InteractiveFly: GeneBrief

TATA binding protein: Biological Overview | References


Gene name - TATA binding protein

Synonyms -

Cytological map position - 57F8-57F8

Function - Transcription factor

Keywords - Initiation of transcription, RNA polymerase II core promoter binding factor

Symbol - Tbp

FlyBase ID: FBgn0003687

Genetic map position - 2R:17,573,705..17,575,145 [-]

Classification - eukaryotic TATA box binding protein

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional module. RNAi depletion and overexpression experiments revealed a genetic circuit that controls the balance of transcription from two core promoter motifs, the TATA box and the downstream core promoter element (DPE). In this circuit, TBP activates TATA-dependent transcription and represses DPE-dependent transcription, whereas Mot1 and NC2 block TBP function and thus repress TATA-dependent transcription and activate DPE-dependent transcription. This regulatory circuit is likely to be one means by which biological networks can transmit transcriptional signals, such as those from DPE-specific and TATA-specific enhancers, via distinct pathways (Hsu, 2008).

The RNA polymerase II core promoter comprises the sequences that direct the initiation of transcription. Although it has often been presumed that the core promoter is a generic entity, current evidence indicates that there is considerable diversity in core promoter structure and function. Hence, the core promoter is a regulatory element (for reviews, see Smale, 2003; Sandelin, 2007; Juven-Gershon, 2008; Hsu, 2008 and references therein).

This study focuses on the relation between two core promoter motifs: the downstream core promoter element (DPE) and the TATA box. The TATA box is the most ancient core promoter motif, as it is conserved from archaebacteria to humans. It has a consensus of TATAWAAR, where the upstream T nucleotide is typically located about -31 or -30 relative to the A + 1 in the Initiator (Inr) element. The DPE appears to be conserved among metazoans. It is strictly located from +28 to +33 relative to the A + 1 in the Inr, and has a consensus of RGWYVT in Drosophila (Hsu, 2008).

Both the TATA box and DPE are binding sites for the TFIID basal transcription factor, but TFIID appears to have distinct modes of binding to the two core promoter motifs. The TBP subunit of TFIID binds to the TATA box, whereas the TAF6 and TAF9 subunits of TFIID are in close proximity to the DPE. In addition, the DNase I footprinting patterns on TATA-containing versus DPE-containing promoters are different (for example, see Burke, 1996). In particular, TFIID footprints of DPE-dependent core promoters exhibit a periodic 10-bp DNase I digestion pattern that suggests an extended, close interaction of TFIID from the Inr through the DPE (Burke, 1996; Kutach, 2000; Hsu, 2008 and references therein).

There are differences in the functional properties of DPE-dependent versus TATA-dependent core promoters. For instance, an enhancer-trapping analysis in Drosophila revealed the existence of DPE-specific as well as TATA-specific transcriptional enhancers (Butler, 2001). It was also found that a set of factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNA polymerase II, PC4, and Sp1) that is sufficient for transcription of promoters containing both TATA and DCE (downstream core element; Lee, 2005) motifs is not able to transcribe a DPE-dependent promoter (Lewis, 2005). In that case, DPE-dependent transcription was additionally found to require casein kinase II (CKII) and Mediator. In other studies, NC2 (also known as Dr1-Drap1), which was originally identified as a repressor of TATA-dependent transcription, was found to activate transcription from five different DPE-dependent core promoters in reactions performed with a nuclear extract (Willy, 2000). With a purified transcription system, however, NC2 activation of a DPE-dependent core promoter was not observed (Lewis, 2005; Hsu, 2008).

To determine the nature of the factors that promote DPE-dependent versus TATA-dependent transcription, the properties of key transcription factors was investigated by RNAi depletion, overexpression, and chromatin immunoprecipitation (ChIP) analyses with multiple DPE-dependent and TATA-dependent promoters. The new findings reveal a regulatory circuit that controls the balance between DPE-dependent versus TATA-dependent transcription (Hsu, 2008).

This study used cultured Drosophila cells as the experimental system to investigate DPE versus TATA function. Two sets of reporter constructs were created that contain either TATA or DPE motifs driving a luciferase reporter gene. The DPE-dependent and TATA-dependent promoters in each set were identical, except for the sequences at the positions of the DPE and TATA motifs, and had comparable transcriptional activities (Hsu, 2008).

The effects of several transcription factors were investigated upon DPE versus TATA transcription by RNAi depletion analysis. The transcription factors were selected on the basis of their fundamental importance as well as their potential role in DPE-dependent transcription. First RNAi depletion of each target factor was carried out, and then one-half of the cells was transfected with the DPE-dependent reporter construct and the other half of the cells with the TATA-dependent reporter. The resulting transcription levels were assessed by measurement of the luciferase activities relative to those in mock RNAi controls (Hsu, 2008).

Depletion of TBP sharply decreases TATA-dependent transcription, but has little effect on DPE-dependent transcription. This effect was observed with a distinct and independent set of DPE-dependent and TATA-dependent reporter constructs as well as with a different nonoverlapping dsRNA probe for TBP. Consistent with the ability of TFIIA to promote TBP binding to DNA (for example, see Buratowski, 1989; Maldonado, 1990), depletion of TFIIA reduces TATA transcription more than DPE transcription with two different sets of reporter constructs. In contrast, no differential DPE versus TATA effects were seen upon RNAi depletion of TAF4 (which is essential for the structural integrity of TFIID), TFIIB, CKIIα, a PC4-like protein, subunits of Mediator (Med17, Med24), or subunits of the SAGA/TFTC complex (Gcn5, Spt3, Ada2b) (Hsu, 2008).

Thus, these findings indicate that TBP and, to a lesser extent, TFIIA have a key role in discriminating between DPE- versus TATA-dependent transcription. The stronger effect of TBP relative to TFIIA is consistent with an auxiliary function of TFIIA, such as its ability to increase the binding of TBP to the TATA box. Because depletion of TBP did not adversely affect DPE-dependent transcription, the possibility was considered that DPE-dependent transcription might involve a factor, such as SAGA/TFTC, that lacks TBP (Wieczorek, 1998; for review, see Nagy, 2007). Therefore the effect of depletion of three SAGA/TFTC subunits (Gcn5, Spt3, and Ada2b) was tested, but no substantial decrease was seen in DPE-dependent transcription or any differential DPE versus TATA effects. Thus, it appears unlikely that SAGA/TFTC is important for DPE-dependent transcription. Lastly, upon depletion of CKII, Mediator, PC4-like, TAF4, and TFIIB, a decrease was observed in both DPE-dependent and TATA-dependent transcription. These results are consistent with a more general transcriptional function rather than a DPE-specific or TATA-specific activity for these factors (Hsu, 2008).

NC2 has been previously found to be a DPE-specific transcriptional activator (Willy, 2000). With a different biochemical system, however, NC2-mediated enhancement of DPE transcription was not observed (Lewis, 2005). Therefore attempts were made to clarify these apparently contrasting results by RNAi analysis of NC2 with DPE versus TATA reporter gene systems. NC2 comprises two subunits, NC2α (Drap1) and NC2β (Dr1). Upon RNAi depletion of either NC2α or NC2β, a more substantial decrease was seen in DPE- relative to TATA-dependent transcription with two different sets of reporter genes as well as with two different dsRNAs. These results therefore indicate that NC2 promotes DPE-dependent transcription relative to TATA-dependent transcription in cultured cells (Hsu, 2008).

Next, the effects were tested of Mot1 (also known as BTAF1 and Hel89B) on DPE versus TATA transcription. Like NC2, Mot1 antagonizes TBP function. NC2 represses TATA-dependent transcription by blocking the association of TBP with other factors such as TFIIA and TFIIB (for review, see Thomas, 2006). Mot1 is an ATPase that removes TBP from DNA by an ATP-dependent mechanism (for example, see Auble, 1994; Pereira, 2003). Genetic studies in Saccharomyces cerevisiae suggest that NC2 and Mot1 have related functions (Prelich 1997; Lemaire, 2000). NC2 and Mot1 bind to overlapping regions in the yeast genome and form a complex with TBP and DNA (Darst, 2003; van Werven, 2008). In addition, although NC2 and Mot1 are often thought to be repressive, a positive function for these factors has been observed in vitro and in vivo (Willy, 2000; Andrau, 2002; Cang, 2002; Dasgupta, 2002; Dasgupta, 2005; Geisberg, 2004; Albert, 2007; van Werven, 2008; Hsu, 2008 and references therein).

It was observed that RNAi depletion of Mot1 has a stronger detrimental effect on DPE-dependent than TATA-dependent transcription. This effect was seen with two different sets of reporter genes as well as with two independent nonoverlapping dsRNA fragments. Thus, like NC2, Mot1 promotes DPE- relative to TATA-dependent transcription (Hsu, 2008).

To investigate the relationship between TBP, NC2, and Mot1 in the regulation of core promoter activity, different combinations of these factors were codepleted and the resulting effects upon DPE versus TATA transcription were determined. Codepletion of both NC2α and Mot1 preferentially decreases DPE relative to TATA transcription to an extent that is similar to that seen upon depletion of either NC2α or Mot1 alone. These results suggest that NC2 and Mot1 promote DPE-dependent transcription via the same pathway. In contrast, when TBP + Mot1 or TBP + NC2α were codepleted, nearly the same effect on DPE versus TATA transcription was seen as that seen upon depletion of TBP alone. These findings suggest that TBP is downstream from NC2 and Mot1 in the pathway that regulates DPE versus TATA transcription. Thus, NC2 and Mot1 appear to modulate DPE versus TATA transcription by acting via TBP (Hsu, 2008).

To complement the RNAi depletion studies, the effects of overexpression of TBP, Mot1, or NC2 was investigated in S2 cells. In these experiments, TBP, Mot1, or NC2 expression vectors were cotransfected along with the DPE-dependent or TATA-dependent reporter constructs. Overexpression of TBP increases TATA-dependent transcription and decreases DPE-dependent transcription. Conversely, overexpression of Mot1 increases DPE-dependent transcription and decreases TATA-dependent transcription. Overexpression of both subunits of NC2 decreases TATA-dependent transcription, but has little effect on DPE-dependent transcription. Consistent with the two NC2 subunits functioning together in a complex, overexpression of NC2α alone or NC2β alone has no effect on DPE-dependent or TATA-dependent transcription. In addition, a parallel set of overexpression experiments weew was carried out with TBP, Mot1, and NC2 with a different set of DPE-dependent and TATA-dependent reporter genes, and nearly identical results were obtained. These findings further demonstrate that TBP favors TATA relative to DPE transcription, whereas Mot1 and NC2 favor DPE relative to TATA transcription (Hsu, 2008).

To examine the functions of TBP, Mot1, and NC2 in a more natural context, the effects of RNAi depletion of TBP, Mot1, or NC2 upon transcription of endogenous DPE- or TATA-containing genes was tested in Drosophila Kc cells. In these experiments, secondary/late ecdysone-responsive genes, that are activated upon ecdysone induction, were employed. In this manner, it was possible to characterize the requirements for TBP, Mot1, and NC2 for transcriptional activation (Hsu, 2008).

Many genes in Drosophila are activated by the steroid hormone 20-hydroxyecdysone (20HE). A list of genes was obtained that was induced by 20HE in Drosophila Kc cells. From this list, secondary/late-response genes were identified with DPE+Inr motifs (CG9511, CG16876, Glut1) or TATA + Inr motifs (Obp99c, CG4500) in their core promoters. The 20HE induction of these genes in Kc cells was confirmed by using real-time RT-PCR. In addition, the transcription start sites of each of these genes was verified by primer extension analysis of mRNA isolated from Kc cells (Hsu, 2008).

The RNAi analysis of the endogenous secondary/late-response genes was carried out as follows: TBP, TAF4, NC2α, and Mot1 were each individually depleted by RNAi in Kc cells for 4 d, and then the ecdysone-responsive genes were induced with 20HE for 24 h. The total RNA was isolated, and the transcript levels of the selected genes were determined by real-time RT-PCR. It was observed that depletion of TBP decreases transcription of the TATA-containing promoters and increases transcription of the DPE-containing promoters. Thus, these results suggest not only that TBP activates TATA-dependent promoters, but also that it represses DPE-dependent promoters. Conversely, it was found that depletion of Mot1 or NC2α decreases transcription of DPE-containing promoters and increases transcription of TATA-containing promoters. These findings suggest a positive function of Mot1 and NC2 at DPE-dependent promoters and a negative function at TATA-containing promoters. RNAi depletion of TAF4 causes a substantial decrease in transcription from both DPE-containing and TATA-containing promoters. These results further support the conclusion that TAF4 is required for both DPE-dependent and TATA-dependent transcription (Hsu, 2008).

The RNAi depletion analysis with the endogenous genes leads to nearly the same conclusions as the experiments with the transfected luciferase reporter genes. Both sets of experiments indicate that TBP favors TATA-dependent relative to DPE-dependent transcription, and that Mot1 and NC2 favor DPE-dependent relative to TATA-dependent transcription. However, it is useful to note the two distinctions. First, TBP depletion results in an increase in transcription from endogenous DPE-containing genes, but does not alter transcription from transfected DPE-dependent reporter genes. Second, depletion of Mot1 or NC2α causes an increase in transcription from endogenous TATA-containing genes, but results in a slight decrease in transcription from transfected TATA-dependent reporter genes. The analysis of the endogenous genes is likely to provide a more accurate representation of TBP, Mot1, and NC2 activity than the studies with the transfected genes, because the endogenous genes are in their natural context at the normal copy number and the experiments with the endogenous genes do not involve the extra transfection procedure. Thus, the findings from the analysis of the endogenous genes suggest a repressive function of TBP at DPE-dependent promoters as well as a repressive function of Mot1 and NC2 at TATA-dependent promoters (Hsu, 2008).

The secondary/late ecdysone-responsive genes were further characterized by ChIP analysis with TBP and RNA polymerase II (Rpb3 subunit), for which ChIP-quality antibodies were available. With the TATA-containing CG4500 promoter, there is increased ChIP signal for both TBP and Rpb3 in the promoter region upon 20HE induction. In the control/reference TATA-containing hsp70 promoter, an increase in ChIP of TBP and Rpb3 was also observed in the promoter region (Lebedeva, 2005). By comparison, with the DPE-containing Glut1 and CG16876 promoters, there is increased ChIP of Rpb3 in the promoter region upon 20HE induction; however, the ChIP signal for TBP does not increase under the same conditions. The absence of an increased ChIP signal for TBP with the DPE-containing promoters does not necessarily indicate that TBP is not present at the promoter; for instance, it is possible that TBP may be in an altered configuration that masks the accessibility of the antibodies. Yet, whether or not TBP is in close proximity to the DPE-containing promoters, these results show that there are differences in the nature of the interaction of TBP with TATA-containing versus DPE-containing promoters (Hsu, 2008).

It is also relevant to note that secondary/late-response genes were chosen in these studies, because secondary/late genes are more likely than primary/early-response genes to be in a naïve state prior to ecdysone induction. To test this notion, RNAi depletion analyses was carried out with two primary/early-response genes, E74A and E75B, both of which contain DPE motifs. With these genes, no change was observed in transcription upon RNAi depletion of TBP, TAF4, Mot1, or NC2α. Moreover, ChIP analysis further revealed that both TBP and RNA polymerase II (Rpb3 subunit) are present at the promoters prior to ecdysone induction. Therefore, it appears likely that these primary/early-response genes exist in a preactivated state that does not require the subsequent action of factors such as TFIID, Mot1, or NC2 (Hsu, 2008).

The RNAi depletion and overexpression data reveal a regulatory circuit with the following properties: TBP activates TATA-dependent transcription and represses DPE-transcription; then, Mot1 and NC2 act to block both the activating and repressive functions of TBP. In this model, there are opposing forces that alter the balance between DPE versus TATA transcription. A decrease in TBP or an increase in Mot1/NC2 favors DPE transcription, whereas an increase in TBP or a decrease in Mot1/NC2 favors TATA transcription. Importantly, the functions of Mot1 and NC2 are dependent on TBP. In addition, the proposed circuit is consistent with the known antagonistic relationship between TBP and NC2 as well as between TBP and Mot1 (Hsu, 2008).

How might TBP repress DPE-dependent transcription? Two possible explanations are suggested. (1) In the absence of a TATA box, TBP might interfere with the proper assembly of the transcription initiation complex. (2) There may be an essential DPE-directed transcription factor that is inhibited by TBP. It is possible that DPE-mediated transcription does not directly involve TBP; there is substantial evidence of RNA polymerase II-mediated transcription occurring in the absence of TBP (for example, see Veenstra, 2000; Müller, 2001; Martianov, 2002; Paulson, 2002; Deato, 2007; Ferg, 2007; Hsu, 2008 and references therein).

It was also considered whether either of the TBP-related factors, TRF1 and TRF2, are used instead of TBP at DPE-containing promoters. To this end, the effect of depleting TRF1 or TRF2 was examined upon the expression of DPE-containing versus TATA-containing endogenous genes. TRF1, which is largely involved in RNA polymerase III transcription in Drosophila, has little or no effect on transcription of DPE-containing or TATA-containing genes. TRF2 is important for both DPE-mediated and TATA-mediated transcription. The effect of TRF2 is similar to that of TAF4, which appears to contribute to both DPE-depentend and TATA-dependent transcription. Neither TRF1 nor TRF2 exhibit an opposite effect on DPE-mediated versus TATA-mediated transcription as do TBP, Mot1, and NC2. In addition, a genome-wide ChIP analysis of TRF2 did not reveal an association of TRF2 with DPE-containing genes (Isogai, 2007). Thus, at the present time, there is no evidence suggesting a specific link between either TRF1 or TRF2 and DPE-mdidated or TATA-mediated transcription (Hsu, 2008).

In conclusion, the analysis of TBP, Mot1, and NC2 in the context of DPE-containing versus TATA-containing promoters has revealed a regulatory circuit that controls the balance between DPE-mediated versus TATA-mediated transcription. This circuit may be a key means by which DPE or TATA specificity of transcriptional enhancers is achieved. In the future, it will be interesting and important to build upon this core circuit to identify the connections and mechanisms by which biological networks use DPE and TATA specificity to increase the number of pathways by which signals can be transmitted (Hsu, 2008).

Occupancy of the Drosophila hsp70 promoter by a subset of basal transcription factors diminishes upon transcriptional activation

The presence of general transcription factors and other coactivators at the Drosophila hsp70 gene promoter in vivo has been examined by polytene chromosome immunofluorescence and chromatin immunoprecipitation at endogenous heat-shock loci or at a hsp70 promoter-containing transgene. These studies indicate that the hsp70 promoter is already occupied by TATA-binding protein (TBP) and several TBP-associated factors (TAFs), TFIIB, TFIIF (RAP30), TFIIH (XPB), TBP-free/TAF-containg complex (GCN5 and TRRAP), and the Mediator complex subunit 13 before heat shock. After heat shock, there is a significant recruitment of the heat-shock transcription factor, RNA polymerase II, XPD, GCN5, TRRAP, or Mediator complex 13 to the hsp70 promoter. Surprisingly, upon heat shock, there is a marked diminution in the occupancy of TBP, six different TAFs, TFIIB, and TFIIF, whereas there is no change in the occupancy of these factors at ecdysone-induced loci under the same conditions. Hence, these findings reveal a distinct mechanism of transcriptional induction at the hsp70 promoters, and further indicate that the apparent promoter occupancy of the general transcriptional factors does not necessarily reflect the transcriptional state of a gene (Lebedeva, 2005; full text of article).

An inverse correlation was observed between factor occupancy and transcriptional activation. In the absence of heat shock, it was found that TBP, TAFs, TFIIB, TFIIF, TFIIH, TFTC, and Mediator are present at the hsp70 promoter region. These results are similar to previous observations in which the basal factors have been found to be present at transcriptionally inactive promoters. Surprisingly, however, the apparent occupancy of TBP, several TAFs, TFIIB, and TFIIF significantly decreases upon transcriptional activation. These results could be due to some of the following scenarios: (1) upon activation, the undetected factors are present but adopt a conformation that renders them refractory to polytene chromosome staining and to ChIP analysis; (2) the factors that are not detected are indeed absent and do not participate in the ongoing transcription of the genes; or (3) the factors are present only transiently at the actively transcribed promoter and thus exhibit lower average occupancy upon polytene chromosome staining and ChIP analysis (Lebedeva, 2005).

The first scenario requires that TBP, several TAFs, TFIIB, and TFIIF simultaneously become essentially invisible to polytene immunostaining as well as to ChIP analysis upon transcriptional activation of hsp70 and other heat-shock genes. The observed effects are not a consequence of the heat shock treatment, because these factors are observed at ecdysone-responsive genes that have been subjected to heat shock. Moreover, for several factors (TBP, TAF1, and TAF10), the immunostaining was repeated with two different polyclonal antibodies that were raised against different epitopes, and identical results were obtained after heat-shock treatment. Furthermore, histone H3 K14 acetylation was detected at the hsp70 promoter after heat shock. Thus, the conditions allow the access of antibodies to proteins that are in close proximity to hsp70 promoter DNA. Thus, given that these experiments involve the use of many highly specific polyclonal antibodies and that the effect is observed with multiple polypeptides and is not a consequence of the heat-shock treatment, the first model appears to be unlikely (Lebedeva, 2005).

In the second scenario, TBP, several TAFs, TFIIB, and TFIIF do not participate in the ongoing transcription of heat-shock genes after heat induction. For instance, the factors required for transcription reinitiation may be a subset of those that participate in the first round of transcription. In fact, biochemical studies in yeast have shown that some, but not all, GTFs remain at the promoter after initiation and form a platform for the assembly of subsequent reinitiation complexes. This subset of factors includes TBP, TAF5, TFIIA, TFIIH, TFIIE, and Mediator, but not TFIIB or TFIIF. In accord with those results, this stydy found that TFIIH (XPB subunit) and Mediator (MED13), but not TFIIB or TFIIF remain at the hsp70 promoter after heat induction. In contrast, the apparent occupancy of TFIID (TBP, TAF1, and several other TAFs) is significantly reduced upon heat shock. Thus, for the second scenario to be correct, TBP and several TAFs must be dispensable for transcription reinitiation from heat-induced hsp70 promoters (Lebedeva, 2005).

In the third scenario, the average occupancy of the basal transcription factors at the hsp70 promoters is higher in the inactive gene than in the transcriptionally induced gene. This situation could occur if the basal transcription factors are in a static complex at the inactive hsp70 promoter and in a rapid cycling state of preinitiation-complex assembly and disassembly at the transcriptionally active hsp70 promoter. More specifically, in vivo data in the context of the third scenario suggest that TBP, several TAFs, TFIIB, and TFIIF make a transition from a static state to a rapidly cycling state upon heat-shock induction (Lebedeva, 2005).

It should be considered that the latter two scenarios might appear to be inconsistent with in vivo KMnO4 footprinting data, which suggest that TFIID binds to the Drosophila hsp70 promoters both before and after heat shock. In this regard, it should be noted that ChIP (as well as immunofluorescence) and footprinting experiments yield distinct types of information. ChIP provides data regarding the occupancy of a particular factor at a specific DNA sequence but does not indicate how the factor interacts with DNA or if the factor is biochemically active. Moreover, in some instances, specific DNA-bound factors may not be detectable by ChIP (although, as discussed above, it is unlikely that multiple subunits of a protein complex, such as TFIID, would be invisible in a ChIP assay with multiple polyclonal antibodies). In vivo footprinting, however, shows that a factor is bound to a specific DNA sequence but does not indicate exactly what factor is bound to that sequence. Therefore, the models and data are not necessarily contradictory. For example, it is possible that the factor that is responsible for the TATA footprint in the induced gene is not TBP or TFIID but rather another protein, such as a TBP-related factor, or a TFTC/STAGA-type complex. Alternatively, an induced hsp70 promoter might not contain the complete TFIID complex but rather only a subcomplex or TBP alone that is in a ChIP-invisible state, possibly hidden under other proteins, such as the polymerase. At the present time, however, the resolution of these issues will require the development of more sophisticated assays for the analysis of the functions of transcription factors in vivo (Lebedeva, 2005).

Thus, a model for the activation of hsp70 genes is as follows. First, the inactive gene contains many GTFs (such as TFIIB, TFIID, TFIIF, and TFIIH) as well as the downstream paused RNA Pol II. Upon heat induction, HSF binds to the promoter and recruits coactivators, such as Mediator and SAGA complexes, and these factors promote the release of the paused polymerase and the assembly of a new transcription preinitiation complex. After initiation, the transcription complex might partially disassemble, at which point factors such as TFIIB and TFIID (or many TFIID subunits) dissociate from the template DNA. (TFIIF may remain associated with the elongating polymerase and thus depart the promoter region.) Then, in subsequent rounds of initiation (i.e., reinitiation), the reassociation of TFIIB and TFIID with the template may be fleeting with a low residence time at the promoter (the third scenario described above). Alternatively, TFIIB and TFIID may be dispensable for reinitiation (the second scenario described above). TFIIH, in contrast, is needed to unwind the template DNA for every new round of transcription; thus, the average occupancy of TFIIH at the promoter increases along with the polymerase in proportion to the number of transcription reinitiation events. Thus, upon heat induction, an increase would be observed in HSF, Mediator, SAGA/TFTC, TFIIH, and RNA Pol II as well as a decrease in TFIIB, TFIID (or many TFIID subunits), and TFIIF at the promoter (Lebedeva, 2005).

The specific mechanism of transcriptional activation by HSF at heat shock genes is likely to be one of multiple mechanisms of regulation that are used in vivo. For example, in contrast to what is seen at the hsp70 promoters, the apparent occupancy of TBP, TFIIB, and several TAFs at ecdysone-responsive promoters does not decrease upon transcriptional induction, even if the cells are also subjected to heat shock (Lebedeva, 2005).

In conclusion, these results with the hsp70 promoters provide an example of a transcriptional mechanism wherein the apparent occupancy of TBP, several TAFs, TFIIB, and TFIIF decreases upon gene activation. Therefore, the extent of the apparent occupancy of these factors at a given promoter does not necessarily reflect the transcriptional activity of that promoter. The discovery and analysis of distinct transcriptional mechanisms is a key step toward the ultimate goal of understanding all of many strategies that are used by the cell to control gene activity (Lebedeva, 2005).

Deactivation of TBP contributes to SCA17 pathogenesis

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant cerebellar ataxia caused by the expansion of polyglutamine (polyQ) within the TATA box-binding protein (TBP). Previous studies have shown that polyQ-expanded TBP forms neurotoxic aggregates and alters downstream genes. However, how expanded polyQ tracts affect the function of TBP and the link between dysfunctional TBP and SCA17 is not clearly understood. In this study a novel Drosophila models was generated for SCA17 that recapitulate pathological features such as aggregate formation, mobility defects, and premature death. In addition to forming neurotoxic aggregates, it was determined that polyQ-expanded TBP reduces its own intrinsic DNA-binding and transcription abilities. Dysfunctional TBP also disrupts normal TBP function. Furthermore, heterozygous dTbp amorph mutant flies exhibited SCA17-like phenotypes and flies expressing polyQ-expanded TBP exhibited enhanced retinal degeneration, suggesting that loss of TBP function may contribute to SCA17 pathogenesis. It was further determined that the downregulation of TBP activity enhances retinal degeneration in SCA3 and Huntington's disease fly models, indicating that the deactivation of TBP is likely to play a common role in polyQ-induced neurodegeneration (Hsu, 2014).


REFERENCES

Search PubMed for articles about Drosophila Tbp

Albert, T. K., Grote, K., Boeing, S., Stelzer, G., Schepers, A. and Meisterernst, M. (2007). Global distribution of negative cofactor 2 subunit-α on human promoters. Proc. Natl. Acad. Sci. 104: 10000-10005. PubMed ID: 17548813

Andrau, J. C., et al. (2002). Mot1p is essential for TBP recruitment to selected promoters during in vivo gene activation. EMBO J. 21: 5173-5183. PubMed ID: 12356733

Auble, D. T., et al. (1994). Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism. Genes Dev. 8(16): 1920-34. PubMed ID: 7958867

Buratowski, S., Hahn, S., Guarente, L. and Sharp, P.A. (1989). Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56: 549-561. PubMed ID: 2917366

Burke, T. W. and Kadonaga, J. T. (1996). Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters. Genes Dev. 10: 711-724. PubMed ID: 8598298

Butler, J. E. F. and Kadonaga, J. T. (2001). Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs. Genes Dev. 15: 2515-2519. PubMed ID: 11581157

Cang, Y. and Prelich, G. (2002). Direct stimulation of transcription by negative cofactor 2 (NC2) through TATA-binding protein (TBP). Proc. Natl. Acad. Sci. 99: 12727-12732. PubMed ID: 12237409

Darst, R. P., et al. (2003). Mot1 regulates the DNA binding activity of free TATA-binding protein in an ATP-dependent manner. J. Biol. Chem. 278: 13216-13226. PubMed ID: 12571241

Dasgupta, A., Darst, R. P., Martin, K. J., Afshari, C. A. and Auble, D. T. (2002). Mot1 activates and represses transcription by direct, ATPase-dependent mechanisms. Proc. Natl. Acad. Sci. 99: 2666-2671. PubMed ID: 11880621

Dasgupta, A., Juedes, S. A., Sprouse, R. O. and Auble, D. T. (2005). Mot1-mediated control of transcription complex assembly and activity. EMBO J. 24: 1717-1729. PubMed ID: 15861138

Deato, M. D. and Tjian, R. (2007). Switching of the core transcription machinery during myogenesis. Genes Dev. 21: 2137-2149. PubMed ID: 17704303

Ferg, M., et al. (2007). The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish. EMBO J. 26: 3945-3956. PubMed ID: 17703193

Geisberg, J. V. and Struhl, K. (2004). Cellular stress alters the transcriptional properties of promoter-bound Mot1-TBP complexes. Mol. Cell 14: 479-489. PubMed ID: 15149597

Hsu, J.-Y., et al. (2008). TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription. Genes Dev. 22: 2353-2358. PubMed ID: 18703680

Hsu, T. C., Wang, C. K., Yang, C. Y., Lee, L. C., Hsieh-Li, H. M., Ro, L. S., Chen, C. M., Lee-Chen, G. J. and Su, M. T. (2014). Deactivation of TBP contributes to SCA17 pathogenesis. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25104854

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Biological Overview

date revised: 25 October 2014

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