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 sFecific activity was close to -100%. Electrophoretic mobility-shift analysis demonstrated a single major DNA-protein complex. This holo-TFIID contains TAFs of -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-Iess promoter.

Signal transduction within the canonical Wnt/␤-catenin pathway drives development and carcinogenesis through programmed or unprogrammed changes in gene transcription. Although the upstream events linked to signal-induced activation of ␤-catenin in the cytoplasm have been deciphered in considerable detail, much less is known regarding the mechanism by which ␤-catenin stimulates target gene transcription in the nucleus. Here, we show that ␤-catenin physically and functionally targets the MED12 subunit in Mediator to activate transcription. The ␤-catenin transactivation domain bound directly to isolated MED12 and intact Mediator both in vitro and in vivo, and Mediator was recruited to Wnt-responsive genes in a ␤-catenin-dependent manner. Disruption of the ␤-catenin/MED12 interaction through dominant-negative interference-or RNA interference-mediated MED12 suppression inhibited ␤-catenin transactivation in response to Wnt signaling. This study thus identifies the MED12 interface within Mediator as a new component and a potential therapeutic target in the Wnt/␤-catenin pathway.

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.

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.

SUMMARY Mediator occupies a central role in RNA polymerase II transcription as a sensor, integrator, and processor of regulatory signals that converge on protein-coding gene promoters. Compared to its role in gene activation, little is known regarding the molecular mechanisms and biological implications of Mediator as a transducer of repressive signals. Here, we describe a protein interaction network required for extra-neuronal gene silencing comprising Mediator, G9a histone methyltransferase, and the RE1 silencing transcription factor (REST; also known as neuron restrictive silencing factor, NRSF). We show that the MED12 interface in Mediator links REST with G9a-dependent histone H3K9 di-methylation to suppress neuronal genes in non-neuronal cells. Notably, missense mutations in MED12 causing the X-linked mental retardation (XLMR) disorders FG syndrome and Lujan syndrome disrupt its REST corepressor function. These findings implicate Mediator in epigenetic restriction of neuronal gene expression to the nervous system and suggest a pathologic basis for MED12-associated XLMR involving impaired REST-dependent neuronal gene regulation.

Summary Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (∼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8 driven cancers.

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 ligandindependent transcriptional repression of the estrogen receptor ␣ (ER␣), 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, ER␣ 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 ER␣ 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 ER␣ 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 ER␣ 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. G ermline inactivation of the gene that encodes BRCA1represents a predisposing genetic factor in Ϸ15-45% of hereditary breast cancers, and minimally 80% of combined hereditary breast and ovarian cancer cases (1). Functionally, BRCA1 has been implicated in the maintenance of global genome stability (2-4), and the underlying basis for this activity likely derives from its central role in the cellular response to DNA damage, wherein it controls both DNA damage repair and the transcription of DNA damage-inducible genes (5-14).Because the DNA damage-induced signaling pathways that converge on BRCA1 are likely to be conserved in most cell types, BRCA1 is likely to occupy a fundamental and universally conserved role in the mammalian DNA damage response. Nonetheless, germ-line inactivation of BRCA1 leads predominantly to cancer of the breast and ovary, and the underlying basis for its tissue-restricted tumor-suppressive properties thus remains undefined.At least two hypotheses have been proposed to explain the tissue-specific nature of BRCA1-mediated tumor suppression, both of which invoke a role for estrogen in either the initiation or promotion of tumor formation (15). According to one model, the tissue-specific tumor-suppressive properties of BRCA1 derive, at least in part, from its response to tissue-specific DNA damage. In this regard, certain oxidative metabolites of estrogen itself have been documented to be genotoxic in nature (16), and BRCA1 may therefore play a role in protecting breast and ovarian tissue from estrogen-induced DNA damage.A second model, not mutually exclusive with the one described above, to account for the...

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.Regulated transcription initiation of protein-coding genes by RNA polymerase II requires the assembly of a large preinitiation complex composed of the polymerase and general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH at the transcription start site (for a review, see reference 55). The recent discovery in Saccharomyces cerevisiae of a specialized form of RNA polymerase involved in regulated initiation, a holoenzyme associated with an additional ϳ20 polypeptides (32, 33), suggests that a similarly complex holoenzyme occurs in higher eukaryotes. Thus, the preinitiation complex is an extremely complicated macromolecular assembly. Counting the additional polypeptides found in the yeast holoenzyme, it contains 60 to 70 polypeptides and has a total mass in excess of 4 MDa, roughly the size of a eukaryotic ribosome.For promoters containing a TATA box, the binding of TFIID to this promoter element is the first step in assembly of the preinitiation complex on promoter DNA (6, 63). TFIID is a multisubunit protein of ϳ750 kDa, consisting of a TATAbinding polypeptide (TBP) and several additional subunits referred to as TBP-associated factors (TAFs) (19). TAFs are required for activated transcription. If the TBP subunit alone is substituted for TFIID in an in vitro reaction, a preinitiation complex can assemble and initiate from the correct start site but transcription is not stimulated by activator proteins (13, 58). The unregulated transcription observed in reactions in which TBP is substituted for TFIID is often referred to as basal transcription.Activators bind to DNA tens to tens of thousands of base pairs from the transcription initiation site and increase the rate of transcription. They are often modular proteins with distinct DNA binding and activation domains (21, 43). Activators probably function by interacting with components of the preinitiation complex. Since the preinitiation complex is indeed complex, there are many potential s...