The experiments described in this report highlight a fundamental role for a T. brucei TBP-related factor (TRF4) in transcription mediated by Pol I, Pol II, and Pol III. Although we cannot discount the possibility that another TBP-related gene exists in the trypanosome genome, we think this is highly unlikely for the following reasons. First, the coverage of the trypanosomatid genome is quite extensive and includes not only three finished trypanosomatid protozoan genomes, namely, T. brucei, L. major, and T. cruzi, but also considerable coverage of the genomes of two other African trypanosomes, which are closely related to T. brucei, namely, Trypanosoma congolense and Trypanosoma vivax. Thus, we think it is significant that we could identify only TRF4 in the combined genomic information of the family Trypanosomatidae. Second, by performing low-stringency Southern hybridization and PCR amplification with degenerate oligonucleotides, we did not obtain evidence for the presence of TBP or an additional TBP-related factor (unpublished data).
For Pol I, the ChIP experiments placed TRF4 at the promoter region of the developmentally regulated PARP genes, but interestingly, we could not detect TRF4 at the promoters of the rRNA genes. The latter result was already apparent in RNAi experiments, where downregulation of TRF4 did not have a noticeable effect on rRNA gene transcription (Fig. ). One concept emerging from these observations is that the composition of the transcriptional machineries assembling on these two promoters is distinct. Surveying the current T. brucei
database did not produce candidate factors that could be tested by ChIP for their involvement in Pol I transcription (C. Tschudi, unpublished data). Thus, resolution of this intriguing possibility will have to come from a biochemical analysis of the available in vitro transcription system (23
To assess whether TRF4 is involved in Pol II-dependent transcription in T. brucei
, we chose the SL RNA gene, which remains the only Pol II unit in trypanosomatids characterized in some detail (8
). Initial experiments in a variety of systems identified two regulatory elements by mutational analysis: one close to the transcription start site and the other located between 60 and 80 bp upstream (35
). Elegant biochemical experiments in V. Bellofatto's laboratory have extended these studies to isolate a transcription factor, referred to as PBP-1, which specifically binds to the upstream element (8
). This factor is composed of three polypeptides, and one of these proteins, with a molecular mass of 57 kDa, is an orthologue of the human 50-kDa subunit of SNAPc
, also known as PBP or PTF. SNAPc
binds to the proximal sequence element of Pol II- and Pol III-transcribed snRNA genes. Thus, the transcriptional machinery assembling on the SL RNA promoter appears to resemble snRNA transcription in higher eukaryotes. It is well established that TBP is required for both Pol II- and Pol III-transcribed snRNA genes (14
). The results of our experiments presented here showed that TRF4 is recruited to the SL RNA genes and thus is involved in the transcription of these genes. This result raises the intriguing possibility that TRF4 has taken the place of TBP in the assembly of a transcription initiation complex on the SL RNA genes. At this time, we do not know whether TRF4 recognizes SL RNA promoter elements directly or whether it is recruited by interaction with other component(s) of the transcriptional machinery, such as PBP-1. Nevertheless, since several key residues, including two of the four highly conserved phenylalanines, known to interact with the TATA box are replaced in the T. brucei
TRF4 (Fig. ), it is likely that TRF4 does not bind to a canonical TATA box. Indeed, studies of a dinoflagellate TBP-like protein, in which all four phenylalanines have been replaced, revealed that a TTTT box was a better binding substrate than a TATA box (12
). So far, we have not succeeded in producing T. brucei
TRF4 in a soluble recombinant form to test its binding to DNA.
We have also shown that TRF4 is recruited to the Pol III-transcribed U-snRNA and 7SL RNA genes. The regulatory elements of these genes are unusual in that a gene internal element acts in concert with upstream A and B boxes located in a divergently oriented tRNA gene (10
). At this time, TRF4 is the only known transcription factor involved in Pol III-mediated transcription in trypanosomatids. Similar to the SL RNA genes, we do not know whether TRF4 binds directly to DNA in the U-snRNA or 7SL RNA promoters or whether the observed recruitment is mediated by auxiliary factors.
Having established that T. brucei
TRF4 binds to promoters mediating transcription by all three nuclear RNA polymerases, we went on to ask whether TRF4 is also involved in Pol II transcription of protein-coding genes. Genome sequencing projects have underscored that protein-coding genes in trypanosomatids are organized in large directional clusters that most likely give rise to polycistronic pre-mRNAs (9
). Although the genome data also exposed putative Pol II promoters at points of diverging clusters, experimental evidence for the existence of promoters in such regions is restricted to a single report (26
). In addition, at this time Pol II is the only known component of the machinery transcribing protein-coding genes in trypanosomes.
Due to the apparent absence of a bona fide TBP in the kinetoplastid databases, we hypothesized that TRF4 might lead us to putative transcription initiation sites by Pol II. Thus, we took a limited global approach and searched for regions in the genome that recruit both Pol II and TRF4. Using ChIP DNA to screen a genomic phage library, we indeed uncovered 27 regions in the T. brucei genome that bind Pol II and TRF4. As predicted, a subset of the positive results corresponded to the SL RNA gene. The regions in the remaining 10 isolates were all mapped to clusters of protein-coding regions.
The unexpected result came when we realized that Pol II and TRF4 were binding to sequences near the 3′ ends of genes. Although perplexing at first, very recent studies in S. cerevisiae
and humans have uncovered scenarios, where transcription can initiate within coding regions. In one study, a mutation in Spt6, believed to be involved in transcription elongation, mRNA processing, and interaction with nucleosomes resulted in aberrant transcription initiation within coding regions (19
). In a second report, conditional inactivation of the Spt16 subunit of the FACT complex (named FACT for facilitates chromatin transcription) increased Pol II density, transcription, and TBP recruitment in the 3′ portion of certain genes (27
). Taken together, these observations were interpreted to mean that these factors contribute to the fidelity of Pol II transcription by repressing initiation at cryptic promoters. In both studies, transcription initiation at these cryptic sites generated a stable poly(A)-containing transcript.
In humans, the mapping of the binding sites for transcription factors Sp1, c-Myc, and p53 on chromosomes 21 and 22 uncovered a surprising number of sites (36%) lying within or near the 3′ ends of genes (4
). Furthermore, these regions produce stable noncoding RNAs, some of them overlapping with protein-coding transcripts. This is quite different from our results, since we have no evidence for the production of stable RNAs initiating at the 3′ ends of the Ptr1 and TR genes. One explanation for this difference could be that Pol II elongation is somehow arrested at these two loci, as evidenced by the synthesis of short nascent transcripts. It is worth pointing out that the Pol II, as well as TRF4, density at the 3′ ends of the Ptr1 and TR genes is extraordinarily high, compared to the surrounding coding regions, which remained at background levels and thus could not be measured (Fig. and data not shown). The significance of this pileup of Pol II and TRF4 is not clear at this time, but it would be interesting to test whether the recruitment of Pol II and TRF4 to these 3′-end regions can be manipulated, for instance by downregulating subunits of the T. brucei
FACT complex. Nevertheless, our results highlight once more that Pol II transcription of protein-coding genes in these organisms is highly unusual and far from understood.