TBP-related factor

Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription

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).

Protein Interactions

Trf can interact directly with TFIIA and TFIIB. While Tbp alone binds very poorly to the adenovirus major late (AdML) TATA box, binding is strongly enhance by the addition fo TFIIB. In contrast to Tbp, Trf alone binds reasonably well to the AdML TATA box; yet, like Tbp, its DNA binding is greatly enhanced by the addition of TFIIB. TFIIA can also strongly stimulate the DNA-binding activity of Trf, probably by stabilizing the Trf-DNA complex. Trf can substitute for Tbp by directly initiating transcription from an adenovirus E1B promoter containing a TATA box. A complete transcription reaction mixture including Trf, TFIIA, TFIIB, TFIIE, TFIIF, TFIIH and RNA polymerase II was prepared. Omission of TFIIA results in a modest decrease in activity. By contrast, transcription is abolished by removal of TFIIB, TFIIE, TFIIF, or RNA polymerase II, while removal of TFIIH results in a residual low level of activity (Hansen, 1997).

Trf can mediate activated transcription. Cotransfection of NTF-1 (also known as Grainy head) and Trf results in a 5-fold activation of a TATA-containing appropriate promoter. Tbp can also serve this function. A Trf with altered DNA binding specificity fails to activate such a promoter, but modification of the TATA box to a form that can bind altered Trf restores activation of potential to altered Trf. Trf fails to function as a general mediator of transcription. For example, Trf fails to stimulate activation by the Sp1A activation domain, while Tbp strongly potentiates activation. Thus the ability of Trf to support transcriptional activation is not general but instead appears to be activator-specific (Hansen, 1997).

Trf is present in a large protein complex that does not contain the ubiquitous TAF subunits found associated with Tbp. The core subunit of TFIID, Tbp-associated factor 250kD, is absent from the Trf complex, as are TAF150, TAF110, TAF80, TAF60, TAF40, TAF30a and TAF30b, all of which serve as TAFs that make up TFIID. Seven polypeptides were identified, ranging in molecular weight from 35 to 180 kDa that reproducibly coprecipitate and elute from anti-Trf affinity resins. This Trf/neuralTAF (nTAF) complex can serve to activate transcription. It is speculated that, like the ubiquitous TAFs, neural TAFs may help direct TRF to a specific promoter and mediate activation by a select subset of enhancer binding factors (Hansen, 1997).

A TRF1:BRF complex directs Drosophila RNA polymerase III transcription

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. B-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).

TRF1 was initially studied as a transcription factor involved in RNA pol II transcription, and in vitro characterization showed its ability to bind TFIIA and IIB, and initiate transcription at promoters of protein coding genes. This study shows that TRF1 can also interact specifically with Drosophila BRF and direct transcription by RNA pol III. Indeed, most of the TRF1 (~90%) 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 TRF1 rather than TBP and is able to respond selectively to RNA pol II transcription initiation complexes directed by TRF1. Thus, it is likely that TRF1 can be involved in transcription by different classes of RNA polymerases. Whether TRF1 is also involved in RNA pol I transcription remains to be determined (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 does not detectably affect pol III transcription of any templates tested, although it does abrogate the pol II transcription from the Adh promoter. In contrast, treatment with anti-TRF1 severely impairs or eliminates transcription by pol III. As expected, depletion of TRF1 had little or no effect on transcription by pol II from the Adh promoter. These results suggest that Drosophila TRF1 but not TBP is required for pol III transcription, at least in an in vitro assay (Takada, 2000).

In parallel with in vitro transcription studies, attempts were made to isolate and characterize proteins that are associated with Trf and are thus possibly involved in directing pol III transcription. The native molecular weight of the major Trf species in crude Schneider cell extracts is about 200 kDa estimated by gel-filtration analyses, and is the same as that of Trf from embryo extracts. Trf from crude Schneider cell extracts is found to consistently coimmunoprecipitate polypeptides of 90, 70, and 50 kDa. Microsequence analyses of these individual polypeptides reveals that the 90 kDa species contains a peptide sequence highly homologous to the C. elegans BRF gene product (named for its relatedness to TFIIB). The 70 and 50 kDa polypeptides have been identified as HSP70 and EF1alpha (a translation elongation factor), two relatively abundant proteins in Drosophila extracts that are likely contaminants in the immunoprecipitates used (Takada, 2000).

Amino acid sequence comparison reveals that the 90 kDa protein associated with Trf is closely related to BRF, a well-characterized RNA pol III transcription factor that had previously not been identified from Drosophila. To further investigate the association of the newly isolated BRF molecule with Trf in Drosophila cells, the specificity of the interaction between endogenous Trf and BRF was determined. Immunoprecipitates generated using a polyclonal antibody were analyzed for the presence of Trf, BRF, and TBP by immunoblotting. As expected, anti-Trf precipitates Trf and coprecipitates BRF efficiently. Likewise, anti-BRF coprecipitates Trf and BRF efficiently. In contrast, no TBP was detected in either the anti-BRF or anti-Trf immunoprecipitates. Similar coprecipitation results were obtained using extracts derived from Drosophila embryos. Thus, Trf and BRF polypeptides display a high propensity to interact with one another to form a stable complex, possibly a heterotetramer (predicted mass of 198,319 Da). The molecular weight of this Trf:BRF complex is similar to a TFIIIB activity that has been characterized and partially purified from Drosophila extracts (Takada, 2000).

If BRF and Trf 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 Trf distribution on salivary gland polytene chromosomes. The BRF antibody shows prominent staining of a set of about 70 chromosomal loci. Most sites labeled with anti-BRF are also stained with anti-Trf, though the ratio of the two signals varies at each locus. Major sites of labeling by anti-Trf antibodies are known to contain pol III genes. While the overlap of Trf and BRF on chromosomes is extensive, a number of sites appear to be labeled only by anti-Trf. This is consistent with the notion that Trf may also function as a pol II transcription factor for a subset of genes (Takada, 2000).

Since Trf, like TBP, may be part of different complexes, attempts were made to determine what proportion of Trf is associated with BRF in Drosophila cells. Either Schneider cell or embryo extracts were immunodepleted of TBP, Trf, or BRF and then the levels of these proteins in the supernatants were examined. After treating extracts with anti-BRF antibodies, most of the Trf (greater that 90% as determined by quantitative Westerns) was found to be codepleted with BRF from the extract. Likewise, treatment of extracts with anti-Trf removes the majority of the BRF from the Schneider cell supernatant (Takada, 2000).

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 et al., 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).

DEVELOPMENTAL BIOLOGY

Embryonic

Trf protein is present throughout most of the embryo until developmental stage 13, when Trf staining is particularly intense in the brain lobe and the ventral cord. At stages 14 and 15, Trf staining decreases dramatically in most cells except the brain lobe, ventral cord, and gonads. Within the CNS, certain regions show particularly strong staining. These include cells in the RP cluster and the anterior corner cells (aCC), both of which form motor neurons. At stage 17, expression in RP neurons is still evident, and strong staining is seen in lateral neuronal cell bodies (LN) in positions typical of motor neurons. By contrast, the longitudinal tracts (LT) that contain large axon bundles as well as the anterior commissures and posterior commissures only show weak staining. Little or no Trf is found in glial cells (Hansen, 1997).

Larval

TRF is present as a constituent of larval salivary gland polytene chromosomes, revealing information about potential TRF target gene selectivity. TRF is found in 17 out of up to 600 resolvable sites, suggesting that TRF is localized in a highly gene-specific pattern at the same time that Tbp is present almost ubiquitously. The identified sites have neural or gonadal functions. For example, 63AB contains the shaker cognate b (shab) gene that encodes a potassium channel. This finding is consistent with the observation that trf mutant flies display a leg shaking phenotype, generally attributed to abnormalities in potassium channel function. TRF also associates with the location of quiver, which is also associated with a shaker phenotype. The genes maleless/no action potential exhibit Trf association as well as a number of chromosomal sites that contain one or more tRNA genes. These data suggests that Trf, like Tbp may also play a role in RNA polymerase III transcription, at least in the salivary gland. It would not be surprising to find that in mammals the number of Tbp-related genes and the complexity of TAF family members is even more extensive than the current limited analysis has revealed (Hansen, 1997).

Effects of mutation or deletion

TBP-related factor (Trf) was discovered in a genetic screen for mutant flies that show leg shaking under ether anesthesia, a phenotype that is attributed to defects in nervous system function. trf mutant flies are also male sterile (Crowley, 1993).

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date revised: 10 April 2008 

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