Mapping the unique activation function 3 in the progesterone B-receptor upstream segment. Two LXXLL motifs and a tryptophan residue are required for activity.

Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF1 and AF2 are common to the two isoforms of PR, PR-A and PR-B, whereas AF3 lies within the N-terminal 164 amino acids unique to PR-B, termed the "B-upstream segment" (BUS). To define the BUS regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DBD), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B. Analyses of these mutants identified two regions in BUS whose loss reduces AF3 activity by more than 90%. These are associated with amino acids 54-90 (R1) and 120-154 (R2). R1 contains a consensus (55)LXXLL(59) motif (L1) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at (115)LXXLL(119) and contains a conserved tryptophan (Trp-140). Their mutation completely disrupts AF3 activity in a promoter and cell type-independent manner. Critical mutations elicited similar effects on all three B-receptor backgrounds. This underscores the probability that these mutations alter a process linking BUS structure to the function of full-length PR-B in a fundamental way.

The N-terminal region of human progesterone B-receptors: biophysical and biochemical comparison to A-receptors.

To understand the basis for functional differences between the two human progesterone receptors (PR), we have carried out a detailed biochemical and biophysical analysis of the N-terminal region of each isoform. Extending our previous work on the A-isoform (Bain, D. L, Franden, M. A., McManaman, J. L., Takimoto, G. S., and Horwitz, K. B. (2000) J. Biol. Chem. 275, 7313-7320), here we present studies on the N-terminal region of the B-isoform (NT-B) and compare its properties to its A-receptor counterpart (NT-A). As seen previously with NT-A, NT-B is quantitatively monomeric in solution, yet undergoes N-terminal-mediated assembly upon DNA binding. Limited proteolysis, microsequencing, and sedimentation analyses indicate that the B-isoform exists in a non-globular, extended conformation very similar to that of NT-A. Additionally, the 164 amino acids unique to the B-isoform (BUS) appear to be in a more extended conformation relative to sequences common to both receptors and do not exist as an independent structural domain. However, sedimentation studies of NT-A and NT-B show differences in the ensemble distribution of their conformational states. We hypothesize that isoform-specific functional differences are not due to structural differences, per se. Rather, the transcriptional element BUS, or possibly other transcription factors, causes a redistribution of the conformational ensemble by stabilizing a more functionally active set of conformations in NT-B.

Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors.

Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD(PR)) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD(PR )fusion protein. Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADP-ribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK(CS). DNA-PK that was 'pulled-down' by DBD(PR) on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK(CS), (2) transphosphorylate DBD(PR), and (3) phosphorylate a DNA-PK-specific p53 peptide substrate. DNA-PK was also able to associate with the DBD of the yeast activator GAL4. However, neither a PR DBD mutant lacking a structured first zinc finger (DBD(CYS)) nor the core DBD of the estrogen receptor (DBD(ER)) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/DNA-PK complex can assemble in vivo. These data show that DNA-PK and DBD(PR) interact, that DBD(PR) is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.

The N-terminal region of the human progesterone A-receptor. Structural analysis and the influence of the DNA binding domain.

The role of the N-terminal region in nuclear receptor function was addressed by a biochemical and biophysical analysis of the progesterone receptor A-isoform lacking only the hormone binding domain (NT-A). Sedimentation studies demonstrate that NT-A is quantitatively monomeric, with a highly asymmetric shape. Contrary to dogma, the N-terminal region is structured as demonstrated by limited proteolysis. However, N-terminal structure is strongly stabilized by the DNA binding domain, possibly explaining the lack of structure seen in isolated activation domains. Upon DNA binding, NT-A undergoes N-terminal mediated assembly, suggestive of DNA-induced allostery, and consistent with changes in protease accessibility of sites outside the DNA binding domain. Microsequencing reveals that protease-accessible regions are limited to previously identified phosphorylation motifs and to functional domain boundaries.

A deterministic genetic model for sympatric speciation by sexual selection.

A deterministic haploid genetic model confirms and explores in more detail the results of our previous individual-based simulation model for sympatric speciation by sexual selection. With the deterministic model, we are able to elucidate parameter dependence by phase plane analysis. We clarify how and why sympatric speciation by sexual selection can happen in a number of ways: (1) Female preferences for or against particular types of males have different effects. Whereas the former affects how readily speciation is invoked, the latter changes the stability of speciation equilibrium. (2) When there is no cost on male ornamentations, speciation is triggered regardless of initial haplotype frequencies if sufficient female preference is provided. (3) There exists a threshold for female initial frequencies for speciation to be invoked, but male initial frequencies have little effect. (4) A small cost on female mate choice does not cancel speciation, but when large, it greatly reduces the possibility of speciation.

Sympatric speciation by sexual selection.

There is increasing evidence for the process of sympatric speciation, in which reproductive isolation of species occurs without physical isolation. Theoretical models have focused on disruptive natural selection as the crucial pressure for splitting a species. Here we report the theoretical finding that sympatric speciation may be caused by sexual selection even without disruptive natural selection. Specifically, we show that variation in a male secondary sexual character with two conspicuous extremes and the corresponding variance in female mating preference around no preference may jointly evolve into bimodal distributions with increasing modal divergence of the male and female traits, pulling a population apart into two prezygotically isolated populations. This mode of speciation, driven by two runaway processes in different directions, is promoted by an increase in the efficiency of females in discriminating among males or a decrease in the cost of male conspicuousness, indicating that sympatric speciation may occur more readily if barrier-free or predator-free conditions arise. Although even a slight cost of female preference would cancel the runaway process of sexual selection, it would not cancel the divergent runaway processes of sympatric speciation.

Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors.

Pharmacological antagonists of steroid receptor action had been thought to exert their effects by a passive mechanism driven principally by the ability of the antagonist to compete with agonist for the ligand binding site. However, recent analyses of antagonist-occupied receptor function suggest a more complex picture. Antagonists can be subdivided into two groups, type I, or pure antagonists, and type II, or mixed antagonists that can have variable transcriptional activity based upon differential dimerization and DNA binding properties. This led us to propose that receptor antagonism may not simply be a passive competition for the ligand binding site, but may, in some cases, involve active recruitment of corepressor or coactivator proteins to produce a mixed transcriptional phenotype. We used a yeast two-hybrid screen to identify proteins that interact specifically with antagonist-occupied receptors. Two proteins have been characterized: L7/SPA, a ribosome-associated protein that is localized in both the cytoplasm and nucleus, but with no known extranucleolar nuclear function; and hN-CoR, the human homolog of the mouse thyroid receptor corepressor mN-CoR. In in vivo transcription assays we show that L7/SPA enhances the partial agonist activity of type II mixed antagonists, and that N-CoR and the related corepressor, SMRT, suppresses it. The coregulators do not affect agonists or pure antagonists. Moreover, the net agonist activity seen with mixed antagonists is a function of the ratio of coactivator to corepressor. Based upon these results, we proposed that in breast tumors the inappropriate agonist activity seen with therapeutic antagonists such as tamoxifen is responsible for the hormone-resistant state. To confirm this, we are quantitating coactivator/corepressor ratios in breast tumor cells lines and clinical breast cancers. Results should provide new insights into the mechanisms underlying the progression of breast cancer to hormone resistance, and may suggest strategies for delaying or reversing this process.

Progesterone regulates transcription of the p21(WAF1) cyclin- dependent kinase inhibitor gene through Sp1 and CBP/p300.

Progesterone has biphasic effects on proliferation of breast cancer cells; it stimulates growth in the first cell cycle, then arrests cells at G1/S of the second cycle accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21. We now show that progesterone regulates transcription of the p21 promoter by an unusual mechanism. This promoter lacks a canonical progesterone response element. Instead, progesterone receptors (PRs) interact with the promoter through the transcription factor Sp1 at the third and fourth of six Sp1 binding sites located downstream of nucleotide 154. Mutation of Sp1 site 3 eliminates basal transcription, and mutation of sites 3 and 4 eliminates transcriptional up-regulation by progesterone. Progesterone-mediated transcription is further prevented by overexpression of E1A, suggesting that CBP/p300 is required. Indeed, in HeLa cells, Sp1 and CBP/p300 associate with stably integrated flag-tagged PRs in a multiprotein complex. Since many signals converge on p21, cross-talk between PRs and other factors co-localized on the p21 promoter, may explain how progesterone can be either proliferative or differentiative in different target cells.

Progesterone receptor variants found in breast cells repress transcription by wild-type receptors.

Progesterone, through its nuclear receptors (PR), regulates the development and growth of breast cancers. PR also serve as markers of hormone dependence and prognosis in patients with this disease, and functional PR are required to mediate the antiproliferative effects of progestin therapies. We find that normal and malignant breast cells and tissues can express anomalous forms of PR transcripts. We have isolated four variant PR mRNAs that contain precise deletions of exons encoding sections of the DNA- and hormone-binding domains. The transcripts lack exon 2 (PRdelta2), exon 4 (PRdelta4), exon 6 (PRdelta6), or exons 5 and 6 (PRdelta5,6). On immunoblots, PRdelta4, delta6. and delta5, 6 cloned into the background of the PR A-isoform comigrate with similar proteins present in breast tumor extracts; delta6 and delta5, 6 are dominant-negative transcriptional inhibitors of wild-type A- and B-receptors. We propose that expression of variant PR can compromise the accuracy of receptor measurements as markers of hormone-dependent cancers, and can modify the responses of tumors to progestin therapies.

An N-terminal inhibitory function, IF, suppresses transcription by the A-isoform but not the B-isoform of human progesterone receptors.

The B-isoform of human progesterone receptors (PR) contains three activation functions (AF3, AF1, and AF2), two of which (AF1 and AF2) are shared with the A-isoform. AF3 is in the B-upstream segment (BUS), the far N-terminal 164 amino acids of B-receptors; AF1 is in the 392-amino acid N-terminal region common to both receptors; and AF2 is in the C-terminal hormone binding domain. B-receptors are usually stronger transactivators than A-receptors due to transcriptional synergism between AF3 and one of the two downstream AFs. We now show that the N terminus of PR common to both isoforms contains an inhibitory function (IF) located in a 292-amino acid segment lying upstream of AF1. IF represses the activity of A-receptors but is not inhibitory in the context of B-receptors due to constraints imparted by BUS. As a result, IF inhibits AF1 or AF2 but not AF3, regardless of the position of IF relative to BUS. IF is functionally independent and strongly represses transcription when it is fused upstream of estrogen receptors. These data demonstrate the existence of a novel, transferable inhibitory function, mapping to the PR N terminus, which begins to assign specific roles to this large undefined region.

BCL3 rearrangements and t(14;19) in chronic lymphocytic leukemia and other B-cell malignancies: a molecular and cytogenetic study.

The t(14;19)(q32.3;q13.1) is a recurring translocation found in the neoplastic cells of some patients with chronic lymphocytic leukemia (CLL) or other B-lymphocytic neoplasms. We previously cloned the translocation breakpoint junctions present in the leukemic cells from three such patients and identified a gene, BCL3, whose transcription is increased as a result of the translocation. In the present paper, we describe three additional patients with the t(14;19), one with lymphoma and two with CLL, and report the cloning and sequencing of the breakpoint junction in one of these patients as well as in a previously reported patient. We and others have found that the breakpoints on chromosome 14, with one exception, fall within the switch region upstream of the immunoglobulin heavy chain C alpha 1 or C alpha 2 sequences. Several of the breaks within chromosome 19 fall immediately upstream of the BCL3 gene, but several others are more than 16 kb 5' of the gene. Most patients with CLL and the t(14;19) also show trisomy 12.

The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT.

Steroid receptor antagonists, such as the antiestrogen tamoxifen or the antiprogestin RU486, can have inappropriate agonist-like effects in tissues and tumors. To explain this paradox we postulated that coactivators are inadvertently brought to the promoters of DNA-bound, antagonist-occupied receptors. The human (h) progesterone receptor (PR) hinge-hormone binding domain (H-HBD) was used as bait in a two-hybrid screen of a HeLa cDNA library, in which the yeast cells were treated with RU486. We have isolated and characterized two interesting steroid receptor-interacting proteins that regulate transcription in opposite directions. The first is L7/SPA, a previously described 27-kDa protein containing a basic region leucine zipper domain, having no known nuclear function. When coexpressed with tamoxifen-occupied estrogen receptors (hER) or RU486-occupied hPR or glucocorticoid receptors (hGR), L7/SPA increases the partial agonist activity of the antagonists by 3- to 10-fold, but it has no effect on agonist-mediated transcription. The interaction of L7/SPA with hPR maps to the hinge region, and indeed, the hPR hinge region squelches L7/SPA-dependent induction of antagonist-mediated transcription. Interestingly, pure antagonists that lack partial agonist effects, such as the antiestrogen ICI164,384 or the antiprogestin ZK98299, cannot be up-regulated by L7/SPA. We also isolated, cloned, and sequenced the human homolog (hN-CoR) of the 270-kDa mouse (m) thyroid/retinoic acid receptor corepressor. Binding of hN-CoR maps to the hPR-HBD. mN-CoR, and a related human corepressor, SMRT, suppress RU486 or tamoxifen-mediated partial agonist activity by more than 90%. This suppression is completely squelched by overexpression of the hPR H-HBD. Additionally, both corepressors reverse the antagonist-dependent transcriptional up-regulation produced by L7/SPA. Our data suggest that the direction of transcription by antagonist-occupied steroid receptors can be controlled by the ratio of coactivators to corepressors recruited to the transcription complex by promoter-bound receptors. In normal tissues and in hormone-resistant breast cancers in which the agonist activity of mixed antagonists predominates, steroid receptors may be preferentially bound by coactivators. This suggests a strategy by which such partial agonist activity can be eliminated and by which candidate receptor ligands can be screened for this activity.

Nuclear receptor coactivators and corepressors.

The nuclear receptors belong to a superfamily of proteins, many of which are ligand-regulated, that bind to specific DNA sequences and control specific gene transcription. Recent data show that, in addition to contacting the basal transcription machinery directly, nuclear receptors inhibit or enhance transcription by recruiting an array of coactivator or corepressor proteins to the transcription complex. In this review we define the properties of these putative coregulatory factors; we describe the basal and coregulatory factors that are currently known to interact with nuclear receptors; we suggest various mechanisms by which coactivators and corepressors act; we discuss issues that are raised by the presence of multiple, perhaps competing, coregulatory factors; and we speculate how these additional regulatory layers may explain the heterogeneity of hormone responses that are observed in normal and malignant tissues.

Role of phosphorylation on DNA binding and transcriptional functions of human progesterone receptors.

To study the function of human progesterone receptor (hPR) phosphorylation, we have tested four sets of serine to alanine substitution mutants: 10 serine clusters, located in regions common to both hPR isoforms (the M-series mutants) were mutated in A-receptors and B-receptors; 6 serine clusters located in the B-upstream segment (BUS; the B-series mutants) were mutated individually and collectively and cloned into B-receptors and into BUS-DBD-NLS, a constitutive transactivator, in which the AF3 function of BUS is fused to the DNA binding domain (DBD) and nuclear localization signal (NLS) of hPR. Transcription by most of the M-series mutants resembles that of wild-type A- or B-receptors. Mutation of 3 sites, Ser190 at the N terminus of A-receptors, a cluster of serines just upstream of the DBD, or Ser676 in the hinge region, inhibits transcription by 20-50% depending on cell or promoter context. These sites lie outside the AF1 activation function. M-series mutants are substrates for a hormone-dependent phosphorylation step, and they all bind well to DNA. Progressive mutation of the B-series clusters leads to the gradual dephosphorylation of BUS, but only the 6-site mutant, involving 10 serine residues, is completely dephosphorylated. These data suggest that in BUS alternate serines are phosphorylated or dephosphorylated at any time. However, even when BUS is completely dephosphorylated, both BUS-DBD-NLS and full-length B-receptors remain strong transactivators. Mutant B-receptors also do not acquire the dominant negative properties of A-receptors, and they retain the ability to activate transcription in synergy with 8-Br-cAMP and antiprogestins. We conclude that phosphorylation has subtle effects on the complex transcriptional repertoire that distinguishes the two hPR isoforms and does not influence transactivation mediated by AF1 or AF3, but subserves other functions.

Direct binding of progesterone receptor to nonconsensus DNA sequences represses rat GnRH.

The mechanisms by which steroid receptors repress gene expression are not well understood. In this report, we show that progesterone receptor (PR), in the presence of progesterone (P) directly represses rat gonadotropin releasing hormone (rGnRH) gene transcription. Deletion analysis studies using transient transfection assays in GT1-7 neuronal cells mapped the effects of P to sequences in the proximal rGnRH promoter between -171 and -73. This DNA sequence lacks any consensus steroid response element binding sites. Cotransfection of a mutant progesterone receptor that lacks a functional DNA binding region (hPRcys) abolished repression of the rGnRH promoter by P. Gel mobility shift assays confirmed that PR directly binds to the DNA fragments -171/-126, -126/-73, and -111/-73, which encompass the negative progesterone response element (nPRE) of the rGnRH promoter. Mutagenesis of the rGnRH nPRE -171/-126 DNA fragment resulted in a loss of PR binding. Thus, direct DNA binding of PR to nonconsensus elements in the proximal rGnRH promoter inhibits rGnRH gene expression.

Novel mechanisms of antiprogestin action.

Endocrine therapy used either prophylactically or therapeutically for the treatment of locally advanced or metastatic breast cancers offers many advantages to patients whose tumors contain functional estrogen (ER) and progesterone (PR) receptors. The range of treatments defined as endocrine include surgical ablation of endocrine glands, administration of pharmacologic doses of steroid hormones, chemical blockade of steroid hormone biosynthesis, and inhibition of endogenous steroid hormone action at the tumor with synthetic antagonists. The last of these approaches is the most widely used, making the antiestrogen tamoxifen the preferred first-line therapeutic agent for treatment of hormone-dependent metastatic breast cancer. The wide-spread use of tamoxifen reflects its efficacy and low toxicity, and the fact that it makes good physiological sense to block the local proliferative effects of estrogens directly at the breast. But are estrogens the only hormones with a proliferative impact on the breast and on breast cancers? This chapter focuses on evidence that progesterone also has proliferative actions in the breast; on preliminary data showing that progesterone antagonists may be new tools for the management of metastatic breast cancer; and on recent data suggesting that antiprogestin-occupied PR have novel mechanisms of action that bear on tissue specificity and development of hormone resistance.

Novel mechanisms of antiprogestin action.

When hormone antagonists have unexpected agonist-like effects, the clinical consequences are grave. We describe novel molecular mechanisms by which antiprogestin-occupied progesterone receptors behave like agonists. These mechanisms include agonist-like transcriptional effects that do not require receptor binding to DNA at progesterone response elements, or that result from cross-talk between progesterone receptor and other signalling pathways. We discuss the complex structural organization of progesterone receptors and demonstrate that the B-receptor isoform has a unique third activation domain that may confer agonist-like properties in the presence of antiprogestins. By contrast, the A-receptor isoform is a dominant-negative inhibitor. We argue that these novel mechanisms play a role in the apparent hormone resistance of breast cancers and the variable tissue-specific responses to progestins.

Surprises with antiprogestins: novel mechanisms of progesterone receptor action.

When hormone antagonists have inappropriate agonist-like effects, the clinical consequences are grave. We describe novel molecular mechanisms by which antiprogestin-occupied progesterone receptors behave like agonists. These mechanisms include agonist-like transcriptional effects that do not require receptor binding to DNA at progesterone response elements, or that result from cross-talk between progesterone receptors and other signalling pathways. We discuss the complex structural organization of progesterone receptors, and demonstrate that the B receptor isoform has a unique third activation domain that may confer agonist-like properties in the presence of antiprogestins, whereas the A receptor isoform is a dominant-negative inhibitor. We argue that these novel mechanisms play a role in the apparent hormone resistance of breast cancers and the variable tissue-specific responses to antagonists.

The leucine zippers of c-fos and c-jun for progesterone receptor dimerization: A-dominance in the A/B heterodimer.

Human progesterone receptors (hPR) exist as two isoforms: 120 kDa B-receptors (hPRB) and N-terminally truncated 94 kDa A-receptors (hPRA). When transfected separately, each isoform exhibits different transcriptional properties that are ligand- and promoter-specific. In human target tissues, both receptor isoforms are present, so that a mixture of three dimeric species, A/A, A/B, and B/B, bind to DNA at progesterone response elements (PRE), and regulate transcription. To study the transcriptional phenotype of pure A/B heterodimers uncontaminated by A/A or B/B homodimers, we exploited the property of the leucine zipper (zip) domains of fos and jun, to form pure heterodimers. Chimeric constructs were made linking the zip of either c-fos or c-jun to the C-terminus of hPRB or hPRA (hPR-zip) to produce A-fos, B-fos, A-jun or B-jun. To determine whether the A- or B-isoform is functionally dominant in the A/B heterodimer, cells expressing hPR-zip chimeras were treated with the progestin antagonist RU486, which produces opposite transcriptional effects with the two isoforms. Gel mobility shift and immune co-precipitation assays show that in the presence of RU486 only pure heterodimers form between A-fos/B-jun or A-jun/B-fos, and bind DNA at PREs. Thus, in these pairs, interactions between the extrinsic fos/jun zipper domains override interactions between the intrinsic hPR dimerization domains. We find that under these conditions, antagonist-occupied B-zip homodimers stimulate transcription, while antagonist-occupied A-zip homodimers are inhibitory, and that pure A/B zip heterodimers have the inhibitory transcriptional phenotype of the A-zip homodimers. We conclude that, in pure heterodimers, A-receptors are dominant negative inhibitors of B-receptors. Additionally, the pure PR-zip heterodimers, unlike wild-type receptors, bind a PRE in the absence of hormone but do not activate transcription. Thus, PR dimerization and PRE binding are necessary but, without hormone, not sufficient to activate transcription.