The conventional model for formation of a preinitiation complex and ordered transcription by RNA polymerase II (pol II) is characterized by a distinct series of events: (1) recognition of core promoter elements by TFIID (containing TBP and several other protein subunits), (2) recognition of and binding to the TFIID-promoter complex by TFIIB, (3) recruitment of a TFIIE/pol II complex by TFIIB, (4) binding of TFIIE (related to bacterial sigma) and TFIIH (containing a helicase required for promoter melting) to complete the preinitiation complex, (5) promoter melting and formation of an "open" initiation complex, (6) synthesis of the first phosphodiester bond of the nascent mRNA transcript, (7) release of pol II contacts with the promoter (promoter clearance, and (8) elongation of the RNA transcript. TFIIA can join the complex at any stage after TFIID binding and stabilizes the initiation complex. TFIID can remain bound to the core promoter supporting reinitiation of transcription. (Orphanides, 1996 and Nikolov, 1997).

This model has been further refined to incorporate known alterations in the level of phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (Cho, 1999). Stable association of RNAPII with promoter sequences requires TFIID (or TBP), TFIIB, and TFIIF. However, the RNAPII transcription system is unique because, after the polymerase has stably associated with promoter sequences, two additional factors, TFIIE and TFIIH, are necessary for transcription. This requirement is likely related to a unique structure found at the carboxyl terminus of the largest subunit of RNAPII known as the carboxy-terminal domain (CTD). This conserved structure consists of multiple tandem repeats of the heptapeptide Tyr-Ser-Pro-Thr-Ser-Pro-Ser, which serves as a substrate for a number of protein kinases. At least two forms of RNAPII have been detected in cells. The most abundant form contains a phosphorylated CTD (RNAPIIO). A second form contains an unphosphorylated CTD and is known as RNAPIIA. The phosphorylation of the CTD has been correlated with function. It was found that the nonphosphorylated form of RNAPII is recruited to the initiation complex, whereas the elongating polymerase is found with a phosphorylated CTD. TFIIH contains a CTD kinase activity and this activity is efficient after RNAPII has associated with promoter sequences. A 150-kD polypeptide termed FCP1 has now been isolated. Together with RNAPII, FCP1 reconstitutes a highly specific CTD phosphatase activity. Functional analysis demonstrates that the CTD phosphatase allows recycling of RNAPII. Upon reaching termination sequences, the CTD becomes dephosphorylated by the FCP1 phosphatase within the ternary complex (consisting of DNA, polymerase and phosphatase) or immediately after the release of RNAPII from the DNA template. The phosphatase dephosphorylates the CTD allowing efficient recycling of RNAPII into transcription initiation complexes, which result in increased transcription. The phosphatase is found to stimulate elongation by RNAPII; however, this function is independent of its catalytic activity (Cho, 1999 and references).

A model is presented detailing the role of cycling of CTD phosphorylation in the function of RNAPII. After the termination of the previous transciption cycle, TBP remains bound to the TATA motif and provides the foundation for association of TFIIB. RNAPII, through its interactions with TFIIF, recognizes the TBP-TFIIB complex association with the TATA motif. Because TFIIF has been found to interact with both the phosphorylated and nonphosphorylated forms of RNAPII and FCP1 and to stimulate FCP1 activity, its association with RNAPII prior to association with the TB complex may be important in attaining an RNAPII that is fully dephosphorylated. The association of RNAPII with promoter sequences provides the foundation for the entry of TFIIE and allows the association of TFIIH, resulting in the formation of a fully competent transcription initiation complex. During the process of initiation and prior to the formation of a fully competent elongation complex, the CTD becomes phosphorylated in a TFIIH-dependent manner. Phosphorylation of the CTD does not affect elongation efficiency, but allows RNAPII to disengage from the promoter and from transcription initiation factors. In the presence of the ribonucleoside triphosphates, the transcription initiation complex disassembles with the release of TFIIB, TFIIE, and TFIIH. CTD phosphorylation provides a foundation for the association of factors involved in RNA processing, such as the capping enzyme, splicing factors, and factors involved in 3'-end formation. Upon transcription of termination/polyadenylation signals, the elongating complex is altered, resulting in the release of RNAPII from the template by an unknown process. It is possible that RNAPII is converted to the nonphosphorylated form prior to, or concomitant with, its release from the DNA template. This possibility is supported by studies demonstrating that FCP1 is capable of dephosphorylating the CTD of RNAPII not only in solution prior to incorporation into transcription initiation complexes, but also in active ternary elongation complexes stalled as a result of nucleotide starvation. The finding that FCP1 also stimulates elongation by RNAPII, independent of its phosphatase activity, suggests that FCP1 may remain associated with RNAPII during elongation. The finding that FCP1 is active in ternary complexes has implications for the mechanism of transcription termination as well as for the down-regulation of RNA processing. Similar to the signal imposed on phosphorylation of the CTD (disengagement of RNAPII from the promoter and from interaction with initiation factors), dephosphorylation of the CTD may result in a signal that releases factors from RNAPII that are involved in RNA maturation (Cho, 1999 and references).

How have the factors required for transcription initiation (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II [pol II]) evolved to accommodate the elaborate transcriptional programs required for growth, differentiation, and development of multicellular organisms? Analysis of the complete Drosophila genome sequence, as well as those of C. elegans, Saccharomyces cerevisiae, and humans sheds light on this well studied question in eukaryotic biology. All four organisms encode single isoforms of RNA pol II, TFIIB, TFIIE, TFIIF, and TFIIH components, but multiple, sequence-related isoforms of TFIID components. In addition, Drosophila and humans encode multiple isoforms of TFIIA components. Current evidence indicates that tissue- and cell type-specific transcription is directed by differentially expressed TFIID and possibly TFIIA isoforms. Thus, in accord with experimental data, this analysis points to TFIIA and TFIID as the factors that help generate the broad transcriptional repertoire of multicellular organisms. The identification of the complete set of TFIIA and TFIID components in a genetically and biochemically tractable organism like Drosophila is an important step toward understanding the mechanisms governing developmentally regulated transcription not only in Drosophila but also in humans (Aoyagia, 2000 and references therein).

Biochemical fractionation of Drosophila embryos, human cells, and yeast cells has defined a set of multiprotein complexes termed general transcription factors (GTFs; TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) required for mRNA transcription initiation in vitro. Transcription is initiated by recognition of core promoter elements by TFIID and sequential or concerted assembly of the other GTFs and RNA pol II to form the preinitiation complex (PIC). Although GTFs play essential roles during transcription initiation, it is the factors that regulate the ability of the GTFs to assemble and stably bind a core promoter that are probably major determinants of gene-specific transcription levels. For example, activators and coactivators are thought to stimulate transcription by recruiting GTFs to a promoter, thereby accelerating PIC assembly (Aoyagia, 2000 and references therein).

The GTF TFIID is composed of TATA-binding protein (TBP) and coactivator subunits termed TBP-associated factors (TAF_IIs). TAF_IIs not only function as 'conventional' coactivators by serving as physical links between DNA-binding activator proteins and the PIC but also possess enzymatic or promoter recognition activities that presumably enhance the efficiency of PIC assembly. TFIIA has also been described as a coactivator and displays a number of TAF_II-like properties: it binds to TBP and TAF_IIs; it interacts with specific transcriptional activators; it is generally required for activated transcription in vitro; and it contributes to promoter selectivity (Aoyagia, 2000 and references therein).

Inactivation of individual TAF_IIs in Drosophila , mammalian, and yeast cells has demonstrated that TAF_IIs are not required for the transcription of all RNA pol II genes, and in fact there is great variation in regard to the identity and number of gene targets for individual TAF_IIs. Furthermore, different domains within a single TAF_II can play gene-specific roles in transcription. The isolation of a human B cell-specific isoform of TAF_II130 (TAF_II105) raises the possibility that substoichiometric subunits of TFIID mediate tissue- or cell type-specific transcription and that additional components of TFIID may have escaped detection because of their low abundance. These possibilities have been born out in Drosophila where isoforms of TAF_II110 and TAF_II80 (No hitter [Nht] and Cannonball [Can], respectively) are expressed exclusively in testis and regulate transcription of a subset of genes required for spermatogenesis, and isoforms of TBP (TBP-related factors [TRF1 and TRF2]) are expressed in a tissue-specific manner and bind different genes in salivary gland cells. Similarly, analysis of the human TFIIA-L isoform ALF (TFIIAalpha/ß-like factor) reveals that its expression is restricted to the testis; however, it remains to be determined if it is used for the transcription of testis-specific genes. In Drosophila , TFIIA-S is expressed in a dynamic pattern during eye development and is transiently upregulated in photoreceptor precursor cells before their fate is determined. Therefore, the role of TFIIA and TFIID in transcription initiation is governed by the expression patterns and activities of their varied components (Aoyagia, 2000 and references therein).

Finally, it is critical to note that analysis of the function of TAF_IIs is complicated by the fact that they are components of at least two other complexes that lack TBP: p300/CBP-associated factor (PCAF) and TBP-free TAF_II-containing complex (TFTC). The human PCAF histone acetyltransferase (HAT) complex contains three TAF_IIs that are shared with TFIID (TAF_II31/32, TAF_II20/15, and TAF_II30) and three TAF_II isoforms (PCAF-associated factor 65ß [PAF65ß], PAF65alpha, and SPT3) related to TAF_II100, TAF_II70/80, and TAF_II18, respectively. Yeast possess an analogous complex, Spr-Ada-Gcn5-acetyltransferase (SAGA), containing TFIID TAF_IIs and the Gcn5 HAT, and Drosophila may also, since it contains a Gcn5/PCAF homolog that interacts with TAF_II24 (Aoyagia, 2000 and references therein).

Searches of the completed Drosophila, C. elegans, and yeast genomes and the partial human genome for sequence homologs of biochemically identified components of the general transcription machinery have led to the following conclusions: (1) all of the components of RNA pol II, TFIIB, TFIIE, TFIIF, and TFIIH are encoded by single copy genes in Drosophila , C. elegans, and yeast;(2) multiple isoforms of TFIID components are encoded in Drosophila , C. elegans, humans, and yeast, and multiple isoforms of TFIIA components are encoded in Drosophila and humans; (3) each organism encodes isoforms of different sets of TFIIA and TFIID components, some which are unique to a particular organism (Aoyagia, 2000 and references therein).

Sequence comparisons uncovered Drosophila homologs of TAF_IIs previously identified in yeast or humans by biochemical means but which had not been described in Drosophila (yeast TAF_II67/human TAF_II55, yeast TAF_II30/ human ENL/AF-9, and yeast TAF_II19/human TAF_II18). Thus, all TAF_IIs present in both yeast and humans are present in Drosophila , as well as C. elegans. In contrast, yeast TAF_II47 and TAF_II65 are absent from Drosophila, C. elegans, and apparently from humans, suggesting that these TAF_IIs perform a yeast-specific role, such as serving as coactivators for DNA-binding activators that are not present in metazoans. Finally, there are TAF_IIs present in Drosophila, C. elegans, and humans that are absent from yeast (human TAF_II68/Drosophila Cabeza and multiple TAF_II isoforms). In addition to Can and Nht, there are alternatively spliced forms of TAF_II30alpha, two genes (TAF_II24 and TAF_II16) that encode Drosophila homologs of human TAF_II30, and TAF_II60 and TAF30alpha isoforms (TAF_II60-2 and TAF30alpha-2, respectively). TFIIA-S and TFIIA-L are the only other GTF components in Drosophila and humans, respectively, that are expressed in multiple isoforms. The fact that these proteins are unique to multicellular organisms suggests that they play cell-specific roles (Aoyagia, 2000 and references therein).

A number of TAF_IIs contain a common structural motif called the histone fold that was originally shown to drive folding and association of each of the core histones (H2A, H2B, H3, and H4) and subsequently shown to play a similar role in association of TAF_IIs. TAF_II pairs, such as Drosophila TAF_II40 and TAF_II60, form heterotetramers, analogous to H3 and H4, and numerous other TAF_II-TAF_II and TAF_II-nonTAF_II interactions have been shown to involve histone fold motifs. The demonstrated histone fold interaction of human TAF_II135 and TAF_II20, predicts that Drosophila isoforms of these proteins, Nht and TAF_II30alpha-2, respectively, may heterodimerize and hints at the existence of a human TAF_II20 isoform that would heterodimerize with the TAF_II135 isoform, TAF_II105. B cell-specific expression of the hypothetical TAF_II20 isoform may explain why TAF_II105 associates with TFIID in B cells but not in other cell types (Aoyagia, 2000 and references therein).

In addition to the TAF_IIs indicated above, other Drosophila transcription factors contain histone fold motifs, including Prodos, NF-YC-like (CG3075), CG11301, CHRAC-14 (CG13399), CHRAC-16 (CG15736), Dr1 (CG4185), NC2alpha (CG10318), and BIP2 (CG2009). It is interesting to speculate that these factors may be unidentified TAF_II components of TFIID or binding partners for known TAF_IIs in complexes that lack TBP (Aoyagia, 2000 and references therein).

Analysis of eukaryotic genomes has defined sets of proteins that are similar in sequence to known components of TFIIA and TFIID. Since known components of TFIIA and TFIID have been shown to play key roles in developmentally regulated transcription, it is exciting to speculate that the newly identified genes will play similar roles and that TFIIA and TFIID components have evolved to support tissue- or cell type-specific transcriptional requirements of individual eukaryotic organisms. The challenge now is to determine if TAF_IIs that have been identified on the basis of their sequence are components of TBP-containing complexes or other TAF_II-containing complexes, whether TAF_IIs and TFIIA isoforms are differentially expressed during development, and how differentially expressed TBP, TAF_II, and TFIIA isoforms function in concert with the ubiquitously expressed form of TFIID and TFIIA to regulate gene expression. The subunit composition of human PCAF complex leads to the prediction that Drosophila TAF_II60-2 and Can and C. elegans Y37E11AL.c are components of PCAF/SAGA and not TFIID. However, protein isoforms that are unique to a particular organism, such as Drosophila TAF_II30alpha-2 and C. elegans F54F7.1 and K10D3.3, may be tissue- or cell type-specific components of TFIID and not of PCAF/SAGA. Drosophila may be the most appropriate organism for these studies since the biochemical activities of these factors can be determined using established TFIIA and TFIID purification schemes and in vitro transcription systems, and developmental requirements for these factors can be determined using existing mutants or mutants generated by traditional mutagenesis schemes, P-element insertion, RNA interference (RNAi), or homologous recombination (Aoyagia, 2000 and references therein).

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).

Cells often fine-tune gene expression at the level of transcription to generate the appropriate response to a given environmental or developmental stimulus. Both positive and negative influences on gene expression must be balanced to produce the correct level of mRNA synthesis. To this end, the cell uses several classes of regulatory coactivator complexes including two central players, TFIID and Mediator (MED), in potentiating activated transcription. Both of these complexes integrate activator signals and convey them to the basal apparatus. Interestingly, many promoters require both regulatory complexes, although at first glance they may seem to be redundant. RNA interference (RNAi) was used in Drosophila cells to selectively deplete subunits of the MED and TFIID complexes to dissect the contribution of each of these complexes in modulating activated transcription. The robust response of the metallothionein genes to heavy metal was used as a model for transcriptional activation by analyzing direct factor recruitment in both heterogeneous cell populations and at the single-cell level. Intriguingly, it was found that MED and TFIID interact functionally to modulate transcriptional response to metal. The metal response element-binding transcription factor-1 (MTF-1) recruits TFIID, which then binds promoter DNA, setting up a 'checkpoint complex' for the initiation of transcription that is subsequently activated upon recruitment of the MED complex. The appropriate expression level of the endogenous metallothionein genes is achieved only when the activities of these two coactivators are balanced. Surprisingly, it was found that the same activator (MTF-1) requires different coactivator subunits depending on the context of the core promoter. Finally, the stability of multi-subunit coactivator complexes can be compromised by loss of a single subunit, underscoring the potential for combinatorial control of transcription activation (Marr, 2006).

There are four known metallothionein genes in Drosophila: MtnA, MtnB, MtnC, and MtnD. Of these, the best characterized is the MtnA gene, which produces a transcript of ~600 bases in length, bearing one intron. All of the regulatory elements required for robust response to heavy metals, including copper, lie within 500 bp of the transcription start site. The gene is controlled by a single activator, metal response element-binding transcription factor 1 (MTF-1), which binds two adjacent metal response elements (MRE) 50 bp upstream of the TATA-box (Zhang, 2001). Quantitative PCR (qPCR) analysis of the endogenous gene in Drosophila S2 cells shows that the gene is highly induced (~250-fold) after a short exposure to copper. The total amount of stable MtnA mRNA approximates the level of the abundant transcript for the ribosomal subunit Rp49. Primer extension analysis confirms that transcriptional activation of the endogenous MtnA gene originates from a unique start site overlapping the core promoter. The transcript accumulates linearly for ~12 h, thus measurements in this time window likely reflect relative levels of transcription of the MtnA gene. Importantly, induction at the endogenous chromosomal locus is easily assayed in order to measure physiologically relevant transcriptional activation in the context of native chromatin. Taken together, these properties establish the endogenous MtnA gene as a useful model for studying transcriptional mechanisms governing an inducible gene (Marr, 2006).

Using chromatin immunoprecipitation (ChIP), it was found that the sequence-specific DNA-binding protein MTF-1 is specifically recruited to the MtnA promoter region in response to copper. Curiously, the ChIP of the promoter region was compared to a region 1 kb downstream, a significant amount of MTF-1 was found to be present on the promoter even in the absence of added copper. Under these conditions, little transcription is detected from this gene. As a preliminary experiment to investigate a potential functional interaction between TFIID and MED, it was first asked whether the two complexes are both recruited in a signal-dependent manner to the MtnA gene. Using ChIP, it was found that both TBP and the TAFs are efficiently recruited to the promoter region in response to copper. In addition, the MED17, MED24, MED26, and MED27 subunits of MED are all recruited to the promoter region in response to copper treatment. Consistent with the high level of induction, RNAPII occupancy at the MtnA promoter is also increased in response to heavy metal treatment. Thus, both core coactivator complexes and RNAPII are efficiently recruited to the promoter region upon induction and resultant binding of MTF-1 to the MREs (Marr, 2006).

Because the ChIP assay is limited to measuring response in a heterogeneous population of cells, a transgenic model system was extablished in Drosophila S2 cells in order to visualize the response at the single-cell level. Such an approach has proved useful in understanding transcription factor dynamics in vivo. By selecting for stably transfected MtnA firefly luciferase reporters, a concatenated transgenic locus was generated in a clonal line of S2 cells. The transgenic locus was assayed for dependence on copper using a luciferase assay. Importantly, transcription initiates a unique site that maps to the correct start site of the MtnA core promoter. With this substantial increase in gene number (~2000) at the integrated transgenic locus, it should now be possible to visualize direct recruitment of specific transcription factors to the MtnA promoter within a single cell (Marr, 2006).

As expected, in the absence of heavy metal, MTF-1 is predominantly cytoplasmic; however, in agreement with ChIP data, some MTF-1 can be detected at the transgenic cluster even in the absence of a metal stimulus. Thus, antibody labeling of MTF-1 provides a useful marker for the subnuclear location of the transgene cluster in both induced and uninduced cells. Notably, the locus is not undergoing transcription (as detected by RNA FISH) in the absence of heavy metal induction despite the presence of some MTF-1 at the transgene cluster. Upon copper induction, MTF-1 vacates the cytoplasm and accumulates selectively at the transgenic locus. Under these same conditions, TBP is also actively recruited to this cluster. Consistent with not only TBP but holo-TFIID complex recruitment, it was found that TAF2 also accumulates at the transgene. Likewise MED components recruited to the transgene were detected using antibodies against MED26. As expected, RNAPII is recruited to the cluster in a copper-dependent manner consistent with the transcriptional induction of the transgene under these conditions. In contrast, TBP-related factor 1 (TRF1), a subunit known to be a key component of the RNA polymerase III core promoter recognition complex, is not recruited to the transgene. This negative control helps rule out the possibility that the tandemly reiterated transgene is simply nonspecifically attracting transcription factors (Marr, 2006).

Having established by two independent methods that both TFIID and MED complexes are recruited to the MtnA promoter in an activator-dependent manner, their role in potentiating transcriptional activation of the endogenous MtnA gene was investigated. The efficient technique of RNAi in Drosophila S2 cells was used to knock down expression of TFIID and MED subunits. In addition, the activator MTF-1 was knocked down to ascertain the extent of the activator’s role in induction. After treatment with copper, total RNA was purified from dsRNA treated and untreated S2 cells and then they were assayed by two independent methods. First, a primer extension analysis was used on equivalent amounts of total RNA. This assay revealed that an accurate transcription is detected from one distinct core promoter start site. Next, qPCR normalized to the Rp49 mRNA was used, to confirm that there is little or no global disturbance of RNAPII transcription (Marr, 2006).

Not surprisingly, depletion of MTF-1 severely reduced transcriptional activation from the MtnA promoter, confirming the central role of this activator. RNAi directed against TBP also had a dramatic inhibitory effect. The MtnA promoter is <10% as active when TBP levels are severely depleted. Surprisingly, knockdown of multiple TAFs had little apparent effect on the ability of MTF-1 to activate MtnA. Indeed, depletion of the TAFs actually stimulated (1.5- to 2-fold) production of RNA. With the exception of TAF11, a reduction of individual TAFs resulted in a remarkably uniform response. The reason for this uniformity became apparent when the stability of the TFIID complex was examined in the RNAi-treated cells. The overall stability of the holo-TFIID complex appears to be coupled to the stability of certain individual TAFs. In the most dramatic example, RNAi-targeted reduction of TAF4 leads to the concomitant loss of TAF1, TAF5, TAF6, and TAF9, as well as a detectable reduction in TBP. Interestingly, TAF2 and TAF11 are largely unaffected by depletion of TAF4. Similar results are observed for the other TAFs as well. When the transcript levels of the TAFs were measure after RNAi treatment, it is clear that the loss of stability occurs at the protein level, since the transcript levels for nontargeted TAFs are unaffected. For example, when TAF4 is targeted, only the TAF4 transcript is depleted (Marr, 2006).

In contrast to the TAFs, RNAi reduction of MED subunits gave striking but variable effects on the ability of MTF-1 to activate transcription from the MtnA promoter. Unlike TFIID, the response is far from uniform. For example, dsRNA directed against MED23 has little effect on induction of MtnA, while loss of MED17, the Drosophila SRB4 homolog, has a strong inhibitory effect. The lack of a uniform response in the MED RNAi led to a further investigation of the potential differential response upon depletion of MED subunits at related promoters activated by MTF-1. As discussed above, Drosophila has four metallothionein genes that respond to heavy metals. Three of these—MtnA, MtnB, and MtnD—are active in S2 cells. All three of these genes are specifically activated by the same factor, MTF-1. All three Mtn genes were examined in a single experiment using qPCR. First, it was confirmed that all three promoters, MtnA, MtnB, and MtnD, require MTF-1 for induction. Remarkably, distinct differential requirements were found for MED subunits depending on the promoter. For example, loss of MED13, a subunit of the larger MED complex (ARC-L) thought to play a repressive role in transcription, is not essential for MtnA induction. In contrast, MED13 was found to be important for both MtnB and MtnD activation by MTF-1. In contrast, the opposite specificity was seen with the MED26 subunit, a component of the smaller MED complex (CRSP), thought to play predominantly a coactivator role in transcription. Interestingly, MED26 is required for full induction of the MtnA promoter but is dispensable for MTF-1 activation of the MtnB and MtnD promoters. Thus, these experiments reveal a remarkable example of differential dependence on cofactor composition even though all three promoters tested use the same activator. Apparently, the precise role of individual MED subunits depends on the promoter context and structure, despite the absence of any evidence of direct binding of DNA by the MED complex (Marr, 2006).

To help rule out nonspecific effects on transcription such as a change in the concentration of free RNA polymerase, representative targets from TFIID and MED were tested in a transient transfection assay where the effect to a second promoter can be normalized. In these experiments, TAF4 and MED17 were chosed as representative targets, since TAF4 compromises much of the TFIID complex and MED 17 is likely a component of the core MED complex. The transient transfection data are largely consistent with the data generated at the endogenous locus and at the transgene (Marr, 2006).

The data presented above suggest that activation of the MtnA gene requires specific MED subunits, and at the same time the TAFs appear to be playing a potential negative regulatory role. Because it is clear that the TAFs are specifically recruited in S2 cells to the MtnA promoter in a copper-dependent manner by MTF-1, whether TFIID recruitment can occur in the absence of the MED complex was examined. To achieve this, RNAi directed against MED17 was used, which results in an almost complete loss of MED activity. Surprisingly, TFIID is still efficiently recruited to the MtnA gene. ChIP experiments confirmed that TBP and TAF2 are still actively (and likely directly) recruited to the endogenous MtnA gene by MTF-1 even when the gene is transcriptionally inactive as measured by qPCR analysis. The MtnA luciferase transgene system was used to investigate this relationship at the single-cell level. Without any RNAi, TBP, TAF2, and RNAPII were all recruited to the transgene. In agreement with the ChIP data above, even in the absence of MED activity, after MED17 depletion, TBP and TAF2 are nevertheless efficiently recruited to the transgene. In contrast, no RNAPII can be detected at the transgene consistent with the loss of transcription activation. Apparently, TFIID is recruited to the promoter, but the promoter is not active in supporting transcription. Importantly, recruitment of this 'inactive TFIID' is dependent on the activator MTF-1. In the absence of MTF-1, no TFIID or RNAPII is recruited to the transgene (Marr, 2006).

This perplexing result of recruiting an apparently 'inactive' TFIID prompted an examination of what happens when both TAFs and MEDs were depleted. Remarkably when both the TAFs and MED complex are depleted and 'removed' from the MtnA promoter, MTF-1-dependent activation of transcription is restored to ~95% the level of untreated cells, which is well above the inhibited level observed when the MEDs alone are depleted. In humans and Drosophila, TAFs can be subunits of other complexes such as TFTC and STAGA, so it is possible that the functional interaction analyzed is not TFIID-specific. To test this, specific subunits of these other complexes were targeted to determine if they would have a similar ability to rescue the MED knockdown. Unlike the TFIID subunits, RNAi against dAda2b, dGCN5, dSPT3, and dTRA1 was unable to rescue the loss of the MED subunits. These findings taken together suggest that most likely the functional relationship revealed by these experiments with the MtnA promoter, indeed, involve some regulatory transaction between TFIID and MED (Marr, 2006).

The requirement for coactivator complexes mediating transcriptional responses to activators has been well documented. However, by using an inducible Drosophila gene as a model system, a previously unknown functional interaction has been uncovered between two coactivator complexes, TFIID and MED. In the absence of TAFs, the cell responds inappropriately to a metal stimulus. The cell synthesizes 50%–200% more mRNA from the MtnA gene than it does in the presence of the TAFs. The data suggest that at this gene, TFIID is recruited in an inactive state, a state that impedes initiation of transcription. It is believed that this sets up a checkpoint early in the initiation process to meter the RNA synthesis. The MED complex must be recruited to get past this checkpoint. It is postulated that the MED complex likely modifies TFIID, converting it to an active state. This could be accomplished either through one of the known enzymatic activities of MED, phosphorylating (cdk8) or ubiquitylating (MED8) TFIID subunits, or through some, as yet undetected, chaperone-like function that remodels TFIID into an active conformation. Not surprisingly then, in the absence of MED subunits the cell cannot mount an appropriate response to environmental signals. In fact, depletions of many of the MED subunits lead to <20% of the normal amount of mRNA. Unlike the uniform response to depletion of TAFs, the response to depletion of MEDs is much less uniform. One possibility is that the MED complex is more functionally and structurally diverse than TFIID. Indeed, alternative subcomplexes of MED have been purified biochemically, whereas no such subcomplexes of TFIID have been reported (Marr, 2006).

By analysis of three different Mtn genes, all of which are dependent on the same single activator, it was found, surprisingly, that there is a differential requirement of specific MED subunits at the three Mtn promoters. This is taken as evidence that, depending on the precise arrangement of cis elements and promoter context, the same activator can require different mediator subunits or modules to transmit its signals to the basal apparatus (Marr, 2006).

Interestingly, the kinase module of the MED complex, previously linked with repression functions, is required for efficient activation at two of the promoters. This result, combined with the finding that at the MtnA promoter the TAFs have a repressive regulatory influence on transcription initiation, underscores the difficulty in assigning black and white functions to the coactivator complexes. It is likely that both TFIID and MED interpret multiple inputs from cellular signals and act either positively or negatively depending on the signals received as well as the specific promoter context. As such, the complexes may better be viewed as coregulators since they can play either a positive or negative role in the process of modulating gene expression. For example, only when both TFIID and MED are intact do Drosophila S2 cells produce the appropriate amounts of MtnA mRNA. In contrast, when either coactivator complex is disrupted, aberrant levels of transcription are seen. However, when both coactivator complexes are depleted, a significant level of metal inducible activation is actually restored. Presumably, in this 'stripped down' system, some portion of the remaining TBP pool can mediate transcription. Curiously, in the absence of TAFs but with a full complement of MEDs, there is also an aberrant level of transcription consistent with the notion that there is some finely tuned codependence between the TBP/TAF complex and the MED complex at this promoter (Marr, 2006).

The results also reinforce the notion that the activator is the primary determinant of the transcriptional response. The MTF-1 depletion experiments were the most detrimental to mRNA induction. In the absence of MTF-1, there is no detectable activation of the Mtn genes. In contrast, there is some residual transcription of MtnA even when either the MEDs or TBP are largely depleted from the Drosophila cells. This remaining activity could be due to incomplete depletion, or it could indicate alternative mechanisms of activation that are activator-dependent but can partially bypass the requirement for the coregulator complexes (Marr, 2006).

In the course of testing the requirement for TAFs in activated transcription, the codependent stability of the TFIID complex was discovered. Particularly striking is the finding that TAF4 depletion destabilizes most of the other TAFs and, to some extent, even TBP. Therefore, the TAF depletion experiments most likely reflect a loss of holo-TFIID rather than just the loss of individual subunits. It is worth noting that metazoan organisms contain multiple variants of TAF4: TAF4b in vertebrates and No-hitter in Drosophila. Both of these have been implicated in tissue-specific gene expression. It is conceivable that substitution of this keystone TAF can provide a mechanism to change the entire coregulator profile of TFIID (Marr, 2006).

One intriguing question this work raises is: Why would an activator recruit an inactive TFIID complex to the promoter? There are several previously described cases in which TFIID occupancy at a promoter does not strictly correlate with transcriptional activity. However, in most of these cases the genes being examined were either in a repressed or an unstimulated state. In contrast, the current studies were designed to specifically measure the role of coactivator complexes such as TFIID and MED in the context of an active gene MtnA upon metal stimulation. The ability to deplete MED activity under these conditions revealed the unexpected finding that although TFIID is dynamically recruited to the MtnA promoter, TFIID is mainly held in an 'inactive' state until the second cofactor complex, MED, is recruited. Perhaps this recruitment of an 'inactive' TFIID is a more common phenomenon that can only be detected in special circumstances and may represent a previously unappreciated control mechanism in transcription activation. If the activator first recruits TFIID, then subsequently recruits MED, and there is a requirement for additional factors to potentiate the secondary recruitment of coregulator assemblies, then this provides a potential checkpoint for fine-tuning the control of gene expression. Alternatively, since the cell invests a significant amount of energy in making a high level of transcript, requirement of continued stimulation (i.e., activator bound at the promoter) for mRNA production would provide the most economical use of resources (Marr, 2006).

list of proteins involved in messenger RNA synthesis