Phosphorylation of the human ubiquitin-conjugating enzyme, CDC34, by casein kinase 2.

The ubiquitin-conjugating enzyme, CDC34, has been implicated in the ubiquitination of a number of vertebrate substrates, including p27(Kip1), IkappaBalpha, Wee1, and MyoD. We show that mammalian CDC34 is a phosphoprotein that is phosphorylated in proliferating cells. By yeast two-hybrid screening, we identified the regulatory (beta) subunit of human casein kinase 2 (CK2) as a CDC34-interacting protein and show that human CDC34 interacts in vivo with CK2beta in transfected cells. CDC34 is specifically phosphorylated in vitro by recombinant CK2 and HeLa nuclear extract at five sites within the carboxyl-terminal 36 amino acids of CDC34. Importantly, this phosphorylation is inhibited by heparin, a substrate-specific inhibitor of CK2. We have also identified a kinase activity associated with CDC34 in proliferating cells, and we show that this kinase is sensitive to heparin and can utilize GTP, strongly suggesting it is CK2. Phosphorylation of CDC34 by the associated kinase maps predominantly to residues 203 and 222. Mutation of CDC34 at CK2-targeted residues, Ser-203, Ser-222, Ser-231, Thr-233, and Ser-236, abolishes the phosphorylation of CDC34 observed in vivo and markedly shifts nuclearly localized CDC34 to the cytoplasm. These results suggest a potential role for CK2-mediated phosphorylation in the regulation of CDC34 cell localization and function.

BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor.

Mutational inactivation of BRCA1 confers a cumulative lifetime risk of breast and ovarian cancers. However, the underlying basis for the tissue-restricted tumor-suppressive properties of BRCA1 remains poorly defined. Here we show that BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor alpha (ERalpha), a principal determinant of the growth, differentiation, and normal functional status of breasts and ovaries. In Brca1-null mouse embryo fibroblasts and BRCA1-deficient human ovarian cancer cells, ERalpha exhibited ligand-independent transcriptional activity that was not observed in Brca1-proficient cells. Ectopic expression in Brca1-deficient cells of wild-type BRCA1, but not clinically validated BRCA1 missense mutants, restored ligand-independent repression of ERalpha in a manner dependent upon apparent histone deacetylase activity. In estrogen-dependent human breast cancer cells, chromatin immunoprecipitation analysis revealed the association of BRCA1 with ERalpha at endogenous estrogen-response elements before, but not after estrogen stimulation. Collectively, these results reveal BRCA1 to be a ligand-reversible barrier to transcriptional activation by unliganded promoter-bound ERalpha and suggest a possible mechanism by which functional inactivation of BRCA1 could promote tumorigenesis through inappropriate hormonal regulation of mammary and ovarian epithelial cell proliferation.

Genome mining for human cancer genes: wherefore art thou?

In an initial data-mining effort, the draft human genome was searched to find paralogs of known tumor suppressor genes, and for gene arrangements, which are typical of oncogenes, in cancer cells. The results were disappointing, indicating that although knowledge of the human genome will undoubtedly be of great help, other approaches to identify new oncogenes are needed.

Sequence-specific transcriptional corepressor function for BRCA1 through a novel zinc finger protein, ZBRK1.

BRCA1 has been implicated in the transcriptional regulation of DNA damage-inducible genes that function in cell cycle arrest. To explore the mechanistic basis for this regulation, a novel human gene, ZBRK1, which encodes a 60 kDa protein with an N-terminal KRAB domain and eight central zinc fingers, was identified by virtue of its interaction with BRCA1 in vitro and in vivo. ZBRK1 binds to a specific sequence, GGGxxx CAGxxxTTT, within GADD45 intron 3 that supports the assembly of a nuclear complex minimally containing both ZBRK1 and BRCA1. ZBRK1 represses transcription through this recognition sequence in a BRCA1-dependent manner. These results thus reveal a novel corepressor function for BRCA1 and provide a mechanistic basis for the biological activity of BRCA1 through sequence-specific transcriptional regulation.

Lessons learned from BRCA1 and BRCA2.

BRCA1 and BRCA2 are breast cancer susceptibility genes. Mutations within BRCA1 and BRCA1 are responsible for most familial breast cancer cases. Targeted deletion of Brca1 or Brca2 in mice has revealed an essential function for their encoded products, BRCA1 and BRCA2, in cell proliferation during embryogenesis. Mouse models established from conditional expression of mutant Brca1 alleles develop mammary gland tumors, providing compelling evidence that BRCA1 functions as a breast cancer suppressor. Human cancer cells and mouse cells deficient in BRCA1 or BRCA2 exhibit radiation hypersensitivity and chromosomal abnormalities, thus revealing a potential role for both BRCA1 and BRCA2 in the maintenance of genetic stability through participation in the cellular response to DNA damage. Functional analyses of the BRCA1 and BRCA2 gene products have established their dual participation in transcription regulation and DNA damage repair. Potential insight into the molecular basis for these functions of BRCA1 and BRCA2 has been provided by studies that implicate these two tumor suppressors in both the maintenance of genetic stability and the regulation of cell growth and differentiation.

Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein.

Adenovirus E1A proteins prepare the host cell for viral replication, stimulating cell cycling and viral transcription through interactions with critical cellular regulatory proteins such as RB and CBP. Here we show that the E1A zinc-finger domain that is required to activate transcription of viral early genes binds to a host-cell multiprotein complex containing homologues of yeast Srb/Mediator proteins. This occurs through a stable interaction with the human homologue of Caenorhabditis elegans SUR-2, a protein required for many developmental processes in the nematode. This human Srb/Mediator complex stimulates transcription in vitro in response to both the E1A zinc-finger and the herpes simplex virus VP16 activation domains. Interaction with human Sur-2 is also required for transcription to be activated by the activation domain of a transcription factor of the ETS-family in response to activated mitogen-activated protein (MAP) kinase.

DA-complex assembly activity required for VP16C transcriptional activation.

One class of transcriptional activation domains stimulates the concerted binding of TFIIA and TFIID to promoter DNA. To test whether this DA-complex assembly activity contributes significantly to the overall mechanism of activation in vivo, we analyzed mutants of the 38-amino-acid residue VP16C activation subdomain from herpes simplex virus. An excellent correlation was observed between the in vivo activation function of these mutants and their in vitro DA-complex assembly activity. Mutants severely defective for in vivo activation also showed reduced in vitro binding to native TFIIA. No significant correlation between in vivo activation function and in vitro binding to human TATA binding protein, human TFIIB, or Drosophila melanogaster TAFII40 was observed for this set of VP16C mutants. These results argue that the ability of VP16C to increase the rate and extent of DA-complex assembly makes a significant contribution to the overall mechanism of transcriptional activation in vivo.

Mechanisms of viral activators.

Adenovirus large E1A, Epstein-Barr virus Zebra, and herpes simplex virus VP16 were studied as models of animal cell transcriptional activators. Large E1A can activate transcription from a TATA box, a result that leads us to suggest that it interacts with a general transcription factor. Initial studies showed that large E1A binds directly to the TBP subunit of TFIID. However, analysis of multiple E1A and TBP mutants failed to support the significance of this in vitro interaction for the mechanism of activation. Recent studies to be reported elsewhere indicate that conserved region 3 of large E1A, which is required for its activation function, binds to one subunit of a multisubunit protein that stimulates in vitro transcription in response to large E1A and other activators. A method was developed for the rapid purification of TFIID approximately 25,000-fold to near homogeneity from a cell line engineered to express an epitope-tagged form of TBP. Purified TFIID contains 11 major TAFs ranging in mass from approximately 250 to 20 kD. Zta and VP16, but not large E1A, greatly stimulate the rate and extent of assembly of a TFIID-TFIIA complex on promoter DNA (DA complex). For VP16, this is a function of the carboxy-terminal activation subdomain. An excellent correlation was found between the ability of VP16C mutants to stimulate DA complex assembly and their ability to activate transcription in vivo. Consequently, for a subset of activation domains, DA complex assembly activity is an important component of the overall mechanism of activation.

A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly.

TFIIA is a general transcription factor that interacts with the TFIID-promoter complex required for transcription initiation by RNA polymerase II. Two lines of evidence suggest that TFIIA is directly involved in the mechanism by which some activators stimulate transcription. First, binding of TFIIA to a TFIID-promoter complex is a rate-limiting step that is enhanced by transcriptional activators GAL4-AH and Zta. Second, recombinant TFIIA greatly enhances activator-dependent transcription. In this study, we found that the activation domains of Zta and VP16 bind directly to TFIIA. Both Zta and VP16 stimulated rapid assembly of a stable TFIID-TFIIA complex on promoter DNA. Analysis of deletion derivatives of the VP16 activation domain indicated that the ability to bind to TFIIA correlates with the ability to enhance TFIID-TFIIA-promoter ternary complex assembly. Thus, we propose that a class of activators stimulate transcription initiation through direct interactions with both TFIIA and TFIID, which stimulate the assembly of an activated TFIIA-TFIID-promoter complex.

Functional interaction of adenovirus E1A with holo-TFIID.

The activation domains of several regulatory transcription factors have been shown to bind directly in vitro to the TATA box-binding protein (TBP). Yet TBP must also interact with multiple associated polypeptides, called TAFs, for these same activators to stimulate transcription. These findings raise the question of how TBP can interact with so many proteins, both activators and TAFs, simultaneously. Here, we show that the activation domain of the adenovirus large E1A protein can bind specifically and stably to isolated holo-TFIID, the multisubunit protein complex consisting of TBP plus TAFs. Consequently, the surface of TBP that interacts with E1A must be exposed in the holo-TFIID complex. To assess the functional significance of this interaction, we established an in vitro transcription system responsive to the E1A activation domain. The addition of excess E1A to this system inhibits (squelches) both E1A-dependent and E1A-independent transcription by sequestering a target factor required for E1A activation. From among the component activities that collectively reconstitute E1A-responsive transcription in this system, holo-TFIID alone is singularly capable of reversing the inhibition of transcription mediated by excess E1A, indicating that holo-TFIID is the direct functional target of the E1A activation domain.

Association between proto-oncoprotein Rel and TATA-binding protein mediates transcriptional activation by NF-kappa B.

The c-Rel protein is able to associate in vitro and in vivo with the TATA-binding protein (TBP) of the TFIID complex. Coexpression of TBP with c-Rel augments transactivation from the kappa B site in Drosophila Schneider cells. DNA-binding mutants of TBP not only fail to cooperate, but they repress transactivation by c-Rel. There may be a direct communication between kappa B enhancer binding proteins and basal transcription factors which leads to enhanced transcription.

Factors (TAFs) required for activated transcription interact with TATA box-binding protein conserved core domain.

TFIID is a multisubunit protein containing the TATA box-binding polypeptide (TBP) and associated factors (TFIID-TAFs) required for activated transcription by RNA polymerase II. TBPs from different eukaryotes contain a highly conserved carboxy-terminal domain and very divergent amino-terminal domains. Earlier studies proposed that the amino-terminal domains of metazoan TBPs are required for activated transcription. However, we report that a human TFIID complex containing an amino-terminal truncated TBP contains all the major TFIID-TAFs and supports in vitro transcriptional stimulation by different classes of activation domains and from a TATA-less promoter. Protein blotting experiments revealed direct interactions between the conserved domain of TBP and the two largest TAFs. The results suggest a model for the interaction of TFIID-TAFs with TBP.

Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter.

Transcription factor IID (TFIID) binds to TATA boxes, nucleating the assembly of initiation complexes containing several general transcription factors and RNA polymerase II. Recently, TFIID was shown to be a multisubunit complex containing a TATA box-binding polypeptide (TBP) and several tightly associated polypeptides (TAFs), which are required for transcriptional stimulation by activator proteins. Here, we report the development of a human cell line expressing an epitope-tagged TBP and the immunopurification of a native, high-molecular-weight form of TFIID that supports transcriptional stimulation by several different classes of activation domains. Recovery of basal and activated TFIID transcriptional specific activity was close to approximately 100%. Electrophoretic mobility-shift analysis demonstrated a single major DNA-protein complex. This holo-TFIID contains TAFs of approximately 250, 125, 95, 78, and 50 kD and sediments at 17S. Holo-TFIID produced an extended footprint over the adenovirus major late promoter TATA box and initiator sequence and supported transcriptional activation from a promoter lacking a TATA box. These results lead us to hypothesize that a single multisubunit TFIID protein supports transcriptional stimulation by diverse activation domains and from a TATA-less promoter.

Modulation of human triosephosphate isomerase gene transcription by serum.

We have monitored the level of mRNA encoding the glycolytic and gluconeogenic enzyme trisephosphate isomerase (TPI) during the growth arrest of cells by serum deprivation and the subsequent growth activation of cells by serum addition. This analysis has demonstrated that the steady state level of TPI mRNA changes 5-20-fold, depending upon the cell type, during the transversal of cells from a proliferative to a nonproliferative state and vice versa. These changes are largely attributable to changes in the rate of TPI gene transcription rather than to alterations in post-transcriptional processes as determined by nuclear run-on measurements. Following serum stimulation, the increase in TPI gene expression is maximal at or around the onset of DNA synthesis. We have also quantitated TPI mRNA throughout the cell cycle following cell synchronization with aphidicolin. Our results indicate that the steady state level of TPI mRNA is relatively constant throughout the division cycle of proliferating cells. Thus, while TPI gene expression is modulated during the traversal of cells to and from a nonproliferative state, it is not significantly modulated during the cycle of events that is characteristic of continuously proliferating cells.

Minimal sequence and factor requirements for the initiation of transcription from an atypical, TATATAA box-containing housekeeping promoter.

We have established the minimal sequence and factor requirements for both constitutive and viral-induced transcription from an atypical, TATATAA box-containing human housekeeping promoter. Utilizing a transient cotransfection protocol, we have found that efficient transactivation of triosephosphate isomerase (TPI) gene transcription by the immediate early proteins of adenovirus and pseudorabies virus is dependent upon the same assembly of sequence elements that collectively confer minimal TPI promoter function in the absence of viral protein. These elements span TPI promoter positions -65 and -6 (where +1 is the transcription initiation site) and include not only a TFIID-responsive TATATAA box (-27 to -21) but a single GC box (-53 to -48) that binds Spl, and a novel cap proximal element (-18 to -6) that binds a 110-kDa nuclear factor that is present in HeLa cells. We demonstrate that these elements function in an interdependent fashion; deleting either GC box 1 or the cap proximal element completely or nearly abolished both basal transcription and viral transactivation. Therefore, these elements and their cognate factors represent the basal transcription initiation complex through which the immediate early protein of adenovirus or pseudorabies virus mediates the stimulation of TPI gene transcription. We discuss the implications of these data for both constitutive and viral-induced transcription.

Transcriptional regulatory sequences of the housekeeping gene for human triosephosphate isomerase.

To examine the functional organization of the human triosephosphate isomerase (TPI) promoter, deletion, insertion, and linker scanning mutations were introduced into the TPI promoter of hybrid TPI/beta-globin genes. These genes were transiently expressed in mouse L and human HeLa cells, and the effect of each mutation on the frequency and position of transcription initiation was assayed by S1 nuclease transcript mapping. Multiple positive regulatory elements reside between positions -595 and +1 in L cells and -920 and -7 in HeLa cells and coordinately promote maximum hybrid gene transcription. These elements include an array of GC boxes (positions -126 to -48) that variably conform to the consensus Sp1-binding site, and a canonical TATA box (positions -27 to -21) that is essential for detectable levels of transcription. In an additive yet position-dependent fashion, the GC boxes function in cis to the TATA box to control both the frequency and position of transcription initiation. Additional positive elements reside upstream of position -131 and are required for full promoter function. Also, an inhibitory element(s) residing between position -7200 and either -2800 in L cells or -920 in HeLa cells reduces transcription approximately 7-fold relative to the level of transcription achieved with the maximally active promoter.