Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone.

The structures of the ligand-binding domains (LBD) of the wild-type androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 A resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wild-type receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

Transforming region of 243R E1A contains two overlapping but distinct transactivation domains.

Conserved regions 1 and 2 as well as the amino terminus of E1A are required for the transforming activity of the E1A oncoprotein. We show here that the amino terminus of 243R E1A has transactivation activity when brought to a promoter in yeast. Recruitment to a specific promoter is essential. Mutagenesis studies correlated the transactivation function with the extreme amino terminus and the conserved region 1 of E1A. Cotransfection assays in rodent cells confirmed that two overlapping but distinguishable domains, amino acids 1-65 and 37-80, can transactivate independently when targeted to a promoter. We also observed that when recruited to the proliferating cell nuclear antigen (PCNA) promoter, the amino-terminal region was sufficient to transactivate the PCNA promoter. On the other hand, deletion of the amino terminus of E1A resulted in failure to induce PCNA expression. Fusion of VP16 with the amino-terminal-deleted E1A mutant was able to restore the ability to induce the PCNA promoter. We further show that the amino-terminal region also is required for 243R E1A to repress the transactivation mediated by a universal transactivator DBD.VP16 and DBD.E1A. This repression could be specifically relieved by overexpression of TBP but not TFIIB. In addition, we show that the amino terminus of E1A is involved in in vitro interaction with the TATA binding protein (TBP). Thus the amino-terminal transforming region of E1A may regulate cellular gene expression in species that are distant in evolution via a common mechanism, functionally targeting TBP.

Interaction of human immunodeficiency virus type 1 Tat with a unique site of TFIID inhibits negative cofactor Dr1 and stabilizes the TFIID-TFIIA complex.

We have previously reported the direct physical interaction between the human immunodeficiency virus (HIV) type I Tat protein and the basal transcription factor TBP/TFIID. Affinity chromatography demonstrated that wild-type Tat, but not a transactivation mutant of Tat, was capable of depleting TBP/TFIID from cell extracts. These experiments represented the first demonstration of a basal transcription factor that binds, in an activation-dependent manner, to Tat. We now report that the Tat-TBP interaction can be detected in HIV type 1-infected cells. The domain of TBP interacting with Tat has been mapped from amino acids 163 to 196 by using deletion and site-specific mutants of TBP. This domain of TBP, which includes the HI and S2 domains, is distinct from the H2 binding site for other activator proteins, such as E1A. The interaction of Tat with TFIID regulates the binding of accessory proteins to TFIID. Tat stabilizes the interaction of TFIID with TFIIA in a gel shift assay. In addition, Tat competes for Dr1 interaction with TBP. Our results suggest that the basal transcription factor TBP/TFIID represents an important regulatory molecule in HIV transcription.

Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation.

Recent results from several laboratories including ours strongly suggest that farnesyltransferase (FT) inhibitors belonging to distinct chemical classes block growth of oncogenic Ras transformed cells at concentrations that do not affect the growth and viability of normal cells. This is despite blocking the farnesylation and thus the membrane association of Ras in both cell types. This is a paradox given the requirement for Ras function in normal cell growth. Recent evidence that R-Ras2/TC21 utilizes components of Ras signal transduction pathways to trigger cellular transformation (Graham et al., MCB 14, 4108-4115, 1994) prompted us to consider the possibility that R-Ras2/TC21 is involved in some aspects of the growth regulation of normal cells. If so, R-Ras2/TC21 may be compensating for Ras function in untransformed cells treated with FT inhibitors. In this study, we demonstrated that a cell active bisubstrate analog FT inhibitor, BMS-186511, completely blocked the function of oncogenic Ras, but did not affect the function of oncogenic R-Ras2/TC21, as determined by several criteria including inhibition of anchorage dependent and independent growth, reversal of transformed morphology and restoration of actin cytoskeleton. While it is known that TC21 protein becomes prenylated, it is not known whether it is farnesylated or geranylgeranylated. Our in vitro prenylation experiments indicate that R-Ras2/TC21 protein serves as a good substrate for FT as well as geranylgeranyltransferase I (GGTI) and thus provide the apparent molecular basis for these differences. Overall, these results, coupled with the ubiquitous expression of R-Ras2/TC21 in many cells including untransformed NIH3T3 cells, are consistent with the possibility that R-Ras2/TC21 may be one of the factors that render normal cells insensitive to the growth inhibitory action of FT inhibitors.

Adenovirus E1A proteins interact with the cellular YY1 transcription factor.

The adenovirus 12S and 13S E1A proteins have been shown to relieve repression mediated by the cellular transcription factor YY1. The 13S E1A protein not only relieves repression but also activates transcription through YY1 binding sites. In this study, using a variety of in vivo and in vitro assays, we demonstrate that both E1A proteins can bind to YY1, although the 13S E1A protein binds more efficiently than the 12S E1A protein. Two domains on the E1A proteins interact with YY1: an amino-terminal sequence (residues 15 to 35) that is present in both E1A proteins and a domain that includes at least a portion of conserved region 3 (residues 140 to 188) that is present in the 13S but not the 12S E1A protein. Two domains on YY1 interact with E1A proteins: one is contained within residues 54 to 260, and the other is contained within the carboxy-terminal domain of YY1 (residues 332 to 414). Cotransfection of a plasmid expressing carboxy-terminal amino acids 332 to 414 of YY1 fused to the GAL4 DNA-binding domain can inhibit expression from a reporter construct with GAL4 DNA binding sites in its promoter, and inclusion of a third plasmid expressing E1A proteins can relieve the repression. Thus, we find a correlation between the ability of E1A to interact with the carboxy-terminal domain of YY1 and its ability to relieve repression caused by the carboxy-terminal domain of YY1. We propose that E1A proteins normally relieve YY1-mediated transcriptional repression by binding directly to the cellular transcription factor.

Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression.

The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.

DNA-binding proteins that interact with the 19-base pair (CRE-like) element from the HCMV major immediate early promoter in differentiating human embryonal carcinoma cells.

The pluripotent human embryonal carcinoma (EC) cell line NTERA-2 provides a useful tool for investigating cell differentiation in a way that is pertinent to the development of the early human embryo. The major immediate early (MIE) gene of human cytomegalovirus (HCMV), which is not transcribed in undifferentiated NTERA-2 EC cells but is transcribed in their differentiated derivatives, offers a model with which to study the developmental regulation of gene activity during the differentiation of these cells. We have investigated the regulatory activity of the cAMP response elements (CRE) and the activation protein (AP1) site found within several repeated 19-base-pair (bp) elements from the HCMV MIE promoter, and the developmental regulation of nuclear DNA-binding factors that interact with these sites. The 19-bp CRE but not the AP1 site is responsive to cAMP in undifferentiated NTERA-2 EC and its activity is enhanced upon differentiation. Nuclear proteins of the CREB, Fos, and Jun families bind to these sites, but, surprisingly, their levels only show limited regulation during NTERA-2 differentiation. This contrasts with results obtained with murine EC cells. However, additional and apparently novel proteins with molecular weights between 80,000 and 90,000, and binding specificities for both CRE and AP1 sites, were detected in undifferentiated EC cells. The activity of these proteins decreased markedly after differentiation, indicating their involvement in negative regulation of the CRE/AP1-like site in undifferentiated EC cells. This suggests novel members able to interact via leucine zippers with other members of the Jun-Fos-CREB family of DNA binding proteins that are also involved in this regulation.

Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells.

Erythropoietin (Epo) production in response to hypoxia or cobalt is primarily mediated by activation of transcription of the Epo gene. Recently an hypoxia responsive enhancer was identified in the 3' flanking region of the mouse and human Epo genes. Using functional analysis in Hep 3B cells we define here the minimal enhancer element as a 29-bp segment starting at the Apa1 site in the 3' flanking region of the human Epo gene. Mutagenesis studies of the minimal element identified three different areas that are necessary for full enhancer activity. Electrophoretic mobility shift assays show the presence of hypoxia- and/or cobalt-inducible nuclear DNA-binding proteins that bind to one of the active sites of the enhancer. Induction of hypoxia-binding activity was abolished by Anisomycin, a potent protein synthesis inhibitor, suggesting that de novo protein synthesis is necessary for the activation process. Further characterization of DNA-binding proteins by use of UV light crosslinking identified a protein of molecular weight of approximately 120-Kd that was present only in hypoxic extracts. This protein was found to be present in hypoxic nuclear extracts from both Epo-producing and non-Epo-producing cells, suggesting that it may be involved in a more generalized mechanism of cellular response to hypoxia.

Analysis of transcription factors binding to the duplicated PEA1 and PEA3 sites that are required for polyomavirus mutant expression in PCC4 embryonic carcinoma cells.

Embryonic carcinoma (EC) cell lines, representative of early embryonic undifferentiated cells, are nonpermissive for polyomavirus (PyV) infection as a result of a blockade of viral DNA early transcription and replication. All enhancers of PyV mutants (Py EC-PCC4), selected for the ability to grow on PCC4 EC cells, display a duplication of PEA1 and PEA3 binding sites (sites 1 and 3). However, the Py EC-PCC4 rearrangement is complex and results in variable mutant enhancer activities. We demonstrate here that duplication of sites 1 and 3 is absolutely required for a cooperative cis activation of early Py EC-PCC4 mutant transcription in PCC4 EC cells. In addition, we detect in PCC4 EC cells significant amounts of site 1- and 3-binding proteins, which we characterize as related to the Fos/Jun and Ets protein families, respectively. Wild-type PyV restriction in PCC4 EC cells may be relieved by a cooperation between site 2- and 3-binding proteins that would thereby be activated. Since site 1- or 3-binding factors could be derepressed, we improved the analysis of UV cross-linked DNA-protein complexes and were able to detect a novel factor, called PEA1/2 (for PyV enhancer A site 1- and 2-binding factor). Its DNA binding sequence overlaps sites 1 and 2 (PEA2 binding site) and is not duplicated in the M1 mutant, which exhibits the highest Py EC-PCC4 enhancer activity. he suggest that PEA1/2 is also involved in the regulation of PyV enhancer activity by repressing the site 1-binding activity.

Wild-type p53 binds to the TATA-binding protein and represses transcription.

p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.

Down-regulation of the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells is accompanied by an increase in factor binding.

In transformed cells, the E1A gene of adenovirus type 12 (Ad12) represses transcription of class I genes of the major histocompatibility complex. The tumorigenic potential of Ad12-transformed cells correlates with this diminished class I expression. In contrast, the E1A gene of the nontumorigenic Ad5 does not affect class I expression. We show here that a transfected reporter chloramphenicol acetyltransferase plasmid driven by an H-2K promoter (-1049 bp) was expressed at much lower levels in Ad12- than in Ad5-transformed mouse cells. Analysis of mutant constructs revealed that only 83 bp of H-2 DNA, consisting of the enhancer juxtaposed to the basal promoter, was sufficient for this differential expression. Whereas the H-2 basal promoter alone was somewhat less active in Ad12-transformed cells, the H-2 TATA box itself did not appear to be important. The H-2 enhancer proved to be the principal element in Ad12 E1A-mediated repression, since (i) substitution of the H-2 enhancer by simian virus 40 enhancers overcame the repression, and (ii) when juxtaposed to either its native or heterologous basal promoters, the H-2 enhancer was functional in Ad5- but not Ad12-transformed cells. Mobility shift assays showed that there is a DNA-binding activity to the 5' site (R2 element) of the enhancer that is significantly higher in Ad12- than in Ad5-transformed cells. These results suggest that decreased class I enhancer activity in Ad12-transformed cells may, at least in part, be due to the higher levels of an enhancer-specific factor, possibly acting as a repressor.

Negative regulation of the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells via a retinoic acid response element.

In cells transformed by the highly oncogenic adenovirus type 12 (Ad12), the viral E1A proteins mediate transcriptional repression of the major histocompatibility class I genes. In contrast, class I transcription is not reduced in cells transformed by the nononcogenic Ad5. The decreased rate of class I transcription is, at least in part, the result of a reduced major histocompatibility complex class I enhancer activity in Ad12-transformed cells and correlates with an increase in the levels of a DNA-binding activity to the R2 element of the enhancer (R. Ge, A. Kralli, R. Weinmann, and R. P. Ricciardi, J. Virol. 66:6969-6978, 1992). Employing transient transfection assays, we now provide direct evidence that the R2 element can confer repression in Ad12- but not Ad5-transformed cells. Repression by R2 was observed only in the presence of the positive enhancer element R1 and was dependent on (i) the number of the R2 elements and (ii) the relative arrangement of R2 and R1 elements. The putative R2-binding repressor protein, R2BF, was similar in molecular weight and binding specificity to members of the thyroid hormone/retinoic acid (RA) receptor family. RA treatment abrogated the R2-mediated repression in Ad12-transformed cells and had no effect on the activity of R2/R1-containing promoters in Ad5-transformed cells. These results are consistent with the presence of an R2-binding repressor in Ad12-transformed cells. In the absence of RA, the repressor compromises enhancer activity by interfering with the activity of the positive cis element R1. RA treatment of Ad12-transformed cells may render the repressor inactive.

A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent.

The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element.

The basic RNA polymerase II transcriptional machinery.

All genes encoding proteins in eukaryotes are transcribed by RNA polymerase II. The first step in analyzing transcriptional regulation requires understanding the general mechanisms of RNA polymerase II-specific gene transcription. The basal promoter, a template containing a TATA box devoid of upstream regulatory sequences, has been used to identify and characterize the factors which, together with RNA polymerase II, govern transcription in mammalian systems: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIG, TFIIH, and TFIIJ. Interactions between regulatory transcription factors and basal elements of the transcriptional machinery affect the transcriptional rate in a positive or negative fashion. As these multiple proteins are purified, and their coding sequences are isolated, we come closer to reproducing these processes in vitro with pure components, and thus to elucidating the complex interactions among them.

The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex.

The two forms of RNA polymerase II that exist in vivo, phosphorylated (IIO) and nonphosphorylated (IIA), were purified to apparent homogeneity from HeLa cells. The nonphosphorylated form preferentially binds to the preinitiation complex. RNA polymerase II in the complex was converted by a cellular protein kinase to the phosphorylated form.

Interactions between adenovirus E1A and members of the AP-1 family of cellular transcription factors.

We have studied interactions between bacterially produced E1A linked to Sepharose and the various DNA-binding proteins present in HeLa cell nuclear extracts (NE). DNA-binding activities and cross-reactive polypeptides recognizing the cAMP-responsive element (CRE) and the activator protein 1 (AP1) sites were bound to the E1A column, whereas nuclear factor 1 (NF1) and the activator protein 2 (AP2) DNA-binding activities were not retained by E1A. The binding activities that were retained belonged to the CRE and JUN protein family, as judged by Western blot analysis. Authentic CRE-BP1, c-Jun and c-Fos proteins produced by in-vitro translation also bound to the E1A column. However, efficient binding of in-vitro-translated CRE-BP1 and c-Fos proteins to E1A required preincubation with NE. We show here that immobilized E1A sequesters several cellular upstream transcription activators, and suggest a role for members of the AP1 family of transcription factors in E1A-mediated gene regulation.