Trypanosoma cruzi TBP shows preference for C/G-rich DNA sequences in vitro

Experimental Parasitology 124 (2010) 346–349

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Research Brief

Trypanosoma cruzi TBP shows preference for C/G-rich DNA sequences in vitro Pamela Cribb a, Luis Esteban b, Andrea Trochine a, Javier Girardini a, Esteban Serra a,* a b

Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 351, 2000 Rosario, Argentina Facultad de Ciencias Médicas, UNR, Santa Fe 3100, 2000 Rosario, Argentina

a r t i c l e

i n f o

Article history: Received 30 July 2009 Received in revised form 28 September 2009 Accepted 12 November 2009 Available online 16 December 2009 Keywords: Trypanosoma cruzi TATA-binding protein SELEX

a b s t r a c t Recent findings associate transcription start in trypanosomatids with chromatin regions containing modified and variant histones. TATA-binding protein (TBP) and other fundamental transcription factors have been also found at these Transcription Start Sites (TSS). Results of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments show that Trypanosoma cruzi TBP (TcTBP) has an in vitro binding preference for G-rich sequences. This finding correlates with the presence of G-rich stretches at the Strand Switch Regions (SSR) and at some putative internal TSS in Trypanosoma brucei and Leishmania. A scanning study of partially assembled T. cruzi genomic contigs determined the presence of Grich stretches in the coding strands. TcTBP affinity for G-rich sequences suggests that this factor could play a role in locating the initiation complex in the right TSS, probably by ‘‘sensing” the G-content on the strand to be transcribed. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction

2. Materials and methods

Trypanosomatid parasites are a group of early divergent protozoa that cause severe diseases in humans, including leishmaniasis, sleeping sickness and Chagas’ disease. In these parasites transcription is polycistronic and seems to be regulated only globally by chromatin-mediated epigenetic events. Nuclear run-on assays demonstrated that RNA pol II transcription starts in both directions within the Strand Switch Region (SSR) (Martínez-Calvillo et al., 2003, 2004). In addition to the RNA polymerases, a reduced number of very divergent transcription factors, an ortholog of TATAbinding protein (TBP) among them, also participate in the transcription process. In Trypanosoma brucei, TBP showed to be essential for RNAP I, II, and III transcription and is recruited to the SL RNA-gene promoter, as well as to RNAP I-transcribed procyclic acidic repetitive genes and RNAP III-transcribed U-rich snRNA and 7SL RNA genes (Ruan et al., 2004; Das et al., 2005). Recruitment of trypanosomatid TBP to the SL RNA-gene promoter was also demonstrated for Leishmania tarentolae and Trypanosoma cruzi (Thomas et al., 2006; Cribb, unpublished results). In this promoter, TBP is part of a complex that includes three divergent components of the SNAP complex, at least one TFIIA orthologous subunit and a very divergent version of TFIIB (Das and Bellofatto, 2003; Ruan et al., 2004; Schimanski et al., 2005, 2006; Das et al., 2005; Palenchar et al., 2006; Cribb and Serra, 2009).

2.1. Expression of recombinant TcTBP

* Corresponding author. Fax: +54 341 4390465 E-mail addresses: [email protected], [email protected] (E. Serra). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.11.003

Trypanosoma cruzi TBP coding region from CL-Brener strain (Tc00.1047053503809.149) was amplified using Pfu polymerase with specific oligonucleotides, cloned into pGEMT-easy plasmid (Promega) and sequenced. TcTBP coding sequence was cloned in pGEX4-T3 and pQE30 (QIAGEN) expression plasmids. Recombinant GST-fusion and His-tagged proteins were purified by affinity chromatography using standard procedures. 2.2. Determination of TcTBP recognition sequence Selection of recognition sequence for TcTBP was performed by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) assay using recombinant TBP, a 54-mer double stranded oligonucleotide containing 12 completely degenerated nucleotides (CHRI, 50 -gATgAAgCTTCCTggACAAT-(N)12-gCAgTCACTgAAgAATTC Tg-30 ) and two primers complementary to the conserved regions on the random primer (CHRIS-1: 50 -gATgAAgCTTCCTggACAAT-30 ; CHRIS-2: 50 -CAgAATTCTTCAgTgACTgC-3 (Oliphant et al., 1989). Before the first binding, CHRI was converted to double stranded DNA with DNA polymerase I Klenow fragment, using CHRIS2 as primer. Agarose-immobilized GST–TcTBP was incubated with 20 pmol of double stranded random oligonucleotide for 30 min at 4 °C in 200 ll binding buffer (20 mM Hepes pH 7, 5 mM MgCl2, 5 mM DTT, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol). After centrifugation, agarose was washed 5 times at 4 °C with 1 ml binding

P. Cribb et al. / Experimental Parasitology 124 (2010) 346–349

buffer containing 0.1 mg/ml sonicated salmon sperm DNA, resuspended in 50 ll TE and boiled. Two microliters of supernatant were used for amplification with CHRIS-1 and CHRIS-2 and amplification products were analyzed by polyacrylamide gel electrophoresis. Further cycles were performed in the same way, using 2 ll of amplification product from the precedent binding assay. After six cycles, the products of amplification reaction were cloned in pGEMT-easy plasmid (Promega). A control assay was performed in parallel using purified GST immobilized to the agarose beads, instead of the fusion protein. 2.3. Analysis of strand switch-containing contigs from T. cruzi Assembled sequences from the T. cruzi genome were recovered from NCBI nucleotide database (CH473309:CH473946[PACC]). Contigs with a Strand Switch (El-Sayed et al., 2005, Supplementary material) were visualized with Artemis and their CG-skews were calculated with the same program. Scanning of the contigs in sliding windows for the calculation of the C or G content was performed with an ad hoc written Pearl script as follows: A 100nucleotides sliding window moves along the contig, one nucleotide by step, and calculates the G (or C) composition at each position. The results of the scanning are recovered in a table where the percentage of the nucleotide obtained for each window is assigned to the first position of the window. The graphics were generated from data in the table using R (http://CRAN.R-project.org). The script is given as Supplementary material (file: CounCG.pl). 3. Results and discussion To explore the TcTBP DNA-binding preference we performed two different SELEX assays. First, we performed a typical gel

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shift-based SELEX, however no shifted band was observed when GST- or His-tagged TcTBP was incubated with the pool of random oligonucleotides, under the tested conditions. Then, we performed a liquid-binding and selection SELEX, using GST–TcTBP fusion protein bound to a GS-agarose column as described in Section 2. A control assay was performed in parallel using purified GST instead of the fusion protein. The results of the amplification reactions from GST–TcTBP- and GST-binding assays were analyzed in parallel by polyacrylamide gel electrophoresis, after washing, at each cycle. No amplification product was observed in control assay after the first cycle. In following cycles amplification products from the GST–TcTBP-binding reaction were also incubated with GST. No amplification product was recovered after anyone of the binding and washing cycles in these control assays. After six rounds, amplified oligonucleotides that bounded to TcTBP were cloned in pGEMT easy plasmid (Promega). Plasmids from 34 randomly chosen colonies were extracted and sequenced. A first analysis showed that sequences obtained were rich (67%) in C/G. Sequences were analyzed both by alignment and visual inspection, and by the Multiple Expectation Maximization for Motif Elicitation (MEME) program (Bailey and Elkan, 1994). Fig. 1 shows sequences obtained after MEME-driven alignment and a consensus motif generated GG(C/ T)G(T/G/A) by the program. For the run, the program was set to align each sequence or its reversed complementary one (which is indicated as + or – strand in the figure). The final alignment is rich in G, which is reflected in the high frequency for this nucleotide at positions 1 (0.70), 2 (0.73) and 4 (0.91) of the motif represented in Fig. 1C. The fact that the interaction between TBP and DNA could not be observed directly suggests that this interaction should be very weak, and most probably mediated and/or stabilized by other factors in the complexes. This could explain why we succeeded in selecting interacting oligonucleotides in a liquid-binding-selection

Fig. 1. (A) Recombinant GST- and His-TcTBP purified from E. coli. 1. Molecular weight marker. 2. E. coli pGEX-TcTBP total extract. 3. Purified GST–TcTBP. 4. E. coli pQE-TcTBP total extract. 5. Purified His-TcTBP. (B) Sequences from oligonucleotides recovered after six SELEX cycles aligned by MEME. Minus () means that the sequence was complementary reverted by the program during the calculations. The P-value generated by MEME estimates the probability for a given sequence to be aligned with the core motif by chance in a dataset of the same size. (C) Logo representation of the 5 nucleotides consensus generated by MEME.

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Fig. 2. CG-skew, C and G content from contig Tc1047053516361. Gray arrows represent the opposite cluster and their direction of transcription. CG-skew were determined in a 5000 nucleotides-wide window (W = 5000) with Artemis and imported into the picture. Percentage of C and G were calculated for a 100 nucleotides-wide (W = 100) sliding window that scan all along the sequence, moving one nucleotide by step. Only peaks higher than 40% are represented in this picture. Similar figures for other 10 contigs and the Pearl script used for G/C percentage calculations are given as Supplementary material.

SELEX assay but not in a gel-shift based assay. The fact that TcTBP binds a sequence different from TATA box is not surprising at all. On one hand, no canonical TATA-boxes have been found in trypanosomatid genomes, on the other, TcTBP has substantial differences in the double repetition region compared to canonical TBPs. The TATA-binding protein of T. cruzi (TcTBP) is a 263 aminoacids polypeptide which contains the conserved double repetition characteristic of TBP-family members. TcTBP conserved region showed nearly 45% sequence identity with metazoan and fungi TBPs, and 36% identity with metazoan TLFs (which do not bind TATA boxes) and archaeal TBPs (aTBPs). The identity between the TcTBP repeats is very low (13%) compared to that from canonical TBPs (30%) and aTBPs (40%), suggesting a more asymmetrical structure. This lack of symmetry may be reinforced by an insertion of 4 amino acids in the carboxy-most repetition. Two asparagines, 167N and 257N (numbers refer to human TBP), conserved in all TBP-family factors, are replaced in TcTBP by glycine and serine, respectively. These residues are located at the centre of the TBP–DNA complex forming hydrogen bonds with the bases’ edges in the middle of the TATA box motif (Dantonel et al., 1999; Patikoglou et al., 1999). Two pairs of phenylalanine residues are usually involved in the interaction between TBPs and the minor groove of DNA. In the amino repeat, 197 F and 214F residues are conserved in TBPs, and aTBPs (Patikoglou et al., 1999) but not in metazoan TBP-like factors (TLFs) where they are replaced mostly by hydrophobic residues. In TcTBP, these Fs are replaced by threonine and arginine residues. Concerning the specificity for non canonical TBPs binding to DNA, only the Drosophila TRF1 binding site has been well determined, being a TC-box at the tudor gene promoter (Holmes and Tjian, 2000). Recent experimental evidences correlate the occurrence of Grich regions with transcription start sites in trypanosomes (Martínez-Calvillo, 2003; Siegel et al., 2009). Leishmania major Chromosome I SSR can enhance the expression of a marker gene 2- to 10-fold and shows a 73 bp region with 69.8% GC and a substantial G/C strand bias (Martínez-Calvillo, 2003). In this parasite, chromatin at SSRs is rich in acetylated H3 and contains both TBP and SNAP50 transcription factors overlapping 1–2 kb upstream of the peak of H3 acetylation (Thomas et al., 2009). In T. brucei, the SSRs are enriched in modified (acetylated H4) and variant histones H2AZ and H2BV, in G-rich zones, near the beginning of the gene clusters (Siegel et al., 2009). Modified histones, variant histones and TBP were also detected in spots within the clusters in both species. In T. brucei, these putative internal transcription start sites also map at G-rich sequences. The presence of modified histones in SSRs has been also reported in T. cruzi (Respuela et al., 2008).

Trypanosoma cruzi genomic data is a collection of not fully assembled contigs showing partial sinteny with T. brucei and L. major chromosomes. We chose a number of T. cruzi large contigs containing SSRs and analyzed their C/G content. As it has been already described, all contigs showed C/G-skew patterns with optima on strand-switch region (Nilsson and Andersson, 2005). The contigs were analyzed for G or C content in a sliding 100 nt-wide window. A typical result is showed in Fig. 2, and results of 10 selected contigs are given as Supplementary material. As it can be observed, coding strand has, all along, G-rich stretches that can reach 50%, in the 100 nt window. The same pattern was observed in the complementary strand as C-rich peaks. When observed in detail, these peaks correspond to G-rich zones that, in average, are no longer than 6 consecutive guanines, resembling the G rich motifs to which TcTBP showed affinity in our SELEX assay. These observations suggest that, in these parasites, chromatin components may delimit DNA zones accessible to transcription factors, and hence to the polymerase. The presence of G-rich tracks correlates with modified chromatin regions. However, there is not conclusive evidence available in favor of a functionality of these tracks in transcription initiation. In this context, our results, even limited, suggest that TBP could have a role in the correct localization of the initiation complex, probably by ‘‘sensing” the G-content on the strand to be transcribed. Acknowledgments E.S. is a carrier member and P.C., A.T. and J.G. are fellows of the National Research Council (CONICET), Argentina. This work was founded by ANPCyT PICT R 300, CONICET PIP 5492. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.exppara.2009.11.003. References Bailey, T.L., Elkan, C., 1994. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. In: Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology. AAAI Press, Menlo Park, California, pp. 28–36. Cribb, P., Serra, E., 2009. One- and two-hybrid analysis of the interactions between components of the Trypanosoma cruzi Spliced Leader (SL) RNA gene promoter binding complex. International Journal for Parasitology 39, 525–532. Dantonel, J.C., Wurtz, J.M., Poch, O., Moras, D., Tora, L., 1999. The TBP-like factor: an alternative transcription factor in metazoa? Trends in Biochemical Sciences 24, 335–339.

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