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The nucleosome is the basic packing unit of the eukaryotic genome. Dynamic interactions between DNA and histones in the nucleosome are the molecular basis of gene accessibility regulation that governs the kinetics of various DNA-templated processes such as transcription elongation by RNA Polymerase II (Pol II). On the basis of single-molecule FRET measurements with chemically modified histones, we investigated the nucleosome dynamics during transcription elongation and how it is affected by histone acetylation at H3 K56 and the histone chaperone Nap1, both of which can affect DNA-histone interactions. We observed that H3K56 acetylation dramatically shortens the pause duration of Pol II near the entry region of the nucleosome, while Nap1 induces no noticeable difference. We also found that the elongation rate of Pol II through the nucleosome is unaffected by the acetylation or Nap1. These results indicate that H3K56 acetylation facilitates Pol II translocation through the nucleosome by assisting paused Pol II to resume and that Nap1 does not affect Pol II progression. Following transcription, only a small fraction of nucleosomes remain intact, which is unaffected by H3K56 acetylation or Nap1. These results suggest that (i) spontaneous nucleosome opening enables Pol II progression, (ii) Pol II mediates nucleosome reassembly very inefficiently, and (iii) Nap1 in the absence of other factors does not promote nucleosome disassembly or reassembly during transcription.

Degradation Factor 1 was discovered 20 years ago as a yeast protein copurifying with Rad26, a helicase involved in transcription-coupled DNA repair. It was subsequently shown to control the ubiquitylation and destruction of the large subunit of DNA damage-arrested RNA Polymerase II. Since that time, much has been learned about Def1's role in polymerase destruction and new functions of the protein have been revealed. We now understand that Def1 is involved in more than just RNA polymerase II regulation. Most of its known functions are associated with maintaining chromosome and genomic integrity, but other exciting activities outside this realm have been suggested. Here we review this fascinating protein, describe its regulation and present a hypothesis that Def1 is a central coordinator of ubiquitin signaling pathways in cells.

Gene expression relies on the balance between mRNA synthesis in the nucleus and decay in the cytoplasm, processes once thought to be separate. We now know that transcription and decay rates are coordinated, but the factors or molecular mechanisms are unclear. The Ccr4-Not complex regulates multiple stages of gene expression, from mRNA synthesis to protein destruction. One of its functions is to promote RNA polymerase II elongation by reactivating arrested elongation complexes. Here we explored the features of polymerase required for Ccr4-Not to promote elongation and found that the Rpb4/7 module is important for Ccr4-Not to associate with elongation complexes and stimulate elongation. Rpb4/7 has also been implicated in coordinating mRNA synthesis and decay, but its role in this process is controversial. The interplay between Ccr4-Not and Rpb4/7 described here suggests a mechanism for how the cell coordinates mRNA synthesis and decay.

The Ccr4-Not complex is a highly conserved nine-subunit protein complex that has been implicated in virtually all aspects of gene control, including transcription, mRNA decay and quality control, RNA export, translational repression and protein ubiquitylation. Understanding its mechanisms of action has been difficult due to the size of the complex and the fact that it regulates mRNAs and proteins at many levels in both the cytoplasm and the nucleus. Recently, biochemical and genetic studies on the yeast Ccr4-Not complex have revealed insights into its role in promoting elongation by RNA polymerase II. This review will describe what is known about the Ccr4-Not complex in regulating transcription elongation and its possible collaboration with other factors traveling with RNAPII across genes. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

The purpose of this review is to provide an analysis of the latest developments on the functions of the carbon catabolite-repression 4-Not (Ccr4-Not) complex in regulating eukaryotic gene expression. Ccr4-Not is a nine-subunit protein complex that is conserved in sequence and function throughout the eukaryotic kingdom. Although Ccr4-Not has been studied since the 1980s, our understanding of what it does is constantly evolving. Once thought to solely regulate transcription, it is now clear that it has much broader roles in gene regulation, such as in mRNA decay and quality control, RNA export, translational repression and protein ubiquitylation. The mechanism of actions for each of its functions is still being debated. Some of the difficulty in drawing a clear picture is that it has been implicated in so many processes that regulate mRNAs and proteins in both the cytoplasm and the nucleus. We will describe what is known about the Ccr4-Not complex in yeast and other eukaryotes in an effort to synthesize a unified model for how this complex coordinates multiple steps in gene regulation and provide insights into what questions will be most exciting to answer in the future.

Gene expression depends upon the antagonistic actions of chromatin remodeling complexes. While this has been studied extensively for the enzymes that covalently modify the tails of histones, the mechanism of how ATP-dependent remodeling complexes antagonize each other to maintain the proper level of gene activity is not known. The gene encoding a large subunit of ribonucleotide reductase, RNR3, is regulated by ISW2 and SWI/SNF, complexes that repress and activate transcription, respectively. Here, we studied the functional interactions of these two complexes at RNR3. Deletion of ISW2 causes constitutive recruitment of SWI/SNF, and conditional reexpression of ISW2 causes the repositioning of nucleosomes and reduced SWI/SNF occupancy at RNR3. Thus, ISW2 is required for restriction of access of SWI/SNF to the RNR3 promoter under the uninduced condition. Interestingly, the binding of sequence-specific DNA binding factors and the general transcription machinery are unaffected by the status of ISW2, suggesting that disruption of nucleosome positioning does not cause a nonspecific increase in cross-linking of all factors to RNR3. We provide evidence that ISW2 does not act on SWI/SNF directly but excludes its occupancy by positioning nucleosomes over the promoter. Genetic disruption of nucleosome positioning by other means led to a similar phenotype, linking repressed chromatin structure to SWI/SNF exclusion. Thus, incorporation of promoters into a repressive chromatin structure is essential for prevention of the opportunistic actions of nucleosome-disrupting activities in vivo, providing a novel mechanism for maintaining tight control of gene expression.

RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.

The Ssn6-Tup1 corepressor complex regulates many genes in Saccharomyces cerevisiae. Three mechanisms have been proposed to explain its repression functions: 1) nucleosome positioning by binding histone tails; 2) recruitment of histone deacetylases; and 3) direct interference with the general transcription machinery or activators. It is unclear if Ssn6-Tup1 utilizes each of these mechanisms at a single gene in a redundant manner or each individually at different loci. A systematic analysis of the contribution of each mechanism at a native promoter has not been reported. Here we employed a genetic strategy to analyze the contributions of nucleosome positioning, histone deacetylation, and Mediator interference in the repression of chromosomal Tup1 target genes in vivo. We exploited the fact that Ssn6-Tup1 requires the ISW2 chromatin remodeling complex to establish nucleosome positioning in vivo to disrupt chromatin structure without affecting other Tup1 repression functions. Deleting ISW2, the histone deacetylase gene HDA1, or genes encoding Mediator subunits individually caused slight or no derepression of RNR3 and HUG1. However, when Mediator mutations were combined with Deltaisw2 or Deltahda1 mutations, enhanced transcription was observed, and the strongest level of derepression was observed in triple Deltaisw2/Deltahda1/Mediator mutants. The increased transcription in the mutants was not due to the loss of Tup1 at the promoter and correlated with increased TBP cross-linking to promoters. Thus, Tup1 utilizes multiple redundant mechanisms to repress transcription of native genes, which may be important for it to act as a global corepressor at a wide variety of promoters.

It is now known that the synthesis and decay of mRNAs are coordinated; however, the factors involved in this process and the mechanism of how these two distinct events are linked are not known. The...

Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions between the N- and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover, RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors to impart stringent control over this very abundant RNA helicase.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labelling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. Cys530A1a mutant), C381 becomes the site of estrogen-competible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed and region-specific chemical mutagenesis have identified a region around C530 important in discrimination between estrogens and antiestrogens, and other mutants have allowed identification of residues important in hormone-dependent transcriptional activation. Some transcriptionally inactive ER mutants also function as potent dominant negative ERs, suppressing the activity of wild-type ERs at low concentrations. These studies are beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer.

Chromatin is regulated by cross talk among different histone modifications, which can occur between residues within the same tail or different tails in the nucleosome. The latter is referred to as trans-tail regulation, and the best-characterized example of this is the dependence of H3 methylation on H2B ubiquitylation. Here we describe a novel form of trans-tail regulation of histone modifications involving the N-terminal tail of histone H2A. Mutating or deleting residues in the N-terminal tail of H2A reduces H2B ubiquitylation and H3K4 methylation but does not affect the recruitment of the modifying enzymes, Rad6/Bre1 and COMPASS, to genes. The H2A tail is required for the incorporation of Cps35 into COMPASS, and increasing the level of ubiquitylated H2B in H2A tail mutants suppresses the H3K4 methylation defect, suggesting that the H2A tail regulates H2B-H3 cross talk. We mapped the region primarily responsible for this regulation to the H2A repression domain, HAR. The HAR and K123 of H2B are in close proximity to each other on the nucleosome, suggesting that they form a docking site for the ubiquitylation machinery. Interestingly, the HAR is partially occluded by nucleosomal DNA, suggesting that the function of the H2A cross talk pathway is to restrict histone modifications to nucleosomes altered by transcription.

We have previously identified cysteine 530 in the human estrogen receptor (ER) as the major site of attachment for covalently binding affinity ligands and have shown that when this cysteine is mutated to alanine (C530A mutant), the affinity ligand [tamoxifen aziridine (TAZ)] can still bind covalently to the ER, presumably by interaction with a different cysteine(s) in the hormone-binding domain (HBD). Using site-directed mutagenesis, we have determined the alternative ligand attachment site and the functional importance of the cysteines (residues 381, 417, 447, and 530) in the HBD of the ER to the hormone-binding and transcriptional responses to estrogens and antiestrogens. Cysteine 530 plus one or more of these other cysteines were mutated to alanines. Analysis of these mutant ERs expressed in Chinese hamster ovary cells provides strong evidence that cysteine 381 is the residue that is preferentially covalently labeled by TAZ in the C530A mutant. Hence, portions of the HBD that are far apart in the linear receptor sequence, namely regions near C381 and C530, are probably closely positioned in the ligand-binding pocket, with the cysteine thiols being 1.1 nm or less apart. The affinity of estradiol binding to receptors was reduced only 2- and 5-fold, respectively, in the double and quadruple Cys to Ala mutants, and estradiol was an effective stimulator of transcription from an estrogen-responsive reporter gene [(ERE)2-TATA-CAT].(ABSTRACT TRUNCATED AT 250 WORDS)

A previous report from this laboratory (Reese, J.C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperature-sensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.

We have carried out experiments to determine the role of the cysteines in the hormone-binding domain (HBD) of the human estrogen receptor (ER) in receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD (cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of the ER cDNA (containing Gly400) from cysteine to alanine; Cys530 was also mutated to a serine. The mutant and wild-type receptor cDNAs were expressed in Chinese hamster ovary cells using an expression vector containing the Rous sarcoma virus promoter. The mutant and wild-type receptors were assayed for hormone binding and for their ability to activate estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2) with affinity similar to wild-type ER, displaying a Kd between 0.3 and 0.8 nM (wild-type ER Kd = 0.45 +/- 0.10 nM). All were capable of covalent labeling by the affinity ligands ketononestrol aziridine, an estrogen agonist, and tamoxifen aziridine, an antagonist. Since in previous work we identified Cys530 as the site of covalent attachment of these ligands (Harlow, K.W., Smith, D.N., Katzenellenbogen, J.A., Greene, G.L., and Katzenellenbogen, B.S. (1989) J. Biol. Chem. 264, 17476-17485) it appears that an alternate residue(s) can be labeled in the absence of a cysteine at position 530; studies with methyl methanethiosulfonate, a cysteine-specific reagent, suggest that this residue is probably another cysteine in the HBD. The C381A, C417A, C530A and C530S ERs showed E2-stimulated transcriptional activation profiles similar to wild-type ER whereas the dose response for E2 for the C447A mutant was shifted to the right, requiring 50 x higher E2 concentrations to achieve half-maximal response. Tamoxifen aziridine inhibited E2-stimulated transcription, and ketononestrol aziridine stimulated transcription by wild-type, C530A, and C530S ER, but the effectiveness of these covalently attaching ligands was altered in the C530A and C530S mutants. Thus, these two mutant receptors are altered in their transactivation response to agonist and antagonist affinity labeling ligands but are unaltered in their response to reversibly binding estrogens and antiestrogens. In addition, we show that a mutant ER (C447A) can have an affinity for E2 similar to that of wild-type ER but differs in its ability to activate transcription in response to E2, indicating a decoupling of the hormone binding and transcriptional activation functions in this receptor.

The gene encoding ribonucleotide reductase 3 (RNR3) is strongly induced in response to DNA damage. Its expression is strictly dependent upon the TAFII subunits of TFIID, which are required for the recruitment of SWI/SNF and nucleosome remodeling. However, full activation of RNR3 also requires GCN5, the catalytic subunit of the SAGA histone acetyltransferase complex. Thus, RNR3 is dependent upon both TFIID and SAGA, two complexes that deliver TATA-binding protein (TBP) to promoters. Furthermore, unlike the majority of TFIID-dominated genes, RNR3 contains a consensus TATA-box, a feature of SAGA-regulated core promoters. Although a large fraction of the genome can be characterized as either TFIID- or SAGA-dominant, it is expected that many genes utilize both. The mechanism of activation and the relative contributions of SAGA and TFIID at genes regulated by both complexes have not been examined. Here we delineated the role of SAGA in the regulation of RNR3 and contrast it to that of TFIID. We find that SAGA components fulfill distinct functions in the regulation of RNR3. The core promoter of RNR3 is SAGA-dependent, and we provide evidence that SAGA, not TAFIIs within TFIID, are largely responsible for TBP recruitment. This taken together with our previous work provides evidence that SAGA recruits TBP, whereas TFIID mediates chromatin remodeling. Thus, we described an unexpected shift in the division of labor between these two complexes and provide the first characterization of a gene that requires both SAGA and TFIID.

In yeast, inactivation of certain TBP-associated factors (TAFIIs) results in arrest at specific stages of the cell cycle. In some cases, cell cycle arrest is not observed because overlapping defects in other cellular processes precludes the manifestation of an arrest phenotype. In the latter situation, genetic analysis has the potential to reveal the involvement of TAFIIs in cell cycle regulation. In this report, a temperature-sensitive mutant of TAF68/61 was used to screen for high-copy dosage suppressors of its growth defect. Ten genes were isolated: TAF suppressor genes, TSGs 1–10. Remarkably, most TSGs have either a genetic or a direct link to control of the G2/M transition. Moreover, eight of the 10 TSGs can suppress a CDC28 mutant specifically defective for mitosis (cdc28-1N) but not an allele defective for passage through start. The identification of these genes as suppressors of cdc28-1N has identified four unreported suppressors of this allele. Moreover, synthetic lethality is observed between taf68-9 and cdc28-1N. The isolation of multiple genes involved in the control of a specific phase of the cell cycle argue that the arrest phenotypes of certain TAFII mutants reflect their role in specifically regulating cell cycle functions. Copyright © 2001 John Wiley & Sons, Ltd.

The budding yeast Saccharomyces cerevisiae was used as a model system to study the function of the adenovirus E1A oncoprotein. Previously we demonstrated that expression of the N-terminal 82 amino acids of E1A in yeast causes pronounced growth inhibition and specifically interferes with SWI/SNF-dependent transcriptional activation. Further genetic analysis identified the yeast transcription factor Adr1 as a high copy suppressor of E1A function. Transcriptional activation by Adr1 requires interaction with co-activator proteins Ada2 and Gcn5, components of histone acetyltransferase complexes including ADA and SAGA. Analysis of mutant alleles revealed that several components of the SAGA complex, including proteins from the Ada, Spt, and Taf classes were required for E1A-induced growth inhibition. Growth inhibition also depended on the Gcn5 histone acetyltransferase, and point mutations within the Gcn5 HAT domain rendered cells E1A-resistant. Also required was SAGA component Tra1, a homologue of the mammalian TRRAP protein which is required for c-myc and E1A induced cellular transformation. Additionally, Gcn5 protein could associate with E1A in vitro in a manner that depended on the N-terminal domain of E1A, and Tra1 protein was co-immunoprecipitated with E1A in vivo. These results indicate a strong requirement for intact SAGA complex for E1A to function in yeast, and suggest a role for SAGA-like complexes in mammalian cell transformation.

The DNA damage inducible gene ribonucleotide reductase (RNR3) is regulated by a transcriptional repression mechanism by the recruitment of the Ssn6-Tup1 corepressor complex to its promoter by the sequence-specific DNA-binding protein Crt1. Ssn6-Tup1 is reported to represses transcription by interfering with transcription factors, recruiting histone deacetylases, and positioning nucleosomes at the promoter of its target genes. Two of the three mechanisms involve effects on chromatin structure, and therefore, we have delineated the nucleosomal structure of RNR3 in the repressed and derepressed state using multiple nuclease mapping strategies. A regular array of positioned nucleosomes is detected over the repressed RNR3 promoter that extends into the coding sequence. Treating cells with DNA damaging agents or deleting CRT1, SSN6, or TUP1 derepresses RNR3 transcription, and causes a dramatic disruption of nucleosome positioning over its promoter. Furthermore, derepression of RNR3 correlated with changes in nuclease sensitivity within the upstream repression sequence (URS) region. Specifically, the loss of a MNase-hypersensitive site, and the appearance of strong DNase I hypersensitivity, was observed over the URS. Interestingly, we find that the binding of Crt1 to the promoter in the absence of Ssn6 or Tup1 is insufficient for nucleosome positioning or regulating chromatin structure at the URS; thus, these two functions are strictly dependent upon Ssn6-Tup1. We propose that RNR3 is regulated by changes in nucleosome positioning and chromatin structure that are mediated by Ssn6, Tup1, and Crt1.

The proteasome is well known for its regulation of the cell cycle and degradation of mis-folded proteins, yet many of its functions are still unknown. We show that RPN11, a gene encoding a subunit of the regulatory cap of the proteasome, is required for UV-stimulated activation of Gcn4p target genes, but is dispensable for their activation by the general control pathway. We provide evidence thatRPN11 functions downstream of RAS2, and show that mutation of two additional proteasome subunits results in identical phenotypes. Our analysis defines a novel function of the proteasome: regulation of the RAS- and AP-1 transcription factor-dependent UV resistance pathway.

The RNA polymerase II general transcription factor TFIID is a complex containing the TATA-binding protein (TBP) and associated factors (TAFs). We have used a mutant allele of the gene encoding yeast TAF(II)68/61p to analyze its function in vivo. We provide biochemical and genetic evidence that the C-terminal alpha-helix of TAF(II)68/61p is required for its direct interaction with TBP, the stable incorporation of TBP into the TFIID complex, the integrity of the TFIID complex, and the transcription of most genes in vivo. This is the first evidence that a yeast TAF(II) other than TAF(II)145/130 interacts with TBP, and the implications of this on the interpretation of data obtained studying TAF(II) mutants in vivo are discussed. We have identified a high copy suppressor of the TAF68/61 mutation, TSG2, that has sequence similarity to a region of the SAGA subunit Ada1. We demonstrate that it directly interacts with TAF(II)68/61p in vitro, is a component of TFIID, is required for the stability of the complex in vivo, and is necessary for the transcription of many yeast genes. On the basis of these functions, we propose that Tsg2/TAF(II)48p is the histone 2A-like dimerization partner for the histone 2B-like TAF(II)68/61p in the yeast TFIID complex.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530 mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.

We have shown that charged amino acids near C530 of the human estrogen receptor (ER) are involved in receptor discrimination between estrogen and antiestrogen. We now examine the role of charged residues, adjacent to the three other cysteines (381, 417, and 447) in the hormone-binding domain of the human ER, in the hormone-binding, DNA-binding, and transcription activation abilities of the receptor. Mutation of the one charged amino acid nearest to C381 gave a mutant receptor (E380Q) requiring two to three times less estradiol (E2) than wild type (WT) ER to achieve maximal activity and having activity in the absence of added estrogen that was 6-fold higher than that of WT receptor. The enhanced ability of this mutant to bind to estrogen response element DNA in the absence and presence of estrogen may, at least in part, explain its elevated, seemingly constitutive trans-activation activity and its increased sensitivity to estrogen. While more sensitive to E2, this E380Q mutant was less sensitive than WT ER to antiestrogen for suppression of transcriptional activity. Mutation of all three charged residues nearest to C381 (the triple mutant D374N, E380Q, and E385Q) resulted in a greatly reduced potency of the receptor in trans-activation with no change in estrogen-binding affinity. When K449 (near C447), highly conserved among steroid receptors, was mutated to Q, 400-fold more E2 was required for maximal reporter gene trans-activation due to an unstable, temperature-sensitive hormone-receptor complex. In contrast, the mutant K416Q (near C417) was unaltered in E2-binding or receptor transcriptional activity. These studies reveal a region in the N-terminal portion of the hormone-binding domain (ca. amino acids 374-385) where alterations in charged residues result in either increases or decreases in receptor transcriptional activity with no change in receptor affinity for hormone. Our findings suggest that this region may be important in DNA binding and protein-protein interactions that modulate transcriptional activity of the ER. In addition, the region near C447, which is well conserved among steroid receptors, appears to be important in maintaining the receptor in a conformation that is stable at physiological (37 C) temperatures. To our knowledge, this is the first report of an ER (E380Q) with a sensitivity to E2 for trans-activation greater than that of WT receptor and having high trans-activation activity in the absence of added hormone.

In this study we report the first estrogen receptor to be characterized in an oviparous elasmobranch. The skate receptor has high affinity for estradiol (Kd = 0.7 nM), binds both estradiol and the synthetic estrogen DES, and exists in low quantities (50-100 fmol/g oviduct). The receptor displays rapid binding kinetics with half-times of 5 min at 22 degrees and 77 min at 4 degrees. DEAE-Sepharose chromatography reveals one receptor moiety which elutes between 0.13 and 0.14 M KCl. By sucrose gradient ultracentrifugation sedimentation coefficients of 3.6 S under high-salt (0.5 M KCl) and 6.0 S under low salt (0.01 M KCl) conditions were obtained. Using Sephadex G200 gel filtration chromatography, a Stokes radius (Rs) of 2.8 nm and an estimated molecular weight of 43 kDa were calculated. DNA-cellulose elution profiles reveal that the receptor elutes as one peak between 0.34 and 0.36 M NaCl (as compared to 0.20-0.22 M NaCl in mammals and birds and 0.55 M for dogfish). Although some differences are noted between the elasmobranch ER and those of other vertebrates (e.g., dissociation kinetics, DNA affinity), in general it can be said that the skate ER is a "classical" ER in most respects. It is suggested that this steroid receptor has played a key role in the reproductive tract functions of nutrient provision, embryo protection, and as a conduit to the external environment since the earliest chordate era, approximately 400 million years ago.

Two progesterone receptors in the oviduct of the freshwater turtle Chrysemys picta: possible homology to mammalian and avian receptor systems. Here we report the characterization of two specific progesterone receptors in nuclear extracts of the turtle oviduct. The receptors differ in dissociation constants (2.8 nM vs 27 nM) which can be separated on DEAE-Sepharose, the former eluting at 0.08 M KCl and the latter at 0.20 M KCl. [3H]R5020 photoaffinity labeling SDS-PAGE revealed that the 2.8 nM moiety migrates with an apparent molecular weight of 80 ± 5 kDa and the 27 nM moiety migrates with an apparent molecular weight of 120 ± 5 kDa. These receptors are termed PR-A and PR-B due to their molecular mass and elution profiles. DNA-cellulose Chromatographic studies show that both bind DNA-cellulose with the PR-A eluting at 0.09 M NaCl and PR-B eluting between 0.20–0.21 M NaCl. In reproductively inactive turtles (from the months of January and February) estradiol is undetectable, and PR-B is absent as determined by Scatchard analysis, [3H]R5020 photoaffinity labeling electrophoretic studies and DEAE-Sepharose and DNA-cellulose chromatography. In these animals PR-B can be replenished by estrogen treatment, suggesting a physiological role for both PR-A and PR-B and dependence of PR-B on estradiol.