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

Abstract

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. Here, we report the genome-wide identification of TRF1-binding sites using high-resolution genome tiling microarrays. We found 354 TRF1-binding sites genome-wide with B78% of these sites displaying colocalization with BRF. Strikingly, the majority of TRF1 target genes are Pol IIIdependent small noncoding RNAs such as tRNAs and small nonmessenger RNAs. We provide direct evidence that the TRF1/BRF complex is functionally required for the activity of two novel TRF1 targets (7SL RNA and small nucleolar RNAs). Our 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.

Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters

“…2B, lane 9), but depletion of TBP had no discernable effect (compare lanes 7 and 8). This agrees with findings by others that TRF1 is required for tRNA transcription in D. melanogaster (17,18). Altogether, these results, along with the ChIP results shown in Fig.…”

“…In contrast, however, our data revealed that TBP is significantly more enriched than TRF1 at U6 promoters. These latter findings are consistent with the failure to detect TRF1 at U6 promoters in genome-wide ChIP-on-chip experiments performed by Isogai et al (18). Furthermore, immunodepletion of TBP (but not TRF1) from a nuclear extract greatly inhibited U6 transcription in vitro (Fig.…”

“…Finally, in vitro transcription experiments with plasmid templates containing 5 S rRNA, tRNA, or U6 snRNA genes led the authors to conclude that TRF1 was responsible for transcription from all three major types of Pol III promoters in D. melanogaster (17). This notion has since become widely accepted in the scientific literature (10,13,18).…”

TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription

“…10). TRF1, which is largely involved in RNA polymerase III transcription in Drosophila (Takada et al 2000;Isogai et al 2007b), has little or no effect on transcription of DPE-containing or TATA-containing genes. TRF2 is important for both DPE-mediated and TATA-mediated transcription.…”

Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells

“…At all these genes, binding of Pol III correlates with gene activity as measured by RNA levels. In Drosophila Schneider line 2 (S2) cells, a recent analysis has identified 359 BRF1 and 354 TRF1 (which replaces TBP in Drosophila TFIIIB) genomic binding sites, corresponding mostly to tRNA genes, as well as to other RNA genes such as the RN7SL1 (7SL) RNA gene and some snoRNA genes (Isogai et al 2007). In mammalian cells, genomewide studies have been limited by the lack of a technology to study repeated sequences.…”