Identification of direct DAF-16 targets controlling longevity, metabolism and diapause by chromatin immunoprecipitation.

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.

Initiation of zebrafish haematopoiesis by the TATA-box-binding protein-related factor Trf3.

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 carboxy-terminal region and DNA-binding domain virtually identical to that of TBP (ref. 1). TRF3 is highly expressed during embryonic development, and studies in zebrafish and Xenopus have shown that it is required for normal embryogenesis. Here we show that zebrafish embryos depleted of Trf3 exhibit multiple developmental defects and, in particular, fail to undergo haematopoiesis. Expression profiling for Trf3-dependent genes identified mespa, which encodes a transcription factor whose murine orthologue is required for mesoderm specification, and chromatin immunoprecipitation verified that Trf3 binds to the mespa promoter. Depletion of Mespa resulted in developmental and haematopoietic defects markedly similar to those induced by Trf3 depletion. Injection of mespa messenger RNA (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 caudal-related gene required for haematopoiesis. Mespa binds to the cdx4 promoter, and epistasis analysis revealed an ordered trf3-mespa-cdx4 pathway. Thus, in zebrafish, commitment of mesoderm to the haematopoietic lineage occurs through a transcription factor pathway initiated by a TBP-related factor.

Selective interaction between Trf3 and Taf3 required for early development and hematopoiesis.

In zebrafish, TATA-box-binding protein (TBP)-related factor 3, Trf3, is required for early development and initiation of hematopoiesis, and functions by promoting expression of a single target gene, mespa. Recent studies have shown that in murine muscle cells, TRF3 interacts with the TBP-associated factor TAF3. Here we investigate the role of Taf3 in zebrafish embryogenesis. We find that like Trf3-depleted zebrafish embryos, Taf3-depleted embryos exhibit multiple developmental defects and fail to undergo hematopoiesis. Both Trf3 and Taf3 are selectively bound to the mespa promoter and are required for mespa expression. Significantly, Taf3 interacts with Trf3 but not Tbp, and a Trf3 mutant that disrupts this interaction fails to support mespa transcription, early development, and hematopoiesis. Thus, a selective interaction between Trf3 and Taf3 is required for early zebrafish development and initiation of hematopoiesis. Finally, we provide evidence that TRF3 and TAF3 are also required for hematopoiesis initiation in the mouse.

HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs.

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.

Development of a reconstitution system for Rinderpest virus RNA synthesis in vitro.

The RNA dependent RNA polymerase of Rinderpest virus consists of two subunits-the large protein (L) and the phosphoprotein (P), where L is thought to be responsible for the catalytic activities in association with P protein which plays multiple roles in transcription and replication. The nucleocapsid protein (N) is necessary for encapsidation of genomic RNA, which is required as N-P complex. To understand the different steps of transcription and replication as well as the roles played by the three proteins, an in vitro reconstitution system for RNA synthesis is necessary which is not available for any morbillivirus. We describe here, an in vitro reconstitution system for transcription and replication of Rinderpest virus utilizing a synthetic, positive sense N-RNA minigenome template, free of endogenous viral polymerase proteins and recombinant viral proteins (P+L and P+N) expressed in insect cells by recombinant baculoviruses. We show that although L-P complex is sufficient to synthesize negative sense minigenome RNA, soluble N protein is necessary for encapsidation of RNA as well as synthesis of (+) sense leader RNA and (+) sense minigenome RNA.

Effect of single amino acid mutations in the conserved GDNQ motif of L protein of Rinderpest virus on RNA synthesis in vitro and in vivo.

The paramyxovirus RNA-dependent RNA polymerase consists of two subunits, the transcription co-factor phosphoprotein P and the large protein L, which possesses all the catalytic functions such as RNA synthesis (both transcription replication), methylation, capping and polyadenylation. The L protein has high sequence homology among the negative sense RNA viruses. The domains and residues on the L protein involved in the above-mentioned activities are not well defined, although the role of conserved GDNQ motif of the putative catalytic centre of L protein of few related viruses have been examined. In order to gain insight into the role played by the GDNQ motif of the L protein of Rinderpest virus (RPV), we have examined mutations at each amino acid in this motif of the L protein of Rinderpest virus and tested the biological activity in vivo and in vitro. Site directed mutants were generated and transiently expressed in mammalian cells and were shown to interact with P protein similar to wild type L. The biological activity of mutant L proteins has been tested in an in vitro reconstituted system capable of carrying out cell-free RNA synthesis on synthetic Rinderpest N-RNA template. Further, the role played by individual amino acids has also been defined in vivo using an in vivo minigenome replication/transcription system which indicated the importance of this conserved sequence in viral RNA synthesis.

Phosphoprotein P of Rinderpest virus binds to plus sense leader RNA: regulation by phosphorylation.

The negative sense genome RNA of Rinderpest virus, a Paramyxoviridae, is encapsidated with the nucleocapsid protein N and serves as a template for the viral RNA dependent RNA polymerase for transcription and replication. The viral RNA polymerase consists of the large protein L and the phosphoprotein P functioning as the P-L complex. We provide in this report, evidences for specific binding of P protein of Rinderpest virus to the plus sense leader RNA depending on its phosphorylation status. We have also demonstrated that P protein is released from the le RNA:P protein complex upon phosphorylation in vitro. Finally, we have identified that the C-terminal 358-389 amino acid residues of P protein is involved in le RNA binding. The leader RNA binding may signify a hitherto unidentified role for P protein in the viral RNA synthesis. Moreover, our results indicate a possible role for P protein in the transcription-replication switch through leader RNA binding.

Leader RNA of Rinderpest virus binds specifically with cellular La protein: a possible role in virus replication.

Rinderpest virus (RPV) is an important member of the Morbillivirus genus in the family Paramyxoviridae and employs a similar strategy for transcription and replication of its genome as that of other negative sense RNA viruses. Cellular proteins have earlier been shown to stimulate viral RNA synthesis by isolated nucleocapsids from purified virus or from virus-infected cells. In the present work, we show that plus sense leader RNA of RPV, transcribed from 3' end of genomic RNA, specifically interacts with cellular La protein employing gel mobility shift assay as well as UV cross-linking of leader RNA with La protein. The leader RNA synthesized in virus-infected cells was shown to interact with La protein by immunoprecipitation of leader RNA bound to La protein and detecting the leader RNA in the immunoprecipitate by Northern hybridization with labeled antisense leader RNA. Employing a minireplicon system, we demonstrate that transiently expressed La protein enhances the replication/transcription of the RPV minigenome in cells. Sub-cellular immunolocalization shows that La protein is redistributed from nucleus to the cytoplasm upon infection. Our results strongly suggest that La protein may be involved in regulation of Rinderpest virus replication.

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.

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.

Leader RNA binding ability of Chandipura virus P protein is regulated by its phosphorylation status: a possible role in genome transcription-replication switch.

The molecular events associated with the transcriptive and replicative cycle of negative-stranded RNA viruses are still an enigma. We took Chandipura virus, a member of the Rhabdoviridae family, as our model system to demonstrate that Phosphoprotein P, besides Nucleocapsid protein N, also acts as a leader RNA-binding protein in its unphosphorylated form, whereas CKII-mediated phosphorylation totally abrogates its RNA-binding ability. However, interaction between P protein and leader RNA can be distinguished from N-mediated encapsidation of viral sequences. Furthermore, P protein bound to leader chain can successively recruit N protein on RNA while itself being replaced. We also observed that the accumulation of phosphorylation null mutant of P protein in cells results in enhanced genome RNA replication with concurrent increase in the viral yield. All these results led us to propose a model explaining viral transcription-replication switch where Phosphoprotein P acts as a modulator of genome transcription and replication by its ability to bind to the nascent leader RNA in its unphosphorylated form, promoting read-through of the transcription termination signals and initiating nucleocapsid assembly on the nascent RNA chain.