TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish.

General transcription factors TFIIA, B, D, E, F, H, and RNA polymerase II (Pol II) are required for accurate initiation of Pol II transcription. The TATA binding protein (TBP), a subunit of TFIID, is responsible for recognition of the TATA box, a core element shared by a category of class II promoters [1]. Recently, novel TBP-like factors (TLFs) have been described in metazoan organisms [2]. In spite of the numerous in vitro studies describing the general role of TBP in RNA polymerase II (Pol lI) transcription initiation, the precise function of TBP and the newly described TLF is poorly understood in vivo. We inhibited TBP and TLF function in zebrafish embryos to study the role of these factors during zygotic transcription. A dominant-negative variant of TLF mRNA and a TBP morpholino antisense oligo was used to block either TLF or TBP function. Both TBP- or TLF-blocked embryos developed normally until the midblastula stage; however, they then failed to gastrulate. Several zygotic regulatory genes were downregulated by a block in either TBP or TLF function, while others were differentially affected. These results suggest that TBP is not universally required for Pol II transcription in vertebrates and that there is a differential requirement for TBP and TLF during early embryogenesis.

TBP-like factor is required for embryonic RNA polymerase II transcription in C. elegans.

Recently, a novel family of TATA binding protein (TBP)-like factors (TLFs) have been described in metazoan organisms; however, their function has not yet been elucidated. Using Caenorhabditis elegans (Ce) as a model, we demonstrate that CeTLF is required in vivo for zygotic transcription during embryogenesis. Elimination of CeTLF expression by RNA interference caused embryonic lethality either due to the lack of expression of early patterning genes or to their ectopic expression. Moreover, the absence of CeTLF in vivo prevented the correct soma-specific phosphorylation of RNA polymerase II (Pol II). Thus, CeTLF may positively or negatively regulate Pol II transcription, depending on the developmental stage of the embryo.

The TBP-like factor: an alternative transcription factor in metazoa?

Protein sequence analysis has revealed a family of TATA-binding-protein (TBP)-like factors (TLFs) in metazoan organisms. Modelling of the three-dimensional structure of these TLFs suggests that they form an asymmetric saddle-like structure and that, unlike TBP, TLFs might bind to DNA sequences other than classical TATA boxes. Thus, the existence of TLFs presents a challenge to the doctrine that TBP is a universal regulator of transcription in metazoans.

The TATA-binding protein and its associated factors are differentially expressed in adult mouse tissues.

We have investigated the expression levels of the TATA-binding protein (TBP) and several TBP-associated factors (TAFIIs) in differentiated adult mouse tissues. Immunoblots performed using monoclonal antibodies show that there are considerable variations in the levels of TBP and many TAFII proteins present in various tissues. Consequently, the relative levels of TAFIIs and TBP vary significantly from one tissue to another. TBP and several TAFIIs are overexpressed in both testis and small intestine, while in marked contrast, many of these proteins, including TBP itself, were substantially down-regulated in nervous tissues and in the heart. These tissues do, however, show a high expression level of the TBP-like factor, which thus may represent an alternative factor for the specialized transcription program in some differentiated tissues. While there are significant variations in the levels of TAFII28 protein, reverse transcription-coupled polymerase chain reaction shows similar expression of the TAFII28 mRNA in different tissues. The variations in TAFII28 protein levels therefore result from post-transcriptional regulatory events.

Transcription factor TFIID recruits factor CPSF for formation of 3' end of mRNA.

Initiation of transcription by RNA polymerase II from a promoter region on DNA requires the assembly of several initiation factors to form a preinitiation complex. Assembly of this complex is initiated by the binding of the transcription factor TFIID, composed of the TATA-box binding protein (TBP) and TBP-associated factors (TAF[II]s), to the promoter. We have now characterized an immunopurified TFIID complex which we unexpectedly find contains the cleavage-polyadenylation specificity factor (CPSF), one of the factors required for formation of the 3' end of messenger RNA. CPSF is brought to the preinitiation complex by TFIID, but after transcription starts, CPSF dissociates from TFIID and becomes associated with the elongating polymerase. We also show that overexpression of recombinant TBP in HeLa cells decreases polyadenylation without affecting the correct initiation of transcription of the reporter gene. This indicates that, owing to incomplete assembly of TFIID on recombinant TBP, CPSF is not brought to the promoter and therefore polyadenylation becomes less efficient. Our observations have thus revealed a link between transcription initiation and elongation by RNA polymerase II and processing of the 3' end of mRNA.

CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues.

Both the TATA and CCAAT boxes are widespread promoter elements and their binding proteins, TBP and NF-Y, are extremely conserved in evolution. NF-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YB and NF-YC contain a putative histone-like motif, a domain also present in TBP-associated factors (TAFIIs) and in the subunits of the transcriptional repressor NC2. Immunopurification of holo-TFIID with anti-TBP and anti-TAFII100 antibodies indicates that a fraction of NF-YB associates with TFIID in the absence of NF-YA. Sedimentation velocity centrifugation experiments confirm that two pools of NF-YB, and most likely NF-YC, exist: one associated with NF-YA and binding to the CCAAT box; another involved in high molecular weight complexes. We started to dissect NF-Y-TFIID interactions by showing that: (i) NF-YB and NF-YC interact with TBP in solution, both separately and once bound to each other; (ii) short stretches of both NF-YB and NF-YC located within the evolutionary conserved domains, adjacent to the putative histone fold motifs, are necessary for TBP binding; (iii) TBP single amino acid mutants in the HS2 helix, previously shown to be defective in NC2 binding, are also unable to bind NF-YB and NF-YC.

The major histocompatibility complex class II Ea promoter requires TFIID binding to an initiator sequence.

The major histocompatibility complex (MHC) class II Ea promoter is dependent on the presence of conserved upstream X and Y boxes and of initiator (Inr) sequences. In vitro transcription analysis of the Inr region with linker-scanning mutants pinpoints a functionally essential element that shows homology to the terminal deoxynucleotidyltransferase (TdT) Inr; contrary to the TdT Inr and other Inrs identified so far, the key sequence, between positions +5 and +12, is located within a transcribed area. Swapping the TdT sequence into the corresponding Ea position leads to a fivefold increase in transcription rate, without altering start site selection. Inr-binding proteins LBP-1/CP2 and TIP--a TdT Inr-binding protein unrelated to YY1--recognize the Ea Inr; they interact with overlapping yet distinct sequences around the Cap site, but their binding does not coincide with Ea Inr activity. A good correlation is, rather, found with binding of immunopurified holo-TFIID to this element. TFIID interacts both with Ea TATA-like and Inr sequences, but only the latter is functionally relevant. Unlike TBP, TFIID binds in the absence of TFIIA, indicating a stabilizing role for TBP-associated factors in Ea promoter recognition. Sequence comparison with other mouse and human MHC class II promoters suggests a common mechanism of start site(s) selection for the MHC class II gene family.