Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription
2006; Springer Nature; Volume: 26; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7601448
ISSN1460-2075
AutoresYoh Isogai, Shinako Takada, Robert Tjian, Sündüz Keleş,
Tópico(s)Chromosomal and Genetic Variations
ResumoArticle14 December 2006free access Novel TRF1/BRF target genes revealed by genome-wide analysis of Drosophila Pol III transcription Yoh Isogai Yoh Isogai Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Shinako Takada Shinako Takada Department of Biochemistry and Molecular Biology, Gene and Development, Program of Graduate School of Biomedical Science, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Robert Tjian Corresponding Author Robert Tjian Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Howard Hughes Medical Institute, UC Berkeley, Department of Molecular and Cell Biology, Berkeley, CA, USA Search for more papers by this author Sündüz Keleş Sündüz Keleş Departments of Statistics and Biostatistics and Medical Informatics, University of Wisconsin, Madison, WI, USA Search for more papers by this author Yoh Isogai Yoh Isogai Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Shinako Takada Shinako Takada Department of Biochemistry and Molecular Biology, Gene and Development, Program of Graduate School of Biomedical Science, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Robert Tjian Corresponding Author Robert Tjian Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Howard Hughes Medical Institute, UC Berkeley, Department of Molecular and Cell Biology, Berkeley, CA, USA Search for more papers by this author Sündüz Keleş Sündüz Keleş Departments of Statistics and Biostatistics and Medical Informatics, University of Wisconsin, Madison, WI, USA Search for more papers by this author Author Information Yoh Isogai1, Shinako Takada2, Robert Tjian 1,3 and Sündüz Keleş4 1Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA 2Department of Biochemistry and Molecular Biology, Gene and Development, Program of Graduate School of Biomedical Science, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA 3Howard Hughes Medical Institute, UC Berkeley, Department of Molecular and Cell Biology, Berkeley, CA, USA 4Departments of Statistics and Biostatistics and Medical Informatics, University of Wisconsin, Madison, WI, USA *Corresponding author. Department of Molecular and Cell Biology, University of California, Berkeley/HHMI, 16 Barker Hall, Berkeley, CA 94720-3204, USA. Tel.: +1 510 642 0884; Fax: +1 510 643 9547; E-mail: [email protected] The EMBO Journal (2007)26:79-89https://doi.org/10.1038/sj.emboj.7601448 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 ∼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. 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. Introduction It is essential for multicellular organisms to support a complex network of gene expression patterns that are highly regulated during development and responsive to a variety of physiological stimuli. Under the current working hypothesis of eukaryotic transcriptional initiation, mechanisms that support the specificity of transcriptional activation are largely attributed to enhancer–core-promoter interactions. The protein machinery that enables these interactions includes sequence-specific enhancer DNA-binding proteins and the core-promoter recognition machinery, which is generally believed to be universal at all promoters and largely invariant. However, a number of recent studies have revealed that there are alternatives of core-promoter complexes that have evolved in multicellular organisms. It appears that mixing and matching these two classes of transcriptional factors represent a significant mechanism by which activation and repression at specific promoters are achieved. Although activators that direct specific transcriptional responses have been amply documented, functions of alternative core-promoter recognition complexes remained poorly understood. Diversified core-promoter machinery such as variant TFIID and TATA binding protein (TBP)-related factors (TRFs) are found in many metazoan species (Hochheimer and Tjian, 2003), but it remains unclear how these alternative core-promoter recognition complexes contribute to mechanisms of transcriptional regulation. Early studies addressing individual genes have been conducted largely using in vitro biochemical approaches to dissect the role of these alternative core-promoter recognition factors (Hansen et al, 1997; Holmes and Tjian, 2000; Takada et al, 2000; Hochheimer et al, 2002). However, a bottleneck to progress further has been the identification of genome-wide in vivo targets for these factors. Here, we describe the use of chromatin immunoprecipitation (ChIP) assays combined with genome tiling microarrays (ChIP-on-chip) coupled with a new computational tool to more accurately identify, in an unbiased manner, genome-wide targets of core-promoter recognition factors. To test the usefulness of this strategy, we have applied this methodology to the mapping of specific promoters targeted by the TRF1/BRF complex. TRF1 represents a unique class of TRF found in insect species such as Drosophila and Anopheles. TRF1 is ubiquitously expressed, although it is upregulated in the central nervous system during embryogenesis and in primary spermatocytes in adults (Crowley et al, 1993; Hansen et al, 1997). Extensive sequence conservation between TBP and TRF1 is found within the core DNA-binding domains, whereas significant divergence is seen in the N-terminal domain. Previously, in vitro biochemical approaches established that TRF1 is likely involved in transcription of both Pol II and Pol III genes (Hansen et al, 1997; Holmes and Tjian, 2000). Importantly, a majority of the TRF1 in Drosophila appears to form a complex with BRF (Takada et al, 2000). In vitro transcription assays revealed that the TRF1/BRF complex plays a critical role in the transcription of several tRNA, 5S rRNA and U6 snRNA genes. Salivary gland polytene chromosome staining suggested that TRF1 can occupy a few hundred genomic sites, the majority of which are co-occupied by BRF (Takada et al, 2000). Our findings also suggested that the TRF1/BRF complex at these few promoters displayed an apparent dominance over TBP-containing complexes. To gain a more comprehensive of the transcriptional role played by TRF1, it would be advantageous to decipher the potential utilization of TRF1 on a genome-wide scale. In particular, we hoped to discover how TRF1 might be utilized for transcription mediated by different types of RNA polymerases. However, inherent limitations of resolution have precluded the analysis of polytene chromosome staining as a means to unambiguously identify specific promoters recognized and regulated by TRF1/BRF. Here, we report the higher resolution and genome-wide mapping of TRF1/BRF-binding sites using ChIP-on-chip assays. Using this experimental platform, we obtained a high-resolution (35 bp) in vivo map of TRF1- and BRF-binding sites throughout the Drosophila genome. Consistent with our previous in vitro biochemical findings, a major class of TRF1/BRF targets represents Pol III genes such as tRNAs. A small percentage of sites bound by TRF1 were mapped to Pol II promoters. In addition, we report two new classes of TRF1/BRF targets, 7SL RNA and small nucleolar RNAs (snoRNAs), which are small nonmessenger RNAs (snmRNAs). In vitro transcription assays were used to verify that the TRF1/BRF complex is functionally required for accurate transcription initiation of these new target genes. Taken together, these results strongly support a global role of the TRF1/BRF complex in Drosophila Pol III transcription. Results Genome-wide colocalization of TRF1 and BRF at noncoding small RNA promoters In order to determine high-resolution in vivo target genes of the TRF1/BRF complex, we performed ChIP-on-chip analyses using Drosophila genome tiling arrays (Affymetrix). This high-density oligonucleotide array covers the entire genome of Drosophila melanogaster at 35 bp resolution with the notable exception of repeat regions such as transposons and 28S and 5S rRNA genes. We first established robust ChIP assays using affinity-purified anti-TRF1 and anti-BRF antibodies that efficiently co-precipitate specific genomic fragments such as 5S rRNA and tRNA genes. These few genes had previously been characterized as targets of the TRF1/BRF complex in vitro and are typically precipitated by the specific antibodies at a level 20- to 100-fold above nonspecific IgG controls (Figure 2A). These co-precipitated genomic fragments were amplified and subsequently hybridized to the microarrays in duplicate. The data were extensively analysed using a newly developed statistical platform (Tiling Hierarchical Gamma Mixture Model, TileHGMM). This statistical approach explicitly modeled binding of the probes in the control sample and TRF1/BRF-enriched samples (Figure 1A). The fitting of this statistical model provided us with probabilities of binding that is specific to a genomic region of interest. We then identified TRF1- and BRF-bound regions by thresholding these probabilities while controlling the false discovery rate using a false discovery rate calculation (Newton et al, 2004). Initially, 215 genomic regions (2 kb on average) were identified as bound by TRF1 and 211 by BRF. Some of the genomic regions contained multiple peaks indicative of multiple target sites, which were identified as part of postprocessing by calculating the odds of binding according to our statistical model (Supplementary data). Overall, we identified 354 binding sites for TRF1 and 359 binding sites for BRF, which appear largely colocalized and uniformly distributed on each chromosome (Figure 1B and C). Figure 1.Overview of ChIP-on-chip data analysis. (A) TileHGMM: a statistical framework for the analysis of Chip-on-chip data. Our pipeline for the Chip-on-chip data analysis involves: (1) preprocessing and normalization; (2) performing diagnostic checks for validating statistical model assumptions; (3) partitioning each chromosome into genomic regions of approximately 2000 base pairs; (4) fitting a hierarchical gamma mixture model that models probe-level occupancy measures while allowing information sharing across probes to accommodate small sample sizes; (5) identifying a final set of bound regions by thresholding posterior probability of binding estimated by the statistical model fit; (6) annotation of the genomic regions. In (4), TileHGMM assumes that each genomic partition has at most one peak. Within each unbound genomic region, probe-specific control and IP-enriched observations follow different Gamma distributions conditional on latent (unobserved) mean binding measure. Let μj1(i) and μj2(i) represent the latent control and IP-enriched means for probe j in genomic region i. The model assumes that control binding measurements for probe j form a random sample from a Gamma distribution with scale and shape parameters equal to a1 and μj1(i)/a1, respectively. This ensures probe-specific control binding distributions with mean μj1 while avoiding overparameterization through a common scale parameter. Similarly, IP-enriched binding measurements for probe j form a random sample from a Gamma distribution with scale and shape parameters equal to a2 and μj2(i)/a2, respectively. If region i is unbound, μj1(i)=μj2(i). Otherwise, we have μj2(i)>μj1(i), reflecting transcription factor–DNA interactions, as we expect the IP-hybridizations to be greater than the control hybridizations. (B) Genome-wide colocalization of TRF1/BRF-binding sites. For each chromosome, the top graph represents TRF1, and the bottom graph represents BRF. Chromosome 4 was omitted as no binding sites were observed. The X-axis represents the genomic location in Mbp, and the Y-axis represents the binding efficiency indicated by likelihood ratio score. (C) Distribution of TRF1/BRF bound regions across the genome. Number of total TRF/BRF-binding sites per each chromosome is plotted on the graph. Download figure Download PowerPoint Figure 2.Spatial structure of enrichment by TRF1/BRF ChIP. (A) (top) Quantitative PCR detection of specific enrichment of small noncoding RNA promoters by TRF1/BRF ChIP. 5S rRNA, CR30206 (tRNA), snoRNA:644, and 7SL RNA promoter regions are significantly enriched by TRF1/BRF ChIP whereas the promoter region of a Pol II gene (CG11700) is not. (Bottom) ChIP assay using S2 cells expressing V5-tagged Pol III-specific subunit RPIII128. Download figure Download PowerPoint In order to visualize the spatial topology of the enrichment revealed by TRF1/BRF immunoprecipitations, the signal intensity of each probe was plotted over the chromosomal locations for the selection of genomic regions. Figure 2B displays a region on the X chromosome that encodes three tRNA genes (CR30208, CR30206, and CR30207) displaying equally strong hybridization signals, indicating that all three sites are bound by TRF1 and BRF with comparable efficiency. The second region represents chromosome 3R with multiple TRF1/BRF occupancies at five tRNA genes (CR31485, CR31490, CR31487, CR31489, and CR31486) and two noncoding RNA genes corresponding to 7SL RNA (Figure 2C). The third example illustrates snoRNA:644 gene on chromosome 2R (Figure 2D). In all three cases, it is evident that the hybridization signals peak precisely in register with these noncoding RNA genes. We further verified these microarray results by conventional quantitative PCR detection of co-immunoprecipitated fragments. All sites except for CG11700, a control locus that is not bound by TRF1 and BRF, displayed a significant enrichment with antibodies against TRF1 and BRF, but not with control IgG (Figure 2A). These results confirm that our microarray detection methodology faithfully recapitulates conventional ChIP detected by quantitative PCR. Figure 3.(B–D) High-resolution detection of TRF1/BRF ChIP by genome tiling microarrays. Top plot is the averaged log ratios of ChIP and control binding intensities over two TRF1 replicate experiments, bottom plot is the same for BRF1. Dashed lines mark the predicted peak start and end positions. All the annotations are based on the 4.2.1 version of the D. melanogaster genome. Brown bars indicate tRNA genes, and light blue bars indicate snmRNA genes. (B) Cytosolic tRNA genes (CR30208 on the + strand; tRNAs CR30206, CR30207) on chromosome 2R are bound by the TRF1/BRF complex. The yellow bar indicates the Pol II gene CG4266 on the − strand. (C) 7SL RNA locus (from left to right, CR31490, CR31489, 7SL RNA, CR32864 on the + strand; CR31485, 7SL RNA, CR31487, CR31486 on the − strand) on chromosome 3R is bound by the TRF1/BRF complex. We identified the second 7SL RNA on the − strand by a BLAST search. (D) snoRNA:644 locus on chromosome X is bound by the TRF1/BRF complex. Download figure Download PowerPoint We next annotated the identified TRF1/BRF-binding sites using the 4.2.1 version of the D. melanogaster genome from FlyBase. We classified the target genes of the TRF1/BRF complex into several categories based on the available annotation (Figure 3 and Table I, full lists of the identified targets are in Supplementary Tables). Strikingly, we found that 77.7% of all the identified sites are shared by TRF1 and BRF, consistent with our previous biochemical observation that the majority of TRF1 in Drosophila cells appears to be in a complex with BRF (Takada et al, 2000). The major class of these colocalized sites corresponded to tRNA genes. Importantly, we found that 93% of all known tRNA genes in Drosophila are bound by both TRF1 and BRF. In addition, a minor fraction (4%) is bound only by BRF. For the 20 tRNA genes that are not identified as TRF1-bound, two corresponded to regions that are not tiled on the array, and six can be identified with a less stringent threshold on the binding probabilities. Likewise, for the seven tRNA genes not identified as BRF-bound, two corresponded to regions not tiled on the array, and three can be identified using a lower probability threshold (Supplementary data). Therefore, our methods not only identified conventional targets of the TRF1/BRF complex with good sensitivity but also provided a clear correlation between tRNA genes and the TRF1/BRF complex throughout the entire genome. Figure 4.Classification of the TRF1 and BRF occupied regions. TRF1/BRF-binding sites are annotated using version 4.2.1 of the D. melanogaster genome and classified according to the type of noncoding RNAs. Percentage and number of target sites are indicated. (A) TRF1/BRF-tRNA: tRNA promoter regions bound by both TRF1 and BRF (77.4%); TRF1/BRF/snmRNA: snmRNA promoters bound by TRF1 and BRF (2.8%); TRF1/BRF/pseudo: pseudo-gene promoters bound by TRF1 and BRF (0.3%); TRF1/BRF/Pol II: Pol II promoters bound by TRF1 and BRF (0.6%); TRF1/BRF: unannotated regions bound by both TRF1 and BRF (0.3%); TRF1/Pol II: Pol II promoters bound only by TRF1 (1.4%); TRF1: unannotated regions bound only by TRF1 (17.2%). (B) BRF/TRF1-tRNA: tRNA promoter regions bound both by BRF and TRF1 (76.3%); BRF/TRF1/snmRNA: snmRNA promoters bound by BRF and TRF1 (2.8%); BRF/TRF1/pseudo: pseudo gene promoters bound by BRF and TRF1 (0.3%); BRF/TRF1/Pol II: Pol II promoters bound by BRF and TRF1 (0.6%); BRF/tRNA: tRNA promoters bound only by BRF (3.6%); BRF/TRF1: unannotated regions bound by both TRF1 and BRF (0.3%); BRF/snmRNA: snmRNA promoters bound only by BRF (1.9%); BRF/Pol II: Pol II promoters bound only by BRF (1.4%); BRF: unannotated regions bound only by BRF (12.8%). Download figure Download PowerPoint Table 1. Representative genomic regions with significant TRF1/BRF binding Rank Start End Chr Score Class Genes 1/1 8952366 8954214 chr2R 41.8 I CR30509 2/2 16687498 16691515 chr2R 34.3 I CR33539, CR30210, CR30209 3/3 14928678 14930667 chr2R 33.1 I CR30224, CR30225, CR30326 4/5 7167448 7169438 chr2R 32.5 I CR30255, CR30254 11/6 8944250 8950296 chr2R 31.2 I CR30244, CR32842, CR32841, CR30521, CR30246, CR30247, CR30508 15/7 18578516 18580486 chr2R 29.0 I CR30202, CR30201 24/9 14637600 14641600 chr3L 28.3 I CR32144, CR32142 5/10 3077634 3081648 chr3L 27.4 I CR32288, CR32289, CR32287, CR32285, CR32286, CR32272 10/11 10498592 10500568 chr2R 27.2 I CR30241 14/12 22781067 22783043 chr3L 26.7 I CR32460 13/21 2581411 2583397 chr2R 24.9 I CR30298, CR30299 14/12 1362366 1364341 chr3L 24.9 I CR32330, CR32328, CR32329 15/23 8039304 8043250 chr3L 24.7 I CR32361, CR32362, CR32363 17/22 7175511 7177469 chr2R 24.4 I CR32844 18/27 21041337 21043328 chrX 24.0 I CR32526, CR32518 19/13 16667371 16669351 chr2R 23.9 I CR30208, CR30206, CR30207 20/7 752349 756360 chr3L 23.7 I CR32480, CR32481 32/16 1647601 1651588 chr2R 14.0 I CR30304, CR32837 22/17 20582213 20586210 chr2R 13.8 I CR30198, CR30199, CR30200 35/19 8000971 8004963 chr3L 12.7 I CR32370 42/20 14153176 14155175 chr2R 12.6 I CR30227, CR30228 21/8 12657949 12662132 chr2R 16.3 I, II CR30234, CR30235, CR33921 (snoRNA:U3:54Aa), CR33628 (snoRNA:U3:54Ab) 85/87 20379116 20383094 chr3R 13.1 I, II CR31540, CR31379** (snRNA:U6:96Aa), CR32867** (snRNA:U6:96Ab), CR31539** (snRNA:U6:96Ac) 109/108 2643770 2649796 chr3R 8.8 I, II CR31490, CR31485, CR31489, CR31487, CR31486, CR32864 (7SLRNA) 143/142 3297494 3303497 chr3R 1.7 I, II CR31500, CR31502, CR33925** (snmRNA:331) 162/167 15238196 15244198 chr2R 2.4 I, II CR30218, CR30452, CR30451, CR30455, CR30453, CR30454, CR30220, CR33930** (snoRNA:185) 23/41 5369112 5370107 chrX 22.9 II CR33787 (snoRNA:644) 24/34 1470403 1473835 chr3L 22.9 II CR33656 (snoRNA:3) 34/— 19555572 19557535 chr3R 20.7 II CR33682** (snmRNA:342) 92/129 18860853 18862812 chr3L 11.8 II CR33686 (snoRNA:269) 93/141 7106831 7108826 chr2R 11.7 II CR33661 (snoRNA:535) 102/— 8389072 8391062 chr2L 10.0 II CR32989** (snRNA:U6atac:29B) 111/138 3877139 3879109 chr3L 8.5 II CR33708 (snmRNA:149) 119/127 19345596 19349590 chr2R 7.2 II CR33913 (snoRNA:314) 121/109 10192815 10194807 chrX 7.0 II CR33662 (snoRNA:U3:9B) 6/4 6919489 6923502 chr2R 32.6 III CG7759 16/30 12370895 12372879 chr2R 24.5 III CG5935 18/8 13130719 13132695 chr2R 28.7 IV 7th intron of CG10936 —/9 4236361 4238331 chr2R 16.0 IV 1st intron of CG8411-RA Highly statistically significant TRF1/BRF binding regions were categorized into four classes: I, tRNA, II, snmRNA, III, Pol II genes, IV, unannotated regions. A statistical score was used to list the top binding regions, and the coordinates for start and end sites of the binding regions are indicated. Double asterisks indicate that the binding was observed only in the BRF dataset. Approximately 20% of the binding sites corresponded to non-tRNA sites. These non-tRNA target regions of the TRF1/BRF complex contained promoters of snmRNA genes (such as 7SL RNA and snoRNAs) and Pol II genes, in addition to genomic loci with no current annotation. Interestingly, we observed some snmRNA genes that are only bound by BRF under our experimental conditions. This could be due to the differences in antibody affinity, or it could indicate that some BRF can associate with subsets of promoters in the absence of TRF1, possibly in association with other, as yet, uncharacterized partners. Functional characterization of novel TRF1/BRF targets Our ChIP-on-chip analysis has provided not only a comprehensive high-resolution profile of TRF1/BRF target sites but also revealed several potentially novel target genes. We were particularly intrigued by the finding that the TRF1/BRF complex was associated with 7SL RNA and snoRNA genes. Therefore, we wanted to determine whether the binding of TRF1/BRF to these sites has functional relevance for the activity of these genes. We selected several representative promoters from these newly identified, putative targets of the TRF1/BRF complex to further characterize by direct in vitro transcription reactions. Figure 4 shows in vitro transcribed RNA products using S2 cell extracts directed by templates containing promoters for tRNA (CR30206), 7SL RNA, and snoRNA genes. As expected, transcription from all three of these classes of genes was resistant to low concentrations of α-amanitin (25 ng/μl) typically used to inhibit Pol II transcription (Takada et al, 2000). By contrast, addition of tagetin (0.5 U/μl), a Pol III-specific inhibitor, completely abolished transcription from all three templates. tRNA and 7SL RNA have previously been established as Pol III genes in Drosophila, human, and plants (Ullu and Weiner, 1985; Takada et al, 2000; Yukawa et al, 2005). Interestingly, snoRNAs have been reported to rely on either Pol II or Pol III for transcription (Antal et al, 2000; Kiss, 2002; Harismendy et al, 2003; Roberts et al, 2003; Moqtaderi and Struhl, 2004). Here, we found that snoRNA:314 and snoRNA:644 are specifically targeted by the TRF1/BRF complex and appear to depend exclusively on the Pol III machinery. This finding is consistent with the observation that these two genes are localized to intergenic regions, rather than as part of introns of host Pol II genes (Yuan et al, 2003), suggesting that at least this class of snoRNAs may consist of functionally independent Pol III transcription units. Indeed, our bioinformatic analysis of novel snoRNA targets revealed conserved B-box sequences, which serve as binding sites for TFIIIC (Figure 4B). Figure 5.In vitro transcription of tRNA, 7SL RNA, and snoRNA genes. (A) In vitro transcription assays were carried out using templates for the putative targets of the TRF1/BRF complex, CR30206 (tRNA), 7SL RNA, snoRNA:314, and snoRNA:644. Transcription from these promoters gave template-dependent transcription products that are resistant to a low concentration of α-amanitin (25 ng/μl), which is sufficient to inhibit Pol II transcription, but displayed sensitivity to tagetin (0.5 U/μl), a Pol III-specific inhibitor. (B) Alignment of conserved B-box sequences found in snoRNAs and tRNAs bound by TRF1 and BRF. The conserved sequences are boxed in black. The consensus B-box sequence is represented as a logo at the bottom. The start sites indicated are the distances from the annotated gene start sites in FlyBase. As most annotated snoRNAs appear to be derived from processed transcripts, the identified B-boxes typically reside upstream of the mature snoRNA sequences. Download figure Download PowerPoint To further establish that these novel TRF1/BRF target genes are indeed bona fide Pol III transcribed genes, we conducted ChIP assays probing directly for the presence of RNA polymerase III at these novel TRF1/BRF targets in vivo. Using S2 cells expressing a V5-tagged RpIII128, the second largest subunit of Drosophila RNA Pol III that is a unique class III subunit, we found that 5S rRNA, tRNA, snoRNA, and 7SL RNA genes are all specifically precipitated via anti-V5 antibody whereas CG11700, a Pol II gene, failed to exhibit any enrichment (Figure 2A, bottom). This result strongly corroborates our finding that the TRF1/BRF complex is responsible for regulating the transcription of both of these novel target genes (7SL and snoRNA) and that this diversified TRF1 containing initiation complex indeed works in conjunction with RNA Pol III in Drosophila. snoRNA:644 is transcribed as a longer precursor Although we observed robust, template-dependent as well as Pol III-dependent transcription from snoRNA templates, we noticed that the size of the in vitro products were significantly longer than what had been reported previously. For example, the snoRNA:314 template produced a ∼250 bp RNA product in vitro whereas stable transcripts detected by Northern blot were only 130 bp in length. Likewise, snoRNA:644 transcribed in vitro produced a ∼300 bp transcription product instead of the 170 bp RNA observed in cells (Yuan et al, 2003). We hypothesized that this discrepancy may be due to RNA processing events that occur at the 5′ and 3′ ends of putative precursor-snoRNAs (Kiss, 2002). Therefore, we tested whether these in vitro products accurately reflect primary transcripts of pre-snoRNAs. To address this, we performed primer extension analysis to compare the in vitro transcription products with the primary in vivo transcripts present in total RNA extracted from S2 cells (Figure 5). Primer extension products using two different primers complementary to different segments of the snoRNA:644 transcript confirmed that the vast majority of snoRNA:644 transcripts exists as two distinct processed RNA species (Figure 5A). Importantly, however, a fraction of the in vivo snoRNA:644 contains a start site that matches perfectly with the long in vitro transcription products (Figure 5B). Similarly, the in vitro transcription start site of snoRNA:314 gene matched exactly the in vivo start site (data not shown). Thus, we conclude that the in vitro products likely represent the unprocessed 'primary transcript' and that the in vitro transcription start site we observe accurately reflects the in vivo transcription start site. Figure 6.Mapping the in vivo transcriptional start site of snoRNA:644. (A, B) In vitro transcription products directed by the snoRNA:644 template were subject to primer extension analysis using two independent primers (lanes 1 and 2) hybridizing to mature and precursor snoRNA:644 transcripts. Control mock transcription reactions without the template did not yield any primer extension products (lanes 3 and 4). The primer extension products with identical sizes with the in vitro products were obtained using S2 total RNA (lanes 5 and 6). Arrowheads represent two major processed forms of snoRNA:644 in lane 5 using Primer 1. (B) Longer exposure of (A) in top region of the gel. Arrowheads represent unprocessed nascent transcripts. (Bottom) Schematic diagram of primers used for the primer extension analysis. Download figure Download PowerPoint TRF1/BRF complex is required for transcription of novel targets Having established an efficient in vitro transcription system that accurately reflects in vivo transcription start sites, we next asked whether the TRF1/BRF complex is required to potentiate activation of these novel target promoters. To test this, we first prepared transcription extracts depleted of the TRF1/BRF complex by preincubating S2 extracts with protein A beads conjugated with affinity-purified anti-BRF antibody (Figure 6A). Importantly, we c
Referência(s)