Faculty Bibliography

The mammalian transcription factor E2F plays a critical role in the expression of genes required for cellular proliferation. To understand how E2F is regulated, we have developed a reconstituted in vitro transcription assay. Using this E2F-responsive assay, we can demonstrate that E2F-mediated transcription can be directly repressed by the tumor suppressor protein pRB. This inhibition is abolished by phosphorylation of pRB with either cyclin A/cdk2 or cyclin E/cdk2. However, these cyclin/kinase complexes exhibit differences in the ability to phosphorylate E2F. Only cyclin A/cdk2 can phosphorylate E2F effectively, and this phosphorylation abolishes its ability to bind DNA and mediate trans-activation. Thus, this in vitro transcriptional assay allows activation and inactivation of E2F transcription, and our findings demonstrate how transcriptional regulation of E2F can be linked to cell cycle-dependent activation of kinases

The temporal activation of E2F transcriptional activity appears to be an important component of the mechanisms that prepare mammalian cells for DNA replication. Regulation of E2F activity appears to be a highly complex process, and the dissection of the E2F pathway will be greatly facilitated by the ability to use genetic approaches. We report the isolation of two Drosophila genes that can stimulate E2F-dependent transcription in Drosophila cells. One of these genes, dE2F, contains three domains that are highly conserved in the human homologs E2F-1, E2F-2, and E2F-3. Interestingly, one of these domains is highly homologous to the retinoblastoma protein (RB)-binding sequences of human E2F genes. The other gene, dDP, is closely related to the human DP-1 and DP-2 genes. We demonstrate that dDP and dE2F interact and cooperate to give sequence-specific DNA binding and optimal trans-activation. These features suggest that endogenous Drosophila E2F, like human E2F, may be composed of heterodimers and may be regulated by RB-like proteins. The isolation of these genes will provide important reagents for the genetic analysis of the E2F pathway

Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit complex composed of the TATA binding protein (TBP) and at least seven tightly associated factors (TAFs). Some TAFs act as direct targets or coactivators for promoter-specific activators while others serve as interfaces for TAF-TAF interactions. Here, we report the molecular cloning, expression and characterization of Drosophila dTAFII60 and its human homolog, hTAFII70. Recombinant TAFII60/70 binds weakly to TBP and tightly to the largest subunit of TFIID, TAFII250. In the presence of TAFII60/70, TBP and TAFII250, a stable ternary complex is formed. Both the human and Drosophila proteins directly interact with another TFIID subunit, dTAFII40. Our findings reveal that Drosophila TAFII60 and human TAFII70 share a high degree of structural similarity and that their interactions with other subunits of TFIID are conserved

A key component of the RNA polymerase II transcriptional apparatus, TFIID, is a multi-protein complex containing the TATA box-binding protein (TBP) and at least seven tightly associated factors (TAFs). Although the functions of most TFIID subunits are unknown, it is clear that TAFs are not necessary for basal activity but that one or more are required for regulated transcription, and so behave as coactivators. The presence of multiple subunits indicates that there is an intricate assembly process and that TAFs may be responsible for other activities. We have described the properties of the subunit dTAFII110, which can interact directly with the transcriptional activator Sp1 (ref. 5). In addition, the largest subunit, dTAFII250, binds directly to TBP and links other TAFs to the complex. Here we describe the cloning, expression and partial characterization of the Drosophila TAF of M(r) 80,000, dTAFII80. Sequence analysis reveals that dTAFII80 contains several copies of the WD40 (beta-transducin) repeat. Moreover, dTAFII80 shares extended sequence similarity with an Arabidopsis gene, COP1, which encodes a putative transcription factor that is though to regulate development. We have expressed recombinant dTAFII80 and begun to characterize its interaction with other members of the TFIID complex. Purified recombinant dTAFII80 is unable to bind TBP directly or to interact strongly with the C-terminal domain of dTAFII250 (delta N250). Instead, dTAFII80 is only able to recognize and interact with a higher-order complex containing TBP, delta N250, 110 and 60. These findings suggest the formation of TFIID may require an ordered assembly of the TAFs, some of which bind directly to TBP and others that are tethered to the complex as a result of specific TAF/TAF interactions

The TFIID complex consists of the TATA-binding protein (TBP) and associated factors (TAFs) serving to mediate transcriptional activation by promoter-specific regulators. Here we report the cloning of Drosophila TAFII250 and the assembly of a partial complex containing recombinant TBP, TAFII110 and the C-terminal domain of TAFII250. This triple complex supports Sp1 activation and reveals specific interactions between TAFII250, TBP and TAFII110

The general transcription factor TFIID is a multiprotein complex containing the TATA-binding protein and several associated factors (TAFs), some of which may function as coactivators that are essential for activated, but not basal, transcription. Here we describe the isolation and characterization of the first gene encoding a TAF protein. The deduced amino acid sequence of TAF110 revealed the presence of several glutamine- and serine/threonine-rich regions reminiscent of the protein-protein interaction domains of the regulatory transcription factor Sp1 that are involved in transcription activation and multimerization. In both Drosophila cells and yeast, TAF110 specifically interacts with the glutamine-rich activation domains of Sp1. Moreover, purified Sp1 selectively binds recombinant TAF110 in vitro. These findings taken together suggest that TAF110 may function as a coactivator by serving as a site of protein-protein contact between activators like Sp1 and the TFIID complex

To investigate the biochemical mechanisms involved in interactions between regulatory factors and the general transcription complex, we have cloned, expressed, and characterized the Drosophila gene encoding the TATA binding protein, dTFIID. Comparison of the protein sequences of the Drosophila and yeast TATA binding proteins reveals a bipartite organization consisting of a highly conserved, basic carboxy-terminal domain and a nonconserved amino-terminal region rich in Gln, Gly, Ser, and Met residues. Purified dTFIID protein binds specifically to the TATA sequence and activates basal-level transcription, and the conserved carboxy-terminal half of the molecule is sufficient for both activities. Partially purified TFIID from Drosophila cells mediates activation by the transcription factor Sp1. In contrast, purified dTFIID expressed from the cloned gene is unable to support Sp1-dependent activation, suggesting that other factors may be required to mediate interactions between upstream activators like Sp1 and the TATA binding protein

In an effort to characterize sequence-specific transcription factors that regulate gene expression during Drosophila development, we identified and purified a novel DNA-binding activity (NTF-1). The purified protein consists of several polypeptides that bind selectively to a functionally important cis-control element of the Ultrabithorax (Ubx) promoter and to the neurogenic elements of both the dopa decarboxylase (Ddc) and fushi tarazu (ftz) promoter/enhancer regions. Purified NTF-1 activates transcription in vitro in a binding site-dependent manner through upstream sequences of the Ubx promoter. A cDNA clone encoding the open reading frame of NTF-1 was isolated, and the deduced primary amino acid sequence of NTF-1 includes a glutamine-rich region reminiscent of the transcriptional activation domains found in Sp1 but no recognizable DNA-binding domain. NTF-1 expression is temporally regulated during embryonic development. In addition, in situ hybridization experiments revealed that NTF-1 is transcribed in a spatially restricted pattern in the embryo, with the highest level of expression observed in the epidermis and a subset of cells in the CNS. Expression of the NTF-1 cDNA in mammalian cells yields a protein that displays DNA-binding and transcriptional activities indistinguishable from that of the collection of proteins isolated from Drosophila embryos. These findings suggest that NTF-1 is a member of a family of developmentally regulated transcription factors that may be involved in directing the expression of genes such as Ubx, Ddc, and ftz in neuronal cells