Brf: Biological Overview | References
Gene name - Brf
Synonyms - TFIIB-related factor
Cytological map position - 90A3-90A4
Function - RNA polymerase III transcription factor
Symbol - Brf
FlyBase ID: FBgn0038499
Genetic map position - 3R:13,200,555..13,217,054 [-]
Cellular location - nuclear
It has been generally accepted that the TATA binding protein (TBP) is a universal mediator of transcription by RNA polymerase I, II, and III. TBP-related factor TRF1 rather than TBP is responsible for RNA polymerase III transcription in Drosophila. Immunoprecipitation and in vitro transcription assays using immunodepleted extracts supplemented with recombinant proteins reveals that a TRF1:BRF complex is required to reconstitute transcription of tRNA, 5S and U6 RNA genes. TFIIB-related factor (BRF) gets its name from its amino-terminal half which is is homologous to the Pol II transcription factor IIB (TFIIB). In vivo, the majority of TRF1 is complexed with BRF and these two proteins colocalize at many polytene chromosome sites containing RNA pol III genes. These data suggest that in Drosophila, TRF1 rather than TBP forms a complex with BRF that plays a major role in RNA pol III transcription (Takada, 2000).
Although it is now evident that eukaryotic organisms have evolved elaborate molecular machines to deal with the control of gene expression, some of the key components and mechanisms that regulate transcription remain unclear. Even the most familiar and seemingly well established paradigms such as the universality of the TATA binding protein (TBP) continue to unveil surprises. For example, recent studies have demonstrated the existence of TBP-related factors (TRF1 and TRF2) in metazoans that appear to regulate the expression of specific subsets of genes. Another surprising discovery was that at least in vitro, transcriptionally competent initiation complexes lacking any known member of the TBP/TRF family have been observed. As more extensive analyses of tissue-specific and developmentally regulated transcription mechanisms are carried out, it would not be surprising to discover additional levels of specificity and specialization in the components that have been traditionally considered as part of an invariant and universal 'basal transcriptional apparatus'. In fact, multiple cell type-selective TBP-associated factors (TAFs) that make up variant TFIID complexes have been discovered in both mammals and insects (Takada, 2000).
Several years ago, studies indicated that the Drosophila TBP-related factor TRF1 might substitute for TBP and play a role in directing transcription from different sets of genes. Biochemical studies indicated that TRF1 could interact specifically with TFIIA and TFIIB to form a functional complex capable of binding to core promoter DNAs and initiating RNA pol II transcription in vitro. Interestingly, both RNA and antibody in situ staining experiments suggested that TRF1 might be expressed in a cell type-specific fashion during development, although in early embryos expression was widespread. Particularly intriguing was the finding that TRF1 was associated in vivo with only a limited subset of loci as revealed by antibody staining of polytene chromosomes. These results suggested that TRF1 might selectively target a limited subset of genes unlike TBP which was found to associate with the majority of genes in the Drosophila genome. The TRF1-positive polytene chromosome sites appeared to include many genes related to male-sterility or neuronal functions. However, many of the TRF1-positive sites also contained tRNA and 5S RNA genes. These early in situ staining experiments and genetic correlation suggested at least two possible functions for TRF1 including neuronal- and/or testis-specific RNA pol II transcription, and/or a function in RNA pol III transcription (Takada, 2000).
In human and yeast, TBP is an essential component of transcription initiation complexes utilized by all three classes of RNA polymerases. Therefore, it was expected that TBP would also play an important role in Drosophila pol III transcription. However, there was no direct biochemical or functional evidence to establish the involvement of either TBP or TRF1 in RNA pol III transcription. Given the sites of TRF1 localization on polytene chromosomes, it was asked whether TBP, TRF1, or both molecules in fact are required for RNA pol III transcription in Drosophila. First, a Drosophila RNA pol III in vitro transcription system was developed to test whether the depletion of TBP and/or TRF1 might affect pol III transcription. Next, immunoprecipitation was used to determine whether other pol III transcription factors such as BRF are associated with TBP or TRF1. This approach might allow isolation of Drosophila BRF, a core component of the RNA pol III transcription factor TFIIIB, that had thus far eluded molecular characterization. In vitro transcription assays carried out with Drosophila nuclear extracts depleted of TRF1, BRF, or TBP were employed to provide evidence for the involvement of these factors in the transcription of tRNA, 5S RNA, or U6 RNA genes. Reconstitution of such depleted extracts with recombinant factors was subsequently carried out to confirm the importance of potentially novel protein complexes involved in directing transcription by RNA pol III. These studies suggest that TRF1 instead of TBP plays an important role in directing transcription by RNA pol III in Drosophila (Takada, 2000).
In order to determine which factors are responsible for Drosophila RNA pol III transcription, an in vitro reaction was developed responsive to a panel of different DNA templates with multiple representatives from each category of genes (six tRNAs, two U6 RNAs, and six 5S RNAs). The in vitro transcription extract accurately initiates and terminates RNA synthesis to produce a number of discrete RNA products of the expected size at different levels of efficiencies depending on the template used. Results from only three representative RNA pol III templates tRNA (35DArg), U6 (6-1), and 5S RNA (1A8) are present because the results from the other templates were essentially the same (Takada, 2000).
To confirm that the RNA products synthesized by these in vitro reactions represented bona fide RNA pol III transcripts, their sensitivity to α-amanitin and tagetitoxin (a selective inhibitor of RNA pol III) was tested. Drosophila RNA pol III is known to be considerably more resistant to α-amanitin than mammalian or yeast RNA pol III. Consistent with previous observations, transcription from active pol III genes (tRNA, U6, 5S) was found to be resistant to α-amanitin up to 200 μg/ml while tagetitoxin (0.5 units/ml) completely inhibited transcription of the pol III templates. In contrast, RNA pol II transcription directed by the Adh distal promoter was highly sensitive to low levels of α-amanitin (2 μg/ml), but resistant to tagetitoxin. These data indicate that a robust in vitro RNA pol III transcription system has been developed suitable for testing the role of TBP or TRF1 (Takada, 2000).
In order to determine which factors participate in directing RNA pol III transcription, cell extracts of TBP or TRF1 were immunodepleted and assayed for pol III transcription. Surprisingly, depletion of TBP from the extracts did not detectably effect pol III transcription of any templates tested, although it abrogated the pol II transcription from the Adh promoter. In contrast, treatment with anti-TRF1 severely impaired or eliminated transcription by pol III. As expected, depletion of TRF1 had little or no effect on transcription by pol II from the Adh promoter. Control (mock) reactions were performed using nuclear extract that had been treated with preimmune antibodies or beads alone. These results suggest that Drosophila TRF1 but not TBP is required for pol III transcription at least in the in vitro assay (Takada, 2000).
In parallel with these in vitro transcription studies, attempts were made to isolate and characterize proteins that are associated with TRF1 and thus possibly involved in directing pol III transcription. Schneider cells (Schneider's Drosophila line 2) were used as the starting material, since this cell line provided a homogeneous pool of reasonable quantities of TRF1. The native molecular weight of the major TRF1 species in crude Schneider cell extracts was about 200 kDa estimated by gel-filtration analyses, and was the same as that of TRF1 from embryo extracts. TRF1 from crude Schneider cell extracts was found to consistently coimmunoprecipitate polypeptides of 90, 70, and 50 kDa. Microsequence analyses of these individual polypeptides revealed that the 90 kDa species contained a peptide sequence highly homologous to the C. elegans BRF gene product. The 70 and 50 kDa polypeptides were identified as HSP70 and EF1α (a translation elongation factor), two relatively abundant proteins in Drosophila extracts that are likely contaminants in the immunoprecipitates (Takada, 2000).
Based on the peptide sequences, cDNA clones encoding the 90 kDa protein were isolated. The deduced amino acid sequence contained all the peptide sequences obtained from the microsequence analyses, suggesting that the 90 kDa band represented a single polypeptide. The translated gene product has a calculated mass of 73,705 Da and an estimated isoelectric point of 4.79. Amino acid sequence comparison revealed that the 90 kDa protein associated with TRF1 is closely related to BRF, a well-characterized RNA pol III transcription factor that had previously not been identified from Drosophila. Therefore, the 90 kDa protein as Drosophila will be referred to as BRF (Takada, 2000).
To further investigate the association of the newly isolated BRF molecule with TRF1 in Drosophila cells, the specificity of the interaction between endogenous TRF1 and BRF was examined. Polyclonal rabbit antisera were generated against BRF. After antigen affinity purification of this antibody, immunoprecipitation experiments were carried out with Schneider cell extracts. The immunoprecipitates were subsequently analyzed for the presence of TRF1, BRF, and TBP by immunoblotting. As expected, anti-TRF1 precipitated TRF1 and coprecipitated BRF efficiently. Likewise, anti-BRF coprecipitated TRF1 and BRF efficiently. In contrast, no TBP was detected in either the anti-BRF or anti-TRF1 immunoprecipitates. Anti-TBP efficiently precipitated TBP from these extracts whereas only background levels of BRF or TRF1 coimmunoprecipitated with TBP. Similar coprecipitation results were obtained using extracts derived from Drosophila embryos (Takada, 2000).
To further ascertain that endogenous TRF1 and BRF form a stable complex in Drosophila cells, these two proteins were examined by gel filtration chromatography. Immunoblotting with anti-TRF1 and anti-BRF revealed that endogenous TRF1 and BRF from Schneider cell extracts comigrated with an apparent native molecular weight of approximately 200 kDa when fractionated on a Superose 6 column. These results, taken together with the coimmunoprecipitation data, suggest that TRF1 and BRF are largely bound to each other and that the identified BRF molecule does not appear to be associated with TBP in Drosophila cells. The absence of detectable amounts of TBP:BRF complex may explain the lack of TBP contribution in pol III transcription observed in in vitro assays (Takada, 2000).
As an independent test of the interaction between TRF1 and BRF, proteins expressed from bacteria were also characterized. Recombinant TRF1 and BRF were coexpressed in E. coli and purified by several conventional chromatography steps as described in Experimental Procedures. Interestingly, recombinant TRF1 was found to be much more soluble when coexpressed with BRF compared to TRF1 expressed alone. The coexpressed recombinant TRF1 and BRF proteins copurified through Poros HS and HQ ion exchange columns. Their chromatographic behavior as well as solubility characteristics suggested that TRF1 was in complex with BRF. Moreover, Superose 6 gel filtration analysis of these recombinant proteins confirmed that TRF1 and BRF comigrate as a 200 kDa complex with an elution profile very similar to the endogenous Drosophila TRF1:BRF complex. As expected, recombinant TRF1 and BRF could be efficiently coimmunoprecipitated from crude E. coli extracts as a complex using either anti-TRF1 or anti-BRF antibodies. These results, taken together, suggest that both the recombinant and endogenous TRF1 and BRF polypeptides display a high propensity to interact with each other to form a stable complex, possibly a heterotetramer (predicted mass of 198,319 Da). The molecular weight of this TRF1:BRF complex is similar to a TFIIIB activity that has been characterized and partially purified from Drosophila extracts. To further test whether TBP can interact with BRF, TBP and BRF were coexpressed in E. coli. The purification of a TBP:BRF complex by conventional chromatography was unsuccessful because of their poor interaction and apparent degradation of BRF. However, utilizing a reticulocyte lysate in vitro translation system, a very weak interaction was detected between TBP and BRF, but the efficiency was much lower than that observed between TRF1 and BRF (Takada, 2000).
To confirm that the TRF1:BRF complex plays an important functional role during RNA pol III transcription, attempts were made to restore activity to the depleted extracts by addition of purified recombinant proteins. Addition of the TRF1:BRF complex to an extract depleted with anti-TRF1 very efficiently restored transcriptional activity. In contrast, supplementing TRF1 alone weakly restored transcriptional activity. This weak activity was likely due to complex formation between exogenously added recombinant TRF1 and residual endogenous BRF remaining in the extracts. As expected, addition of recombinant BRF alone failed to restore transcriptional activity to the depleted extracts. Likewise, RNA pol III transcriptional activity of extracts was eliminated by depletion with anti-BRF, and could only be restored by addition of the TRF1:BRF complex (lanes 14-16) but not with BRF alone (lanes 11-13) or TRF1 alone (Takada, 2000).
To further assess the factor requirements for the Drosophila pol III transcription system, attempts were made to restore activity to the depleted extracts with the addition of purified recombinant TBP and BRF. In contrast to TRF1, purified TBP preincubated with BRF failed to restore RNA pol III transcription significantly above background levels. As expected, recombinant TBP could restore RNA pol II basal transcription of TBP-depleted nuclear extracts. These results indicate that in Drosophila cells there is a stable complex formed preferentially between TRF1 and BRF rather than TBP and BRF. The active TRF1:BRF complex could be efficiently removed with either anti-TRF1 or anti-BRF antibodies. After depletion of this complex, addition of one or the other subunit alone could not efficiently restore transcriptional activity. However, nearly full RNA pol III transcriptional activity was reconstituted with purified recombinant TRF1 and BRF added to the depleted extracts. In contrast, addition of TBP and BRF did not efficiently restore pol III transcription to depleted extracts. These results strongly suggest that in Drosophila the TRF1:BRF complex rather than a TBP containing complex plays an important role in directing specific transcription by RNA pol III (Takada, 2000).
If BRF and TRF1 exist as a functional complex on pol III-transcribed genes in vivo, these proteins should colocalize to specific chromosomal loci that contain pol III-transcribed genes. This prediction was tested by examining BRF and TRF1 distribution on salivary gland polytene chromosomes. The BRF antibody showed prominent staining of a set of about 70 chromosomal loci. Most sites labeled with anti-BRF were also stained with anti-TRF1, though the ratio of the two signals varied at each locus. Major sites of labeling by anti-TRF1 antibodies have been shown (Hansen, 1997) to contain pol III genes (Takada, 2000).
While the overlap of TRF1 and BRF on chromosomes is extensive, a number of sites appear to be labeled only by anti-TRF1. This is consistent with the notion that TRF1 may also function as a pol II transcription factor for a subset of genes (Hansen, 1997). The affinity-purified TRF mouse antibody labels the major sites seen previously (Hansen, 1997), but also reveals additional bands and a higher background. This difference could be due to antigenic differences in the anti-TRF1 populations. The mouse anti-TRF1 in this study was affinity purified using a full-length TRF1, while the rabbit anti-TRF1 used previously was purified using a peptide corresponding to the N-terminal 50 amino acids of TRF1. Thus, the detection of TRF1 at a majority of chromosomal sites labeled with anti-BRF, and the finding that many of these sites contain genes transcribed by pol III (Hansen, 1997), provide support for a functional role of a TRF1:BRF complex in pol III transcription in vivo (Takada, 2000).
Since TRF1, like TBP, may be part of different complexes, attempts were maed to determine what proportion of TRF1 is associated with BRF in Drosophila cells. Either Schneider cell or embryo extracts were immunodepleted of TBP, TRF1, or BRF and then the levels of these proteins in the supernatants were examined. After treating extracts with anti-BRF antibodies, most of the TRF1 (≥90% as determined by quantitative Westerns) was found to be codepleted with BRF from the extract. Likewise, treatment of extracts with anti-TRF1 removed the majority of the BRF from the Schneider cell supernatant. Although the same trends were observed for both Schneider cell and embryo extracts, it was noticed that cross depletion of BRF by anti-TRF1 was more complete in Schneider extracts than in embryo extracts. It is believed that the difference observed between these two cell extracts may largely be explained by the differential expression of TRF1 among different cell types. Immunoblot analyses of nuclear or whole cell extracts indicate that the levels of TRF1 and BRF in Schneider cells are roughly equivalent while in embryos there is significantly less TRF1 relative to BRF. This difference is consistent with the differential expression patterns of TRF1 in the developing embryos previously observed by in situ hybridization and immunostaining. In contrast, in none of the cross-depletion experiments were anti-BRF or anti-TRF1 able to significantly deplete TBP from either Schneider or embryo extracts. Likewise, anti-TBP did not codeplete BRF. These antibody depletion experiments, taken together, indicate that the majority of the TRF1 is in complex with BRF, and that very little, if any, TBP is associated with the BRF molecule in vivo (Takada, 2000).
It has been widely accepted that TBP is a universal factor that plays a central role in eukaryotic transcription by all three RNA polymerases (I, II, and III). Indeed, TBP is highly conserved among all the eukaryotes studied and its universal involvement in all three RNA polymerases has been established for human and yeast. Contrary to this general expectation, this study revealed that Drosophila TBP may not be a major player in directing RNA pol III transcription. Instead, the TBP-related factor TRF1 was found to be a principal participant in Drosophila pol III transcription. In vitro transcription studies with multiple classes of pol III templates failed to show any significant contribution of TBP, but instead revealed a clear contribution by TRF1. Moreover Drosophila BRF was isolated as a TRF1-associated factor, while no significant amount of TBP:BRF complex was observed. Immunodepletion of Drosophila nuclear extracts followed by reconstitution with purified recombinant TRF1:BRF directly implicated this complex in mediating RNA pol III transcription. Thus, it appears that in Drosophila TRF1:BRF contributes significantly to RNA pol III transcription initiation in place of a TBP:BRF complex (Takada, 2000).
Not all potential RNA pol III genes in the Drosophila genome (600-750 tRNA genes for instance) were assayed nor have extracts from every Drosophila tissue type or developmental stage been tested. Therefore, the possibility that in Drosophila there are different BRF, TBP, or TBP-related factor complexes that may collectively contribute to RNA pol III transcription in a cell type or developmental stage-specific manner cannot be excluded. Indeed, efforts to quantitate TRF1 and BRF in Schneider cells and embryos suggest that the relative amounts of these two transcription factors may vary depending on the cell type. Moreover, an excess pool of BRF molecules was detected that appeared not to be tightly associated with either TRF1 or TBP in extracts derived from embryos. Thus, it is possible that Drosophila BRF can be associated with some other, as yet unidentified factors that contribute to RNA pol III transcription. However, since all the detectable pol III transcription activity in the embryonic extracts was abolished by depletion of TRF1, such putative TRF1-free BRF complexes may not be actively involved in pol III transcription at least in vitro (Takada, 2000).
Although in principle it is possible that there are multiple BRFs or BRF-like factors in Drosophila, the nearly complete genome sequences of Drosophila made available recently failed to reveal any obvious BRF homologs other than TFIIB. Thus, it seems unlikely that there are multiple BRFs or BRF-like factors. It has been reported that a 105 kDa protein could be shown to interact with Drosophila TBP and that an antibody against human BRF cross-reacted with this protein. The relationship between the Drosophila BRF identified in this study and the previously detected putative BRF-like protein is presently unknown. Also, there was no clear evidence that this putative TBP-containing complex contributed to RNA pol III transcription (Takada, 2000 and references therein).
Thus far, a homolog of Drosophila Trf, unlike TBP or TRF2, has not been found in yeast or other metazoans, including C. elegans, mouse, or human; however, for mammals, the genome information is incomplete. Therefore, it is possible that Drosophila (or perhaps insects) have evolved distinct sets of factors for RNA pol III transcription. It has been reported that unlike the RNA pol II basal machinery, which is remarkably conserved across divergent species (yeast to human), the RNA pol III and RNA pol I transcription machinery has diverged significantly from species to species. For example, the Drosophila pol III transcription factors are not interchangeable with the corresponding factors from human cells. Also, the insect RNA pol III subunits themselves display differential sensitivity to alpha-amanitin relative to other species. Likewise, the control elements of promoters in these different organisms seem to have diverged. Transcription of tRNA genes in Drosophila and silk worm is strictly dependent on 5' flanking promoter sequences: this is not the case in other species. Furthermore, the mechanism of RNA polymerase selection for transcription of Drosophila U6 RNA genes is significantly different from that of vertebrates. In vertebrates, the presence of a TATA-box in combination with a proximal sequence element (PSE) located in the upstream region of the promoter specifies the recruitment of RNA polymerase III but not II. Remarkably, in mammals the PSEs of the U1 gene (a pol II template) and the U6 gene (a pol III template) are interchangeable. However, Drosophila U1 and U6 PSEs are not interchangeable and RNA polymerase specificity is determined by a few nucleotide differences that occur within the U1 and U6 PSEs. Thus, previous studies could predict the utilization of species-specific transcription factors, and it appears that both the promoter structure and the machinery responsible for recognizing diverse RNA pol III genes may have diverged significantly from species to species (Takada, 2000).
Why does Drosophila BRF efficiently interact with Trf but not TBP? To address this question, an examination was made of the amino acid sequences of Trf, Drosophila TBP, human TBP, and yeast TBP. Several investigators have reported the important amino acids in yeast and human TBPs for pol III-specific transcription. There are no significant amino acid differences at these loci among yeast, Drosophila , and human TBPs. In contrast, 7 out of the 25 important amino acids were found to be altered in Trf (V64E, P65I, L87S, E108S, K133D, Y224M, and Y231S -- these are the residue numbers in yeast TBP). Interestingly, the K133D substitution in Trf is very similar to a reported mutation R231E in human TBP (corresponding to K133E in yeast TBP) that abolished the capacity of human TBP to bind BRF. These amino acid changes may in part explain the difference between Drosophila Trf and TBP in their capacity to interact with Drosophila BRF. It is interesting that Trf retains the functions to serve as a pol III factor, but has divergent amino acids at the relevant sites. This observation suggests that the other components involved in pol III transcription such as BRF may also have diverged significantly. Indeed, the one domain of yeast BRF involved in interacting with TBP is located in the C-terminal half of the molecule that is quite divergent between species. It will be interesting to test whether Drosophila TBP can interact with human BRF, and whether human TBP can interact with Drosophila BRF. For comparison, the amino acids of TBP important for DNA binding were checked. Out of 25 amino acids, all except one (I214F) are conserved in Trf. Thus, it appears that amino acid alterations in Trf occur preferentially in the region of the molecule important for pol III function (Takada, 2000).
Trf is differentially expressed in developing embryos (Hansen, 1997). Although high levels of expression are observed in the central nervous system (CNS) and gonads in late stage embryos, diffuse expression is detected throughout the embryo at earlier stages. In addition, some Trf is expressed in the salivary gland of larvae as well as primary spermatocytes in adult flies. Thus, it seems reasonable to conclude that Trf is expressed at some basal level in most cell types but at higher levels in specific tissues such as the CNS, brain, and reproductive organs. It has been reported that RNA pol III transcription is often upregulated in rapidly growing cells. The highest levels of RNA pol III activities are observed during the S and G2 phases of the cell cycle and have also been found to be elevated in undifferentiated cells. The observation that Trf is highly expressed in dividing cells in the embryo and during spermatogenesis may be consistent with an elevated level of RNA pol III transcription activity in these rapidly growing cells. It remains unclear why the CNS and brain express particularly high levels of Trf. However, it is interesting to note that there are reports of brain- and neuronal-specific small RNAs that are specifically transcribed by RNA pol III in mammals (Takada, 2000).
Trf was initially studied as a transcription factor involved in RNA pol II transcription, and in vitro characterization has showen its ability to bind TFIIA and IIB, and initiate transcription at promoters of protein coding genes. Trf also interacts specifically with Drosophila BRF and directs transcription by RNA pol III. Indeed, most of the Trf in Drosophila cell extracts appears to be associated with BRF, and thus only a small proportion is likely to be available for RNA pol II transcription. However, recent evidence suggests that some RNA pol II promoters contain a control element that can bind preferentially to Trf rather than TBP and is able to respond selectively to RNA pol II transcription initiation complexes directed by Trf (Holmes, 2000). Thus, it is likely that Trf can be involved in transcription by different classes of RNA polymerases. Whether Trf is also involved in RNA pol I transcription remains to be determined (Takada, 2000).
Metazoans have evolved multiple paralogues of the TATA binding protein (TBP), adding another tunable level of gene control at core promoters. While TBP-related factor 1 (TRF1) shares extensive homology with TBP and can direct both Pol II and Pol III transcription in vitro, TRF1 target sites in vivo have remained elusive. This study reports the genome-wide identification of TRF1-binding sites using high-resolution genome tiling microarrays. 354 TRF1-binding sites were found genome-wide with 78% of these sites displaying colocalization with BRF. Strikingly, the majority of TRF1 target genes are Pol III-dependent small noncoding RNAs such as tRNAs and small nonmessenger RNAs. Direct evidence is provided that the TRF1/BRF complex is functionally required for the activity of two novel TRF1 targets (7SL RNA and small nucleolar RNAs). These studies suggest that unlike most other eukaryotic organisms that rely on TBP for Pol III transcription, in Drosophila and possibly other insects the alternative TRF1/BRF complex appears responsible for the initiation of all known classes of Pol III transcription (Isogai, 2007).
This study attemts to identify at relatively high-resolution specific genome-wide binding sites for the TRF1/BRF core promoter recognition machinery in Drosophila. Previous studies used in vitro biochemical methods to identify a few TRF1 target genes; it was found that this TRF can mediate transcription from both Pol II and Pol III promoters. This observation suggested that at least in Drosophila some of the key promoter recognition functions of TBP are carried out by an alternative core promoter recognition factor TRF1. However, previous studies were hampered by technical limitations that prevented direct comparison of the in vivo role of TRF1 in Drosophila cells with in vitro observations. One problem was the resolution of TRF1 localization on polytene chromosomes that did not allow mapping accurately (10-100 kb) TRF1 target promoters in vivo. Another problem was the finding that TRF1 can drive both Pol II and Pol III-mediated transcription, thus complicating the analysis of identifying bona fide promoters subject to regulation by TRF1. Indeed, given the blunt resolution of polytene sites, one could not distinguish between multiple tRNA sites from adjacent Pol II genes with potential TRF1 target sites. This report employed a range of in vivo and in vitro assays including genome-wide ChIP-on-chip assays to obtain a more accurate and global picture of how the TRF1 factor directs promoter recognition. The present study identified ~350 sites in the Drosophila genome that are specifically targeted by TRF1, BRF or both. These data revealed that, in S2 cells, TRF1 as well as BRF are found in a majority of known Pol III gene promoters whereas Pol II promoters appear to constitute a minor proportion of TRF1 targets. It should also be noted that these classes of small noncoding RNA genes identified here generally pose a particularly difficult challenge in determining the exact binding sites using existing lower resolution tiling arrays as they are on average much smaller than Pol II transcripts. High-resolution (35 bp) oligonucleotide microarrays such as the ones used in this study provide a much more accurate mapping of protein-binding sites than ones that have been typically employed in previous studies (Isogai, 2007).
Surveying all the genomic sites identified by this study, a striking degree (77.7%) of colocalization between TRF1 and BRF was observed. This is entirely consistent with, but also significantly extending, previous biochemical study indicating that most of the TRF1 protein in Drosophila S2 cells appears to be in a complex with BRF. Among the colocalized sites, it was found that by far the most dominant class represents tRNA genes, which is consistent with in vitro studies. Remarkably, 93% of known tRNA genes in the Drosophila genome scored as TRF1/BRF targets. This result indicates that the TRF1/BRF complex in Drosophila is tightly linked to Pol III transcription, in contrast to most other eukaryotes where TBP is the core component of the TFIIIB complex. In addition, recent ChIP-on-chip analysis of Drosophila TBP confirmed that less than 1% of the Pol III genomic sites that are bound by either TRF1 or BRF are also bound by TBP, further supporting the role of TRF1, but not TBP, in Pol III transcription. Importantly, several of the other mapped sites corresponded to genes that had not been previously described as TRF1/BRF targets, including 7SL RNA, snoRNAs, and various functionally uncharacterized snmRNAs. Approximately 19% of the identified sites are occupied only by TRF1 or BRF, but not by both. These 'single-hit' sites could be due to differences in the sensitivity of the assays (i.e., variability in antibody strength) or they could reflect some aspect of TRF1 and BRF functional specificity that are not yet understood (Isogai, 2007).
To date, there have been relatively few studies characterizing snoRNA transcription in Drosophila. In yeast, it has been reported that the majority of snoRNAs are transcribed by Pol II, and only one snoRNA gene (snR52) has been identified as a Pol III target. At least two snoRNA genes are transcribed by the Pol III machinery in Drosophila, snoRNA:314 and snoRNA:644, possess independent transcriptional units that are localized to intergenic regions. By examining other snoRNA targets of the TRF1/BRF complex, it was found that five are localized to intergenic regions whereas three are embedded in the introns of Pol II genes. Therefore, it is likely that at least some of these other uncharacterized intergenic snoRNA targets are also Pol III genes. Moreover, of the two different classes of snoRNAs (box C/D type and box H/ACA type), no bias was found in the list of snoRNA targets. Thus, the chromosomal location and promoter structures, rather than specific types of snoRNAs, may be key determinants for designating the class of transcriptional machinery (Pol II or III) utilized for snoRNA genes. Indeed, these snoRNA targets contain the conserved B-box sequence, underscoring the regulation of these promoters by the Pol III transcription machinery (Isogai, 2007).
Another observation regarding snoRNA transcriptional units revealed by these studies is the apparent production of a larger primary transcript precursor that is then most likely subject to processing at its 5' end. In the latest annotation of the Drosophila genome, snoRNAs are mapped according to the size of the mature forms and therefore may not reflect their true transcriptional start sites. The in vitro transcription assays used in this study provide a powerful complementary approach to mapping the promoter regions of these snoRNAs as this assay correctly predicted the transcriptional start sites that were then confirmed in vivo by primer extension (Isogai, 2007).
Pol III promoters have been subdivided into at least two classes, gene internal (5S rRNA and tRNAs) and gene external (U6 snRNA) promoters. What then is the common structure of snoRNA gene promoters? In the yeast snR52 gene, potential A/B boxes have been mapped suggesting that a gene-internal promoter may be important. Consistent with this observation, the snoRNA:644 gene in Drosophila identified here also appears to require gene-internal elements. In addition, it was found that the bulk of the TRF1/BRF complex binds a region overlapping the transcription start site and extending well into the gene (+6), which is reminiscent of a gene-internal promoter element. Importantly, this element has substantially diverged from the typical upstream TATA box. This finding is also consistent with the observation that, unlike fungi, plants, and mammals, Drosophila Pol III genes generally lack conspicuous TATA box sequences. Thus, the core promoter recognition apparatus consisting of TRF1/BRF in insects has apparently evolved to accommodate a more diversified Pol III promoter structure utilized by Drosophila (Isogai, 2007).
Although the snoRNA:644 gene represents one type of snoRNA promoter structure, it was found that not all the snoRNAs regulated by TRF1/BRF exhibit the same type of promoter structure. In the case of snoRNA:314 gene, it appears that significant gene-external sequences and promoter elements may be necessary for transcriptional initiation as in vitro transcription experiments with promoter deletions of the snoRNA:314 template revealed that at least ~250 bp upstream of the putative transcription start site are essential for efficient initiation. This suggests that the promoter structure of snoRNA:644 gene may resemble tRNAs whereas that of snoRNA:314 is more similar to the 7SL RNA gene in plants wherein both gene-external and -internal sequence elements play a role in directing transcriptional initiation. Interestingly, under the in vitro transcription system, the snoRNA:644 template produced larger amounts of transcripts than the snoRNA:314 template. This observation appears well correlated with the ChIP-on-chip results in which the occupancy score of TRF1/BRF at the snoRNA:644 promoter is significantly higher than at the snoRNA:314 promoter, indicating that the recruitment of the TRF1/BRF complex may be a crucial step for successful initiation of transcription by Pol III. Therefore, the snoRNA:314 promoter may represent a case where the Pol III transcription machinery may be potentially directed by yet unknown DNA binding factors, allowing tight transcriptional control of these snoRNAs (Isogai, 2007).
Small nonmessenger RNAs are abundantly expressed in eukaryotic cells and thought to participate in critical cellular functions. For example, 7SL RNA is part of the signal recognition particle and snoRNAs plays an important role in guiding modification (such as pseudouridylation) of ribosomal RNAs. However, the functional roles of the majority of other snmRNAs remain to be characterized. For example, the putative TRF1/BRF target snmRNA:149 gene appears to be transcribed in the antisense direction to a protein coding gene, CG1079. One proposal is that this class of snmRNAs may play a role in the regulation of the corresponding Pol II genes via splicing or potential RNAi-like mechanisms (Isogai, 2007).
The localization of the TRF1/BRF complexes in different cell types or in different Drosophila tissues has not yet been determined. It may be particularly interesting to examine neural tissues where TRF1 was found to be prominently upregulated. It is possible that TRF1 mediates cell-type-specific transcription in these tissues. Recently, an snoRNA in humans was specifically expressed in the brain and was implicated in alternative splicing of the serotonin receptor. Such post-transcriptional RNA modification events may also occur in the central nervous system of Drosophila. Therefore, the role of the TRF1/BRF complex in snmRNA expression in S2 cells may point to a potential link between the TRF1/BRF complex and the regulation of yet to be identified brain-specific snmRNAs. It is thus tempting to speculate that the TRF1/BRF complex may have broad implications for gene regulation in the Drosophila neural system. The finding that some snoRNA promoters rely on gene-external promoter elements supports a potential tissue or developmental stage-specific expression of these snmRNA by employing additional upstream transcription factors in conjunction with TRF1/BRF (Isogai, 2007).
At least in S2 cells, the majority of the TRF1/BRF complex is found to direct the regulation of small non-coding RNA genes, most of which are transcribed by Pol III. Apparently in Drosophila and other insects, TRF1 has evolved to be responsible for initiating all the known classes of Pol III genes. This presents an interesting functional diversification in insects between TBP and TRF1 that may have implications in other organisms (Isogai, 2007).
The nutrient/target-of-rapamycin (TOR) pathway has emerged as a key regulator of tissue and organismal growth in metazoans. The signalling components of the nutrient/TOR pathway are well defined; however, the downstream effectors are less understood. This study shows that the control of RNA polymerase (Pol) III-dependent transcription is an essential target of TOR in Drosophila. TOR activity controls Pol III in growing larvae via inhibition of the repressor Maf1 and, in part, via the transcription factor Drosophila Myc (dMyc). Moreover, it was shown that loss of the Pol III factor, Brf, leads to reduced tissue and organismal growth and prevents TOR-induced cellular growth. TOR activity in the larval fat body, a tissue equivalent to vertebrate fat or liver, couples nutrition to insulin release from the brain. Accordingly, it was found that fat-specific loss of Brf phenocopies nutrient limitation and TOR inhibition, leading to decreased systemic insulin signalling and reduced organismal growth. Thus, stimulation of Pol III is a key downstream effector of TOR in the control of cellular and systemic growth (Marshall, 2012).
The TOR kinase is one of the best-established growth regulators. In virtually all animals, TOR activity can be stimulated by extracellular cues such as growth factors, nutrients and oxygen to control cell, tissue and organismal growth (Marshall, 2012).
Despite the knowledge of the signalling inputs to TOR, little is known about the mechanisms that allow TOR to modulate cell metabolism and drive growth. Most studies on metabolic functions modulated by TOR have been confined to yeast and mammalian cell culture. These studies have been important in defining roles for TOR in protein synthesis, nutrient uptake and metabolism and autophagy. But they leave open the question of what mechanisms operate in vivo to control tissue and organ growth during animal development. Genetic studies in Drosophila have been pivotal in this regard. This study shows that the ability of the TOR pathway to control transcription through Pol III governs cell, tissue and ultimately organismal growth in Drosophila. Given that Pol III drives transcription of several non-coding RNAs required for mRNA translation, it is suggested that the stimulation of Pol III by TOR enhances the protein synthetic capacity of cells. Previous study have shown that Drosophila TOR also controls synthesis of rRNA synthesis, via the RNA polymerase I factor, TIF-IA (Grewal, 2007
The Pol III transcription factor Brf has been shown to be an essential component of the TFIIIB complex responsible for recruiting Pol III to gene promoters. This work indicates that Brf activity is required for Drosophila development. Patterning and cell fate specification appear normal in brf embryos. However, once these mutants hatch as larvae they fail to grow. The data suggest that this growth arrest phenotype reflects a role for Brf activity downstream of TOR. Brf was found to be cell-autonomously required for growth in both endoreplicating cells, which make up the bulk of larval mass, and the mitotically dividing cells of the imaginal discs. In particular, brf mutant wing disc cell clones were found to be outcompeted by wild-type neighbours. This cell competition phenotype is seen in mutants for other genes required for protein synthesis, such as the ribosomal proteins and Myc. An important finding was that the overgrowth caused by loss of TSC1 (and hence increased TOR activity) was blocked in brf mutant cells. In mammalian cells, Brf activity is induced by cues that promote cell growth (e.g., during hypertrophic growth of cardiac cells) whereas cell differentiation leads to inhibition of Brf. In fact, overexpression of Brf alone can promote proliferation and transformation in immortalized fibroblasts. Mutations in tumour suppressors such as TSC are common in cancer and lead to elevated TOR activity and promotion of tumour growth. Based on the current data, it is suggested that Brf is required in vivo for both normal tissue growth and TOR-induced tumour growth (Marshall, 2012).
This study found that the predominant mechanism by which nutrition/TOR controls Pol III is via Maf1 repression, since Maf1 inhibition completely reverses the decrease in tRNA synthesis caused by reducing TOR activity. These findings extend those observed in both yeast and mammalian cell culture, and suggest an important role for dMaf1 in vivo in developing tissues. The exact mechanism by which Maf1 functions is not clear, but it may involve inhibition of Brf and Pol III recruitment to genes, possibly by direct binding or association with Brf/Pol III. Indeed, an enhanced association was seen between dMaf1 and Brf1 upon TOR inhibition. The role of dMyc was explored as a potential link between nutrient-TOR signalling and Pol III. dMyc was found to be both necessary and sufficient for the control of Pol III activity during development. As previously reported in both mammalian and Drosophila culture, it was possible to identify an interaction between dMyc and Brf (Gomez-Roman, 2003; Steiger, 2008). In addition, a role has been identified for dMyc in controlling the levels of components of the Pol III machinery, including both Trf and Brf which form part of the TFIIIB complex. Thus, dMyc likely has both direct and indirect effects on Pol III activity in Drosophila. These effects are necessary for both dMyc-induced cell growth (Steiger, 2008) and, as is shown in this study, for the non-autonomous increases in body size caused by dMyc in fat cells. Previous studies have shown that, in Drosophila, TOR controls Myc protein levels. But these effects on Myc probably do not play major role in how TOR activates Pol III since the data show that, unlike inhibition of Maf1, maintaining Myc levels and activity cannot reverse the decrease in tRNA synthesis caused by TOR inhibition. Moreover, if Myc protein levels were limiting for TOR-dependent control of Pol III, then it would not be expected that knockdown of Maf1 could completely reverse the effects of rapamycin/starvation. Given that Maf1 inhibition did not influence levels of Pol III factors, pre-rRNA or RP gene mRNA—transcripts that are upregulated by dMyc—it is unlikely that Maf1 influences Myc function. It was found that rapamycin feeding could not exacerbate the reduction of tRNA levels seen in dMyc null mutants. This result in principle may suggest that TOR signalling does not exert any dMyc-independent effects on Pol III function. But, it is suggested that this finding probably occurs because in the absence of Myc, Pol III activity may be approaching basal levels and cannot be significantly decreased much further. Taken together, although these data may not completely rule out some contribution of Myc to TOR-dependent control of Pol III, they do indicate that it is not the major contributor (Marshall, 2012).
It is clear that both TOR and Myc are essential regulators of Pol III. But, it is likely that while TOR can control Myc levels, both TOR and Myc can also function in parallel and independently of each other. Previous studies have shown that overactivation of TOR signalling could not promote growth when Myc was inhibited, but at the same time Myc overexpression could not promote growth when TOR was inhibited. These findings and the current data suggest that TOR and Myc cannot necessarily be placed in a simple, linear pathway. Recent studies in Drosophila have emphasized how other conserved growth-regulatory pathways, particularly those that control growth of the imaginal tissues (such as Wingless, EGF/Ras, the Hippo-Yorkie pathway and Bantam RNAi) function via control of dMyc. Thus, dMyc may play a role in coupling these pathways to the control of Pol III activity to stimulate cell growth and proliferation (Marshall, 2012).
It is interesting to speculate as to which Pol III targets are important for growth control. Pol III regulates the expression of several short non-coding RNAs, such as the tRNAs, 5S rRNA and 7SL RNA. Regulation of 5S rRNA production by Brf could influence ribosome synthesis and hence growth. However, it was found that loss of Brf did not inhibit Pol I activity or alter levels of rRNA, suggesting that Brf probably does not directly influence ribosome numbers. One attractive possibility is that levels of the tRNAs may be limiting for translation and growth. In support of this notion, a recent paper showed that overexpression of Brf increased tRNA levels and promoted proliferation and transformation of cultured mammalian fibroblasts (Marshall, 2008). These effects of Brf were phenocopied by just increasing levels of tRNAiMet, and were associated with augmented mRNA translation and increased protein levels of growth promoters such as c-Myc and cyclin D1. No consistent increase was seen in tRNAs when Brf was overexpressed in larvae, perhaps because levels of other components of the TFIIIB complex are limiting in flies. Nevertheless, by controlling Brf activity and tRNA synthesis, TOR could promote translation of growth regulators and drive larval growth. In fact, a recent paper (Teleman, 2008) indicated that TOR signalling in Drosophila regulates dMyc protein levels, but not dMyc mRNA levels, consistent with a possible role for translational control (Marshall, 2012).
One interesting result of this work was the identification of a non-cell autonomous role for Brf in organismal growth. Specifically, it was found that Brf activity in the fat cells of Drosophila larvae could influence larval growth and final size. A role for TOR in the fat body has been shown to exist as a relay to control peripheral insulin signalling. In feeding larvae, amino-acid input into fat cells activates TOR, leading to transmission of a secreted signal from fat to brain to increase dILP expression and release from brain IPCs. These data suggest that stimulation of Pol III activity may be an important downstream effector of this adipose function of TOR. Thus, adipose-specific silencing of Brf led to reduced peripheral insulin signalling, slower larval growth rate and reduced final body size. As in starved larvae, this study found that loss of brf led to reduced expression of dilp mRNA (seen in both brf mutants and cg>brf RNAi larvae) and reduced dILP release from the brain. Moreover, given that levels of phospho-Akt are lower, and levels of dInR (a FOXO target) are higher in tissues from both brf mutant and r4>brf RNAi larvae it is clear that systemic insulin signalling is reduced when Brf is inhibited in the fat body. This study also found that another fat phenotype associated with starvation and loss of TOR, accumulation of lipid droplets, was phenocopied by loss of Brf. However, the autophagy phenotype of starved larval fat bodies was not phenocopied by loss of Brf. Therefore, Brf and Pol III function in the Drosophila fat body may mediate some, but not all of TOR's effects on growth and metabolism. The exact nature of the fat-to-brain secreted factor that controls insulin release in flies is not yet known, but perhaps translation of this signal, if it is a peptide or secreted protein, is influenced by changes in tRNA synthesis and translation rates. Indeed, it has been shown that dMyc activity in the fat body was also important for controlling systemic insulin signalling, growth and body size. This effect of dMyc correlated with elevated expression of ribosome biogenesis genes and increased nucleolar size, an index of ribosome synthesis. dMyc overexpression can also stimulate Pol III and tRNA levels, and the increase in body size caused by fat body overexpression of dMyc is reversed by knockdown of Brf. These data suggest that regulation of mRNA translational capacity is a key step downstream of TOR and dMyc in fat cells to control signalling to IPCs (Marshall, 2012).
Together, these data suggest that mRNA translational control may underlie a role for the fat body as an endocrine organ. A similar theme is emerging in mouse models. Mammalian adipose tissue is known to secrete adipokines and leptin to influence organismal metabolism and growth. The secretion of many of these factors is influenced by diet, suggesting a regulatory role for TOR signalling. Genetic inhibition of either TOR and S6K in mice leads to alterations in metabolic activity in adipose tissue. Moreover, loss of the translational repressors, 4E-BP1 and 4E-BP2, both of which are downstream TOR effectors, alters lipid and glucose metabolism in mice. To date, there are no mouse models of Pol III. However, it is interesting to speculate that changes in Pol III and tRNA synthesis are involved in mediating effects of TOR in adipose tissue in mice. Regulation of Pol III by TOR may also be important in the metabolic control of other processes. For example, TOR is a conserved regulator of organismal stress responses and lifespan. These stress responses rely on TOR's ability to control translation. It is suggested that regulation of Pol III and tRNA synthesis may also be a mode of control. Further organismal studies, using genetic modulation of Pol III function, should provide additional insights into these points (Marshall, 2012).
Search PubMed for articles about Drosophila Brf
Crowley, T. E., et al. (1993). A new factor related to TATA-binding protein has highly restricted expression patterns in Drosophila. Nature 361(6412): 557-561. PubMed citation: 8429912
Gomez-Roman, N., Grandori, C., Eisenman, R. N. and White, R. J. (2003). Direct activation of RNA polymerase III transcription by c-Myc. Nature 421(6920): 290-4. PubMed ID: 12529648
Grewal, S. S., Evans, J. R. and Edgar, B. A (2007). Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway. J. Cell Biol. 179: 1105-1113. PubMed ID: 18086911
Grewal, S. S. (2009). Insulin/TOR signaling in growth and homeostasis: a view from the fly world. Int J Biochem Cell Biol 41: 1006-1010. PubMed ID: 18992839
Hansen, S. K., et al. (1997). Transcription properties of a cell type-specific TATA-binding protein, TRF. Cell 91(1): 71-83. PubMed citation: 9335336
Holmes, M. C. and Tjian, R. (2000). Promoter-selective properties of the TBP-related factor TRF1. Science 288(5467): 867-70. PubMed citation: 10797011
Isogai, Y., Takada, S., Tjian, R. and Keles, S. (2007). Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription. EMBO J. 26(1): 79-89. PubMed citation: 17170711
Marshall L. (2008). Elevated RNA polymerase III transcription drives proliferation and oncogenic transformation. Cell Cycle 7(21): 3327-9. PubMed ID: 18971635
Marshall, L., Rideout, E. J. and Grewal, S. S. (2012). Nutrient/TOR-dependent regulation of RNA polymerase III controls tissue and organismal growth in Drosophila. EMBO J. [Epub ahead of print]. PubMed ID: 22367393
Steiger, D., Furrer, M., Schwinkendorf, D. and Gallant, P. (2008). Max-independent functions of Myc in Drosophila melanogaster. Nat. Genet. 40(9): 1084-1091. PubMed ID: 19165923
Takada, S., et al. (2000). A TRF1:BRF complex directs Drosophila RNA polymerase III transcription. Cell 101: 459-469. PubMed citation: 10850489
date revised: 20 April 2012
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