 and SRC-1 coactivator-dependent gene activation by the human thyroid hormone receptor.

We have used yeast genetics and in vitro protein-protein interaction experiments to explore the possibility that GCN5 (general control nonrepressed protein 5) and several other ADA (alteration/deficiency in activation) adaptor proteins of the multimeric SAGA complex can regulate T3/GRIP1 (glucocorticoid receptor interacting protein 1) and SRC-1 (steroid receptor coactivator-1) coactivator-dependent activation of transcription by the human T3 receptor beta1 (hTRbeta1). Here, we show that in vivo activation of a T3/GRIP1 or SRC-1 coactivator-dependent T3 hormone response element by hTRbeta1 is dependent upon the presence of yeast GCN5, ADA2, ADA1, or ADA3 adaptor proteins and that the histone acetyltransferase (HAT) domains and bromodomain (BrD) of yGCN5 must be intact for maximal activation of transcription. We also observed that hTRbeta1 can bind directly to yeast or human GCN5 as well as hADA2, and that the hGCN5(387-837) sequence could bind directly to either GRIP1 or SRC-1 coactivator. Importantly, the T3-dependent binding of hTRbeta1 to hGCN5(387-837) could be markedly increased by the presence of GRIP1 or SRC1. Mutagenesis of GRIP1 nuclear receptor (NR) Box II and III LXXLL motifs also substantially decreased both in vivo activation of transcription and in vitro T3-dependent binding of hTRbeta1 to hGCN5. Taken together, these experiments support a multistep model of transcriptional initiation wherein the binding of T3 to hTRbeta1 initiates the recruitment of p160 coactivators and GCN5 to form a trimeric transcriptional complex that activates target genes through interactions with ADA/SAGA adaptor proteins and nucleosomal histones.

Reconstruction of ligand-dependent transactivation of Choristoneura fumiferana ecdysone receptor in yeast.

Ecdysteroids play an important role in regulating development and reproduction in insects. Interaction of 20-hydroxyecdysone (20E) with ecdysone receptor (EcR) as a heterodimer with ultraspiracle (USP) protein triggers the activation of 20E-responsive genes. In this paper we describe a ligand-mediated transactivation system in yeast using the spruce budworm Choristoneura fumiferana ecdysone receptor (CfEcR). Coexpression of C. fumiferana USP (CfUSP) with CfEcR in yeast led to constitutive transcription of the reporter gene. However, deletion of the A/B domain of CfUSP abolished constitutive activity observed for the CfUSP:CfEcR complex. Replacement of USP with its mammalian homolog retinoid X receptors (RXRs) abolished the constitutive activity of the heterodimer but it did not restore EcR ligand-mediated transactivation. These data suggest that USP and its A/B domain play a role in the constitutive function of CfEcR:USP in yeast. A ligand-mediated transactivation was observed when GRIP1, a mouse coactivator gene, was added to EcR:RXR or EcR:DeltaA/BUSP complexes. Deletion of the A/B domain of EcR in the context of DeltaA/BEcR:RXR:GRIP1 or DeltaA/BEcR:DeltaA/BUSP:GRIP1 dramatically improved the ligand-dependent transactivation. This is the first example of highly efficient ligand-dependent transactivation of insect EcR in yeast. Analysis of transactivation activity of different ecdysteroidal compounds showed that the yeast system remarkably mimics the response observed in insect tissue culture cells and whole insect systems. The results open the way to develop assays that can be used to screen novel species-specific ecdysone agonist/antagonist insecticides.

Cloning and expression of a yeast copper metallothionein gene.

The induction of a copper-binding metallothionein (Cu-MT) was studied in yeast, Saccharomyces cerevisiae, and a relationship between copper resistance and intracellular levels of Cu-MT in these eukaryotes was established. Poly(A)-containing RNA from a copper-resistant (Cur) yeast strain, which synthesized abundant quantities of Cu-MT and in which Cu-MT gene transcription was enhanced 50-fold upon exposure to CuSO4, was used to screen yeast genomic DNA clones. Restriction analysis revealed common XbaI and KpnI sites in five genomic clones isolated. The transcription of these clones was regulated by copper. Transformation of a copper-sensitive (Cus) yeast strain by one of these clones confers copper resistance in yeast. The results suggest that the expression of the Cu-MT gene is, in part, responsible for mediating copper resistance in yeast.

Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast.

In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction. The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homologue of the retinoid X receptor (RXR). The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes. Our goal was to establish a ligand-dependent transactivation system in yeast utilizing an insect EcR-USP heterodimer. This has been achieved using mosquito Aedes aegypti AaEcR-USP. Expression of AaEcR alone, but not USP, resulted in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements. Removal of the N-terminal A/B domain of AaEcR abolished its constitutive transcription. Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR. This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex. A ligand-mediated transactivation of Aa(Delta A/B)EcR-USP or Aa(Delta A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator. In the presence of a co-repressor, SMRT, Aa(Delta A/B)EcR-USP heterodimer exhibited a ligand-dependent repression activity. In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors were also reported. This is the first report establishing the requirements of co-factors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast. These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes.

Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists.

An expression system that utilized yeast copper metallothionein promoter and ubiquitin fusion technology to express the human estrogen receptor gene in yeast is described. We have studied the biochemical and transcriptional regulatory properties of the human estrogen receptor. The biochemical properties of the yeast expressed receptors are identical to the receptors isolated from human tissue. Estradiol mediated activation of transcription by the receptor was studied by a reporter beta-galactosidase gene where expression was under the control of estrogen response elements. Using this expression system and a hyperpermeable yeast strain we have studied the effects of various antiestrogens on the regulation of estrogen receptor function. We demonstrate that tamoxifen and ICI 164,384 are capable of binding to the receptor but neither antiestrogen was able to block the estradiol mediated increase in transcription. In fact, both antiestrogens exerted weak agonist activity in this system.

Regulation of human estrogen receptor by phytoestrogens in yeast and human cells.

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estrogen. They are present in many foods and their higher consumption in certain populations has been correlated with protection against many diseases including coronary heart disease, breast cancer and endometrial and ovarian cancer. In this report, ten phytoestrogens with diverse chemical structures were studied for their binding to the human estrogen receptor and their transcription activation properties in yeast and mammalian cells. Our results showed that some of these compounds bind with relatively high affinity to the estrogen receptor and activate the receptor in the yeast and mammalian cell system. In addition, none of these compounds showed anti-estrogenic activity. We conclude that the yeast system accurately predicts the estrogenic activity of compounds with diverse chemical structures in mammalian cells. In addition, our data with phytoestrogens that do not show transcription activation properties raise the possibility that these compounds may exert their biological effects through pathways different from the classical estrogen signalling mechanism.

Structural and functional analysis of N-terminal point mutants of the human estrogen receptor.

Twenty N-terminal point mutations of the human estrogen receptor (hER) were constructed as ubiquitin fusion products and expressed under the control of the copper regulated promoter CUP1 in Saccharomyces cerevisiae. The objective of these studies was to overexpress hER in yeast and also to evaluate the functional properties of the N-terminal variants of hER. Fusion of the C-terminus of ubiquitin to the N-terminus of other proteins has been shown to increase the level of protein expression in yeast. Ubiquitin C-terminal hydrolases (UCHs) in yeast efficiently and precisely cleave at the junction with ubiquitin and render free hER with desired amino termini. The variant hER proteins, that were generated by mutating the N-terminus of hER, showed enormous differences in receptor protein levels and transactivation potential. All variant hER proteins were synthesized as 66 kDa species as identified by Western blotting with the exception of the proline-containing variant (Pro-ER). The UB-Pro-ER variant was cleaved inefficiently by UCHs in yeast. The UB-Pro-hER [correction of UB-Pro-hEr] variant also exhibited a different DNA band-shift profile compared to those of the other receptor variants and the wild-type. Val-, Thr-, and Lys-ER did not express, as measured by enzyme-immunoassay and Western blotting; nor did they transactivate a beta-galactosidase reporter gene in yeast. However, the Glu-ER was 50% more active in transactivation as compared to the wild-type. The results of the receptor content, DNA binding properties and transactivation analysis in yeast demonstrate that the N-terminal residue plays an important role in the structure and function of hER.

Transcription factors as drug targets: opportunities for therapeutic selectivity.

Many traditional drugs target cell surface receptors. Medicinal chemists and pharmacologists have not ventured into the field of transcription regulation due to the fear that drugs that interfere with transcription regulation may not be selective or efficacious. The past 5 years have seen some exciting developments in the field of signal transduction in general, and transcription regulation in particular. Our understanding of mechanisms of regulated and basal transcription is advanced to a degree that it should be possible to selectively modulate a target gene directly. In this review we have argued that sufficient diversity exists in the combinatorial interplay of the transcription factors to offer opportunities for selective therapeutic intervention. We have focused our attention on transcriptional factors that play a role in three different therapeutic areas: osteoporosis, immune modulation, and cardiovascular diseases. Human estrogen receptor is considered as a model transcription factor. The role of estrogen in bone remodeling is discussed. Opportunities for tissue-specific modulation of estrogen receptors are described. For selective immune modulation, we have discussed the role of NF-AT (nuclear factors for activated T cells) transcription factors in interleukin-2 gene regulation. The last section focuses on the transcriptional mechanisms conferring tissue specificity in regulated expression of the apoAI gene, a major component of HDL, in liver. We have highlighted opportunities for rational development of transcription-based drugs useful for raising HDL plasma levels and atherosclerosis prevention.

Human nuclear receptor heterodimers: opportunities for detecting targets of transcriptional regulation using yeast.

Nuclear receptors are model transcription factors. This highly conserved superfamily of ligand binding transcription factors includes estrogen, progesterone, retinoic acid, thyroid hormone, vitamin D receptors, and several orphan receptors. Nuclear receptors function as homodimers, heterodimers, or monomers. Human thyroid hormone, retinoic acid, vitamin D, and several orphan receptors prefer to work as heterodimers with retinoic X receptor (RXR). RXR function is regulated by its cognate ligand 9-cisretinoic acid. In some cases heterodimers of RXR are subject to regulation by two different ligands. Mammalian cells are not entirely suited to study pure heterodimeric functions because they contain a repertoire of endogenous receptors and their ligands. Yeast does not contain nuclear receptors or its ligands. Ligand-dependent function of several human nuclear receptors has been reconstructed in yeast. Yeast can be used as a model system to dissect interaction between various heterodimeric partners. The molecular genetics and the speed of doing the experiments in yeast allows us to rapidly clone mammalian cofactors that prefer to work with different heterodimeric partners. Once the human genome sequence is complete, we predict that the total number of human nuclear receptors will increase from 150 to 500. Novel and efficient cell-based systems will be needed to understand the function of orphan receptors. Yeast is an ideal system to identify pure heterodimeric partners and discover novel ligands for orphan receptors. The advantages and disadvantages of yeast and mammalian system to study nuclear receptor function are discussed.

Cross-talk between thyroid hormone and specific retinoid X receptor subtypes in yeast selectively regulates cognate ligand actions.

Thyroid (T3) hormone beta1 (TR) and 9-cis retinoic acid (9c-RA) retinoid X receptors (RXR) can form heterodimer complexes that bind to hormone response elements (HREs) in target genes to either activate or repress transcription. However, the action of each cognate ligand and the accessory cellular factors that can differentially regulate the transcriptional responses of a heterodimer-DNA complex are not well understood. Studies in most mammalian cell lines have demonstrated that 9c-RA cannot bind or transactivate TR/RXR-T3 response element (TRE) complexes. In contrast, when identical heterodimer complexes were coexpressed in the yeast (Saccharomyces cerevisiae) with single copy typical TREs [i.e., DR+4 (direct repeat), F2 (everted repeat), or PAL (inverted repeat) DNA response elements] we observed that i) unliganded TRbeta1 homodimers had constitutive action on F2 and PAL but not DR4 TREs; ii) TRbeta1 homodimer responsivity to T3 ligand was relatively weak (less than twofold) and was only demonstrable on F2 but not PAL or DR4-TREs, whereas TRbeta1 heterodimers responded to T3 when RXRgamma but not RXR alpha was the heterodimeric partner; iii) RXR responsivity to 9c-RA (three- to sixfold) could be demonstrated only on palindromic TREs that could be enhanced by TRbeta1 on all TREs; iv) T3 + 9c-RA ligands increased (additively or synergistically) transactivation when RXRgamma but not alpha heterodimerized with TRbeta1 on both typical as well as atypical (DR1, DR3, DR5, and F2M) TREs. Substitutions for wild-type TRbeta1 of C-terminus mutants deficient in dimerization with RXRs abrogated the anticipated single and dual cognate ligand-induced effects on TRbeta1/RXRgamma transactivation of DR4 TREs, whereas mutants with preserved dimerization function but impaired T3 transactivation regions could maintain an enhanced 9c-RA response but were devoid of the anticipated T3 and dual (T3 + 9c-RA) cognate ligand-induced effects. Thus, the ligand-inducible response of TR and RXR homodimers expressed in yeast are relatively weak but can be further enhanced by TRbeta1 cross-talk with specific RXR subtypes in the presence of both cognate ligands.

An estrogen sensor for poultry sex sorting.

The need to segregate poultry based on sex is driven by sex-related differences in growth rate, market age, management practices, and nutritional requirements. Each day, poultry industry staff globally would ideally like to determine the sex of >150 million newly hatched birds. Currently, this can be done only manually at the hatchery, which is a virtually impossible undertaking. It is becoming more difficult each year to conduct manual sexing because this skill is disappearing from the workforce, is becoming unaffordable to the industry, and is encumbered by such negative effects as repetitive motion disorder. Automated sex sorting of eggs before hatching could resolve many, if not all, of these problems. We have developed a facile, rapid, and low-cost yeast-based assay that distinguishes male from female embryonated eggs before hatching based on the estrogen concentration of their allantoic fluid. Herein, we describe this novel sex-sorting technology, which we believe offers the potential to standardize and automate sex sorting in the poultry industry.

Interferon and sodium butyrate inhibit the stimulation of poly(ADP-ribose) synthetase in mouse cells stimulated to divide.

The activity of poly(ADP-ribose) synthetase, a chromatin-bound enzyme, increases when quiescent 3T3 cells are stimulated to proliferate. The elevation of enzymatic activity requires de novo RNA and protein synthesis. Interferon (IFN) or sodium butyrate, when added to quiescent cells at the time of stimulation, suppressed the rise of enzymatic activity as well as initiation of DNA synthesis in cells. However, other DNA synthesis inhibitors like methotrexate, FudR and hydroxyurea had little effect on the elevation of poly(ADP-ribose) synthetase in quiescent cells.

Relationship between nicotinamide adenine dinucleotide concentration and in vitro synthesis of poly(adenosine diphosphate ribose) on purified nucleosomes.

When oligonucleosomes (8-10 N) were incubated under conditions favoring poly(ADP-ribosylation) with concentrations of [32P]NAD 10 microM and higher, the labeled chromatin components migrated in 3-8% gradient native chromatin polyacrylamide gels, in positions of far greater size than the starting nucleosomes. Only a small fraction of chromatin and/or oligonucleosome components was found to be involved in this aggregation. This phenomenon could be demonstrated as well by the increased sedimentation of ADP-ribosylated chromatin components in velocity sucrose gradients. In contrast, at [32P]NAD concentrations less than or equal to 1 microM, the modified nuclear proteins were found strictly associated with the original oligonucleosomes. The extent of aggregation was proportional to both substrate concentration and time of incubation with NAD. During this process, progressively longer chains of poly (ADP-Rib) (10-60 N) were generated on chromatin proteins correlating with the level of complex formation. Analysis of protein by either acid-urea or Na-DodS4O-gel electrophoresis indicated that at NAD concentrations favoring aggregation, poly(ADP-Rib) induced ADP-ribosylated complexes of histone H1, and possibly core histones were observed. Poly(ADP-Rib) polymerase requires both DNA and nuclear proteins for activity. The presence of this enzyme in the high molecular weight complexes was demonstrated on sucrose gradients and also by direct enzyme assays, in situ, in regions of 3-8% gradient chromatin gels containing the complexed chromatin components.

Nucleosome periodicity in HeLa cell chromatin as probed by micrococcal nuclease.

When HeLa cell nuclei were treated with micrococcal nuclease (nucleate 3-oligonucleotidohydrolase, EC 3.1.4.7), lysed, and centrifuged, the supernatant from early digests contained two predominant classes of polynucleosomes of repeat size 8N and 16N. With increasing digestion time, the 16 N polynucleosome appeared to be cleaved to the 8N species and finally to the basic subunit of chromatin. The size of the polynucleosomes has been determined by DNA analysis and on polyacrylamide electrophoretic gels of native chromatin particles. The 16N polynucleosome appears to be a unique higher ordered structural component of HeLa cell chromatin. Our recent report, showing that the nuclear protein-modifying enzyme poly(ADP-ribose) polymerase increases in specific activity progressively with increasing nucleosome repeat size up to 8-10N, has been extended in the present study. Activity was also elevated in the polynucleosomes of the 16N structure preferentially cleaved by micrococcal nuclease, although specific activity of the enzyme was highest in octanucleosomes. Acceptors for poly(ADP-ribose) have also been determined in these particles.

Nuclear protein modification and chromatin substructure. 3. Relationship between poly(adenosine diphosphate) ribosylation and different functional forms of chromatin.

The relationship between poly(adenosine diphosphate) ribosylation of nuclear proteins and functionally different forms of chromatin from mid-S-phase HeLa nuclei was investigated. The major observations emerging from this study were that unique nonhistone proteins were modified in mid-S-phase HeLa nuclei. The major acceptor for poly(adenosine diphosphate-ribose) [poly(ADP-Rib)] was an internucleosomal nonhistone protein (protein C; 125 000 molecular weight). Histones H3, H1, H2b, and H2a but not H4 were ADP-ribosylated in S-phase nuclei. Chromatin fragments preferentially released by micrococcal nuclease were enriched in nonhistone proteins, poly(ADP)-ribosylated nuclear proteins, poly(ADP-Rib) polymerase activity and nascent DNA from the DNA replicating fork. In extended forms of chromatin, contiguous to the DNA replicating fork, poly(ADP-Rib) polymerase was maximally active. However, in chromatin distal to the replicating fork (i.e., more condensed structures), nucleosomal histones and histone H1 were not significantly ADP-ribosylated, and poly(ADP-Rib) polymerase activity was depressed two- to threefold. The data suggest that a subset of nucleosomes in extended regions of chromatin is subject to extensive ADP ribosylation.

Libraries of random-sequence polypeptides produced with high yield as carboxy-terminal fusions with ubiquitin.

Libraries of random-sequence polypeptides have been shown to be valuable sources of novel molecules possessing a variety of useful biologic-like activities, some of which may hold promise as potential vaccines and therapeutics. Previous random peptide expression systems were limited to low levels of peptide production and often to short sequences. Here we describe a series of libraries designed for increased polypeptide length. Cloned as carboxy-terminal extensions of ubiquitin, the fusions were produced in E. coli at high levels, and were purified to homogeneity. The majority of the extension proteins examined could be cleaved from ubiquitin by treatment with a ubiquitin-fusion hydrolase. The libraries described here are appropriate sources of novel polypeptides with desired binding or catalytic function, as well as tools with which to examine inherent properties of proteins as a whole. Toward the latter goal, we have examined structural properties of random-sequence proteins purified from these libraries. Quite surprisingly, fluorescence emission spectra of intrinsic tryptophan residues in several purified fusion proteins, under native-like and denaturing conditions, often resemble those expected for folded and unfolded states, respectively. The results presented here detail an important expansion in the range of potential uses for random-sequence polypeptide libraries.