Our understanding of eukaryotic transcriptional activation mechanisms has been hampered by an inability to identify the direct in vivo targets of activator proteins, primarily because of lack of appropriate experimental methods. To circumvent this problem, we have developed a fluorescence resonance energy transfer (FRET) assay to monitor interactions with transcriptional activation domains in living cells. We use this method to show that the Tra1 subunit of the SAGA (Spt/Ada/Gcn5/acetyltransferase) complex is the direct in vivo target of the yeast activator Gal4. Chromatin-immunoprecipitation experiments demonstrate that the Gal4-Tra1 interaction is required for recruitment of SAGA to the upstream activating sequence (UAS), and SAGA, in turn, recruits the Mediator complex to the UAS. The UAS-bound Mediator is required for recruitment of the general transcription factors to the core promoter. Thus, our results identify the in vivo target of an activator and show how the activator-target interaction leads to transcriptional stimulation. The FRET assay we describe is a general method that can be used to identify the in vivo targets of other activators. Transcription initiation by RNA polymerase II involves the assembly of general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). A variety of studies indicate that promoter-specific activator proteins (activators) work, at least in part, by increasing PIC formation (Orphanides et al. 1996;Roeder 1996;Ptashne and Gann 1997;. Activator-mediated stimulation of PIC assembly is believed to result from a direct interaction between the activation domain (AD) and one or more components of the transcription machinery, termed the "target." The unambiguous identification of the direct in vivo targets of activators has been a major challenge in the field.Transcriptional induction of genes involved in galactose utilization (GAL genes) has been a model experimental system for studying transcriptional activation mechanisms. The well-characterized acidic activator Gal4 is responsible for the transcriptional stimulation of GAL genes, such as GAL1, which contain Gal4-binding sites in their promoters (Johnston 1987;Johnston and Carlson 1992;Dudley et al. 1999). A variety of transcriptional components have been proposed to be the target of Gal4 including TBP (Melcher and Johnston 1995;Wu et al. 1996), TFIIB (Wu et al. 1996), Srb4 (Koh et al. 1998Park et al. 2000), Gal11 (Jeong et al. 2001), the Swi/Snf complex (Neely et al. 2002), and the SAGA (Spt/Ada/ Gcn5/acetyltransferase) complex (Bhaumik and Green 2001;Brown et al. 2001;Larschan and Winston 2001). These proposals are based on either in vitro protein interaction studies or inferences from various indirect in vivo experiments, and to date there is no definitive evidence that Gal4 directly interacts with any of these putative targets in vivo. The major obstacle has been the lack of an experimental strategy to measure direct interactions with transcriptional ADs in vivo.The development of spectra...

DAF-16, a forkhead transcription factor, is a key regulator of longevity, metabolism and dauer diapause in Caenorhabditis elegans. The precise mechanism by which DAF-16 regulates multiple functions, however, is poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to identify direct targets of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and selected 33 targets for further analysis. Expression of most of these genes is regulated in a DAF-16-dependent manner, and inactivation of more than half of these genes significantly altered DAF-16-dependent functions, including life span, fat storage and dauer formation. Our results show that the ChIP-based cloning strategy leads to greater enrichment for DAF-16 target genes than previous screening strategies. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream targets. The large number of target genes discovered provides insight into how DAF-16 controls diverse biological functions.

The human immunodeficiency virus type I (HIV-1) transactivator protein Tat is an unusual transcriptional activator that is thought to act solely by promoting RNA polymerase II processivity. Here we study the mechanism of Tat action by analyzing transcription complex (TC) assembly in vivo using chromatin immunoprecipitation assays. We find, unexpectedly, that like typical activators Tat dramatically stimulates TC assembly. Surprisingly, however, the TC formed on the HIV-1 long terminal repeat is atypical and contains TATA-box-binding protein (TBP) but not TBP-associated factors (TAFs). Tat function involves direct interaction with the cellular cofactor positive transcription elongation factor b (P-TEFb). Artificial tethering of P-TEFb subunits to HIV-1 promoter DNA or nascent RNA indicates that P-TEFb is responsible for directing assembly of a TC containing TBP but not TAFs. On the basis of this finding, we identify P-TEFb-dependent cellular promoters that also recruit TBP in the absence of TAFs. Thus, in mammalian cells transcription of protein-coding genes involves alternative TCs that differ by the presence or absence of TAFs.

TATA-box-binding protein (TBP)-related factor 3, TRF3 (also called TBP2), is a vertebrate-specific member of the TBP family that has a conserved C-terminal region and DNA binding domain virtually identical to that of TBP 1 . TRF3 is highly expressed during embryonic development, and studies in zebrafish and Xenopus have shown that TRF3 is required for normal embryogenesis 2,3 . Here we show that Trf3-depleted zebrafish embryos exhibit multiple developmental defects and, in particular, fail to undergo hematopoiesis. Expression profiling for Trf3-dependent genes identified mespa, which encodes a transcription factor whose murine orthologue is required for mesoderm specification 4 , and chromatin immunoprecipitation verified that Trf3 binds to the mespa promoter. Depletion of Mespa resulted in developmental and hematopoietic defects strikingly similar to those induced by Trf3 depletion. Injection of mespa mRNA restored normal development to a Trf3-depleted embryo, indicating mespa is the single Trf3 target gene required for zebrafish embryogenesis. Zebrafish embryos depleted of Trf3 or Mespa also failed to express cdx4, a caudalrelated gene required for hematopoiesis. Mespa binds to the cdx4 promoter, and epistasis analysis revealed an ordered trf3-mespa-cdx4 pathway. Thus, in zebrafish commitment of mesoderm to the hematopoietic lineage occurs through a transcription factor pathway initiated by a TBP-related factor.To analyze the role of TRF3 during embryonic development, we used antisense morpholino oligonucleotides (MOs) to ablate Trf3, and as a control Tbp, function in zebrafish embryos. MOs were injected into wild-type one-cell stage fertilized embryos and depletion of Trf3 and Tbp was analyzed by immunoblotting at 6 hours post-fertilization (hpf), a time at which expression of both proteins was readily detectable (Supplementary Fig. 1a). Immunoblot analysis confirmed that injection of each MO efficiently and specifically depleted its target gene ( Supplementary Fig. 1b). Consistent with previous studies 5 , Tbp-depleted embryos appeared to initiate gastrulation but failed to progress past 50% epiboly (Supplementary Fig. 2; n=122/150). By contrast, Trf3-depleted embryos appeared to develop normally until the tailbud stage, but by 14 hpf exhibited delayed development and necrosis compared to siblings injected with a randomized control MO (n=166/177). Inspection of Trf3-depleted embryos at