Computational Identification of Regulatory Factors Involved in MicroRNA Transcription

Transcription

1 Computational Identification of Regulatory Factors Involved in MicroRNA Transcription Praveen Sethupathy 1,2,5,*, Molly Megraw 1,2,5,*, M. Inmaculada Barrasa 4, and Artemis G. Hatzigeorgiou 1,2,3 1 Center for Bioinformatics, University of Pennsylvania, Philadelphia PA 19104, USA agh@pcbi.upenn.edu 2 Department of Genetics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 3 Department of Computer and Information Science, School of Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA 4 Department of Cancer Biology, Abramson Family Cancer Research Institute, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA ibarrasa@mail.med.upenn.edu 5 Genomics and Computational Biology Graduate Group, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA {praveens, megraw}@mail.med.upenn.edu Abstract. MicroRNAs (mirnas) are non-coding RNA molecules that bind to and translationally repress mrna transcripts. Currently ~1345 mirnas have been identified in at least twelve species through experimental and computational approaches. Here, we report on a field not yet thoroughly investigated: the transcriptional regulation of mirnas. Adequately locating mirna promoter regions will provide a reasonable search space for computational and experimental studies to determine regulatory factors that drive mirna transcription. Insight in to the factors that control mirna transcription may provide clues regarding more complicated mechanisms of mirna expression control in a developing organism. We use a novel Expressed Sequence Tag (EST) based approach to approximate promoter regions for intergenic mirnas in order to detect specific and over-represented regulatory elements. We find that mirna promoter regions may be enriched for binding sites that recruit transcription factors (TFs) involved in development, including several homeobox TFs such as HOXA3 and Ncx. Additionally, we use clustering techniques to cluster mirnas according to tissue specificity to find tissue-specific regulatory elements. We find a few over-represented binding sites in brain-specific mirna promoter regions, some of which recruit TFs involved specifically with the development of the nervous system. Based on the results we suggest an interesting mechanism for in vivo mirna expression control. The EST-based pri-mirna assembly program will be made available at the website of the DIANA-group by the time of publication ( A.G.H, M.M., and P.S. are supported in part by an NSF Career Award Grant (DBI ). * These authors contributed equally to this work. P. Bozanis and E.N. Houstis (Eds.): PCI 2005, LNCS 3746, pp , Springer-Verlag Berlin Heidelberg 2005

2 458 P. Sethupathy et al. 1 Introduction MiRNAs are ~22 nt non-protein-coding RNA molecules that have been identified in at least twelve species. MiRNAs are derived from precursor sequences, often termed hairpins due to their secondary structure. These hairpin structures are derived from a primary transcript, termed pri-mirna, which is the original unprocessed transcript of the mirna gene. These tiny RNAs are known to be involved in important roles in cellular development and differentiation in several species ([1], [4], [19]). They function by binding to portions of particular mrna transcripts and either cleaving them or repressing their translation without cleavage. This binding is generally done with perfect complementarity in plants, and often imperfect complementarity in other species ([4]). Some mirnas can be difficult to clone since it is possible for their expression to be restricted to a particular cell type or to a particular environmental condition ([4]). Therefore, within the last three years, significant efforts have been made to predict mirna genes via computational methods ([9], [15], [17]). To understand the specific functions of these mirnas, recent work also attempts to predict the target genes whose expression profiles are regulated by each mirna ([8], [12], [14], [16], [20], [24]). The transcriptional regulation of mirnas themselves has not yet been thoroughly examined. Although there is evidence that mirnas can be transcribed by both pol-ii and pol-iii ([6], [27]), the locations of the mirna promoters, the identity of the polymerases involved, and the identity of the transcription factors involved are all largely unknown. A few studies of putative cis-elements driving mirna transcription have emerged ([13], [25]), but there are still many open questions and substantial work to be done. For example, adequately locating mirna promoter regions will be an important step toward a more coherent understanding of mirna transcription, and this will allow increased flexibility for medical scientists to research methods of mirna control for the purpose of positively altering gene expression levels in diseased states. MiRNAs, just as protein coding genes, can be characterized as either intergenic or intragenic. It is reasonable to assume that the transcription of an intergenic mirna is activated by promoter elements upstream of the primary-mirna transcript from which the mirna is derived. This assumption is not necessarily appropriate for intragenic mirnas. Some intragenic mirnas could be transcribed via the promoter elements of the host gene ([21]), and others via promoter elements within the intron of the host gene and upstream of the mirna. Given these differences, it is appropriate to consider intergenic and intragenic mirnas separately when studying their transcriptional processes. The purpose of this study is to identify regulatory motifs and putative transcription factor binding sites for intergenic mirnas. The goals of this study are to: (A) locate the likely promoter regions for each mirna considered, (B) find motifs and transcription factor binding sites (TFBSs) enriched in these putative promoter regions, and (C) use recently available mirna expression information to search for binding sites enriched in tissue-specific mirna promoters. In this paper, we present a novel method for approximating the promoter regions. We use these approximations to find regulatory regions that may play a role in

3 Computational Identification of Regulatory Factors Involved in MiRNA Transcription 459 intergenic mirna transcription. Results of this work suggests that mirna promoter regions may be enriched for binding sites of transcription factors known to be involved in development. Additionally, we have found that half of the development related transcription factors with binding sites in the promoters of brain-specific mirnas have detailed involvement in the development of the nervous system. 2 Materials and Methods 2.1 Data Sets MiRNAs and ESTs We downloaded from the Rfam mirna registry all known human and mouse mirnas. We also downloaded the genomic locations of the precursors for these mirnas from the UCSC Genome Browser ( mirna track, using the most current genome builds for human and mouse (hg17 and mm5 respectively). We then determined which of the mirnas were most likely to be intergenic by the following procedure: We downloaded the Known Genes and Genscan Genes lists provided by UCSC (using the hg17 and mm5 builds) and wrote a script to identify all mirnas in human and mouse which do not overlap known or Genscan predicted genes. There are 39 such mirnas in human and 26 in mouse. We then further eliminated a few mirnas in both species which were observed to overlap with a gene from the Ensemble set or other predicted gene sets having strong UCSC-mapped EST support for the gene Promoters and Transcription Factors Human promoter sequences were downloaded from the Eukaryotic Promoter Database (EPD). EPD provides the experimentally verified transcription start sites (TSSs) for ~1,800 human genes. We chose ~100 unrelated human genes and downloaded 4kb upstream of their TSSs to search for ubiquitous motifs and transcription factor binding sites MiRNA Expression Data Dataset #1 Type of data: Northern blot ([23]); Scale: 0-28; Mouse tissues: brain, liver, heart, muscle, lung, kidney and spleen; Human tissues: brain, liver, heart and muscle; Source: Table with northern quantifications, no further processing; Numbers of mirnas assayed for expression: 112 (includes mouse and human mirnas), 86 and 70 in common with Dataset #2 and #3, respectively. Dataset #2 Type of data: Microarray ([26]); Scale: -4 to 4; Mouse Tissues: ES cells, six stages of embryo development, and seven adult tissues (liver, kidney, lung, ovary, heart, brain and thymus); Source: The data was downloaded from GEO-NCBI (accession number GSE1635), the downloaded file contained the normalized log of ratios (R/G) of each of the arrays or hybridizations performed, the normalization used by the authors was a global median centering normalization; Numbers of mirnas assayed for expression: 124 (includes mouse and human mirnas), 70 in common with Dataset #3.

4 460 P. Sethupathy et al. Dataset #3 Type of data: Microarray ([2]); Scale: 0-8; Mouse Tissues: ES cells and 38 tissues, for the clustering we have used ES cells and the following 12 tissues- brain, femur, heart, intestine, liver, lung, mammary gland, muscle, spleen, stomach, testis; Source: The data was downloaded from the GEO-NCBI (accession numbers: GSM30346, GSM30347, GSM30350, GSM30351, GSM30352); Numbers of mirnas assayed for expression: 141 (includes mouse and human mirnas). 2.2 Find Regulatory Elements in MiRNA Promoter Regions Approximation of the Promoter Region for Each MiRNA We created a program which assembles ESTs into putative primary transcripts for intergenic mirnas. Essentially, we built the probable transcribed region around a mirna precursor by piecing together a series of adjacent or overlapping ESTs on the same strand as the precursor. However, it was necessary to keep in mind that working with ESTs can be an error-prone process and we wished to avoid piecing together ESTs which have the pitfall properties described next. It was not desirable to consider ESTs which are too long (much longer than the longest expected primary transcript, we cut off at 10kb) as they may be chimeric, ESTs which map to multiple locations on the genome since they cannot be reliably placed in any single location, or ESTs which overlap a neighboring gene since it is not reasonable to expect that primary mirna transcripts overlap the exonic region of a gene. Therefore, using ESTs from dbest that have been mapped onto the most recent genome builds for human and mouse by UCSC, we performed a putative pri-mirna assembly process by finding ESTs not having any of these pitfall properties which overlap an intergenic mirna precursor, and then sequentially extending the EST assembly in both directions with adjacent or overlapping ESTs until no ESTs free of the pitfall properties can be added. Because we are aware that the strand annotation of ESTs can be unreliable, we also performed the assembly process without the restriction that ESTs added to the assembly be on the same annotated strand as the relevant mirna precursor. We then compared the two results and were comforted to find that generally the length and location of the resulting putative primary transcript was not greatly changed by including oppositely annotated ESTs in the assembly. One reason for this is that there were often many fewer candidate ESTs on the oppositely annotated strand than on the like-annotated strand, providing additional support for the idea that ESTs free of the pitfall properties in the precursor region were transcribed as portions of the primary transcript and for whatever reason exited the nucleus (or represent EST contaminants from nuclear material). The putative primary transcripts are used conservatively in subsequent steps of this study as guides to a minimum length for each primary transcript, as opposed to an absolute representation of the pri-mirnas. Thirteen of the human mirnas and ten of the mouse mirnas in the intergenic subset we studied had precursor coverage by these assembled EST Blocks.

5 Computational Identification of Regulatory Factors Involved in MiRNA Transcription Search for Motifs and TFBSs We considered the 5 upstream regions of the set of putative primary transcripts deduced from as the search space for conventional motif finding algorithms. We used two motif finding algorithms, MEME ([3]) and AlignACE ([10]). MEME (Multiple Em for Motif Elicitation) is a probabilistic local alignment tool useful for conserved motif discovery. We used MEME with the following options: Background- Single-nucleotide frequencies; Motif length bases; Motif mode- Any number of motif repititions ; E-val cutoff- 1. MEME E-values refer to the expected number of motifs of the same width with equal or higher likelihood in the same number of random sequences with the same nucleotide composition as the considered set of sequences. AlignACE is a Gibbssampling based probabilistic approach for motif discovery. We used AlignACE with the default options. AlignACE reports each putative motif with a MAP (Maximum A Priori Log Likelihood) score, a score which measures the motif s degree of overrepresentation. Because motif searching algorithms are heuristic, attention must be given to the false positive rate. We placed more confidence in those motifs which were reported as significant by both programs. The significant motifs reported by these programs were further considered as the search space for an in-house transcription factor binding site (TFBS) scanning program. The scanning was accomplished via a script that performs the following steps: 1. Convert reported motifs in to probability matrices (motif matrices). A probability matrix indicates the probability of each of the four nucleotides occurring in each of the motif s nucleotide positions 2. Convert all TRANSFAC vertebrate transcription factor frequency matrices to probability matrices (TF matrices) 3. Since the reported motifs are generally longer than transcription factor binding sites, perform a local matrix alignment between each motif matrix and each TF matrix and record the score of each alignment ( similarity score ) 4. Generate 100 random probability matrices and perform local alignments between all pairs and record the score of each alignment ( random score ) 5. Set one standard deviation from the mean of the random scores as the cutoff for similarity between any two matrices being aligned (and set two standard deviations from the mean as the cutoff for strong similarity ) 6. Filter the set of similarity scores from step 3 to those that satisfy the cutoff requirements 7. Report this final set of similarity scores as indicators of the presence of putative TFBSs Strong hits found by this program were recorded as putative TFBSs. For example, suppose the mean of the random scores is 1.15 and the standard deviation is We consider any similarity score under 0.90 ( ) as a putative TFBS, and any similarity score under 0.65 ( ) as indicative of a strong candidate TFBS. 2.3 Find Regulatory Elements Specific for Certain Tissues In addition to the possibility that there are motifs and TFBSs common to most or all of the intergenic mirnas, it is also possible that several motifs and TFBSs are found

6 462 P. Sethupathy et al. only in mirnas with a certain tissue-specificity. Therefore, we clustered the mirnas according to tissue-specificity and searched for motifs within the promoters of each cluster Cluster MiRNAs According to Expression Several recent studies have published data on mirna expression profiles based on northern blots and microarrays ([2], [23], [26]). These studies included different sets of mirnas, different sets of tissues, reported expression values in different scales, and used slightly different in-house technologies to measure expression. For these reasons, merging the data from the three studies was a task harboring many complications. Therefore, we considered each dataset separately. We used Hierarchical and K-means clustering to cluster the mirnas from each dataset according to their expression profiles. We then searched for clusters within a dataset which indicated tissue-specificity, and corroborated this finding across the other datasets. These clusters were recorded as tissue-specific clusters for further analysis Search for Motifs and TFBSs Within Each Cluster Within each tissue-specific cluster, we searched the promoters of the mirnas for over-represented motifs, again using MEME and AlignACE. We further scanned these motifs for the presence of putative transcription factor binding sites and recorded the strong hits as described previously. 2.4 Computational Validation of TFBS Predictions Not every TFBS in the set of predicted mirna promoter TFBSs is likely to be functional. Many TFs have binding sites that are non-specific, ubiquitous, and frequently occurring across the genome. We used motif searching and TFBS scanning programs on 91 unrelated human promoters and labeled the resulting frequently occurring TFBS predictions as non-specific. Any TFBS from the set of predicted mirna promoter TFBSs that does not match any TFBS in the nonspecific list is considered as potentially specific. 3 Results 3.1 Find regulatory Elements in MiRNA Promoter Regions Approximation of the Promoter Region for Each mirna Table 1 displays the EST blocks identified by the EST assembly program described in of the Materials and Methods section. Thirteen mirnas were found to have EST blocks. Each EST block is a contiguous assembly of acceptable ESTs (those not having any of the pitfall properties ). Figure 1 provides a visual example of a samestrand EST block. The mirna in consideration is hsa-mir-122a. ESTs shown in red are the members of the block, identified by the program as not having any of the pitfall properties.

7 Computational Identification of Regulatory Factors Involved in MiRNA Transcription 463 Fig. 1. ESTs shown in red are members of hsa-mir-122a s EST block Search for Motifs and TFBSs Motif searching algorithms can generally be grouped into two categories: enumerative, such as the ST algorithm, and alignment-based. The alignment-based approach can itself be grouped in to two categories: Expectation Maximization, such as MEME, and Gibbs-sampling, such as AlignACE. Using MEME ([3]), we searched in the first 4kb upstream of all the 5 EST block ends deduced from for sequence motifs which might be involved in mirna transcription. MEME reported eight significant motifs, each ~20-25 nts long. We repeated this search using AlignACE ([10]) and found that this algorithm reported seven out of the eight of these motifs as highly significant and frequently occurring. Details of the options used with MEME and AlignACE are provided in the Materials and Methods section. The seven significant motifs were scanned for putative vertebrate transcription factor binding sites. Details of the scanning procedure are provided in the Materials and Methods section. Twenty-nine unique putative transcription factor binding sites (TFBSs) were found within the motifs. We surmised that many of the 29 TFBSs recruit TFs that are non-specific in their binding and highly common in promoters of Pol II transcribed genes. To test this, we repeated the above motif searching and TFBS scanning procedure on a set of 91 unique and unrelated Eukaryotic Promoter Database (EPD) promoters from human genes of human promoters. At least 11 of the 29 predicted TFBSs are present in high frequency across this set. The remaining 18 TFBSs are further considered as those sites that are more likely to recruit TFs which are more specific in binding and function. Of these 18, 7 are especially noteworthy (Table 2). Two of the 7 are sites that recruit the proteins C/EBP and CREB, both of which are already predicted as transcriptional activators of mirnas in previous studies ([11], [25]). Interestingly, the remaining five are homeo-box and forkheadbox transcription factors proteins well known for their involvement in development. We repeated the motif searching and TFBS scanning procedures in the regions 4kb to 8kb upstream of the 5 EST block ends deduced from Again, we find several strong transcription factor binding sites that are known to recruit transcription factors involved in development (Table 3).

8 464 P. Sethupathy et al. MiRNAs are known to play a vital role in the proper development of C.elegans ([22]), D.melanogaster ([1]) and plants ([19]). For example, lin-4 and let-7 are two C.elegans mirnas known to control the timing of postembryonic events via the translational repression of target genes ([1]). It is probable that mirnas also play a critical role in human development. We suggest that it is very likely for the aforelisted TFs to be transcriptional activators (such as HOXA7) and repressors (such as Msx1) of mirnas, thereby controlling the precise timing and duration of expression of target genes. 3.2 Find Regulatory Elements Specific for Certain Tissues Cluster MiRNAs According to Expression Details of the clustering techniques and mirna expression datasets are provided in the Materials and Methods section. Section conveys the relationship between the datasets considered in this study. The clustering procedures revealed three notable clusters (Table 4) Search for Motifs and TFBSs Within Each Cluster Among the clusters identified, we chose the brain cluster for further analysis. Genes expressed in the brain are often restricted to specific compartments (substantia nigra, cerebellum, etc), perhaps implying the presence of compartment specific silencers and enhancers in the promoter regions of these genes. So, by considering all brainexpressed mirnas in one cluster, we may lose information about compartment specific cis-elements. However, compartment specific expression data for mirnas are not currently available. Therefore, it is reasonable to consider the brain as a whole entity and search for a higher order group of cis-elements tissue(brain)-specific ciselements. None of the mirnas in the brain cluster had EST block support. Therefore, we approximated the promoter region for each of these mirnas by considering both 1.5kb and 4kb upstream of the 5 end of the precursor. We used MEME and AlignACE to search for motifs in these regions and used the in-house TFBS scanning procedure to search for putative transcription factor binding sites in exactly the same manner as previously described. Of the 20 predicted TFs, nine can be considered in the promiscuous/highly common category. Of the remaining 11 TFs, four are known to play key roles in development: En-1, MZF1, HOXA3, and Ncx.. Also, it is confirmatory that all four have moderate to high levels of expression in the brain ( It is unlikely for MZF1, HOXA3, and Ncx to be brainspecific transcription factors because their binding sites are also found in the putative promoter regions of a set of mirnas with heterogeneous expression profiles (Table 2, 3). We believe that more likely brain-specific mirna transcription factors can be identified if more ESTs are published in dbest such that our EST-based pri-mirna assembly program can build EST blocks and provide more accurate promoter regions for motif and TFBS search. We believe that more likely brain-specific mirna transcription factors can be identified if more ESTs are published in dbest such that our EST-based pri-mirna assembly program can build EST blocks and provide more accurate promoter regions for motif and TFBS search.

9 Computational Identification of Regulatory Factors Involved in MiRNA Transcription 465 Table 1. The results of the EST-based pri-mirna assembly program. The * indicates that the EST Block was determined by using ESTs on both strands. The # indicates that the EST Block was determined by using ESTs on the mirna strand only. Precursor Precursor EST Block EST Block EST Block EST Block EST Block EST Block mirna Name Chr Str Left Right Left* Right* Length* Left# Right# Length# hsa-mir hsa-mir-196b hsa-let-7a hsa-let-7d hsa-mir-223 X hsa-mir-34c hsa-mir-135a hsa-mir hsa-mir hsa-mir-122a hsa-mir-27a hsa-let-7a hsa-let-7b Table 2. The TFs predicted to be enriched in the first 4kb upstream of mirna transcription start sites. The * indicates a homeo-box or forkhead-box transcription factor. TF ID Ncx* HOXA7* HOXA3* Msx1* FOXD3* CREB C/EBP Gene Ontology (GO) Function T-cell leukemia homeobox 2; development Homeobox A7; development Homeobox A3; development Muscle-Seg. Homeobox 1; development Forkhead Box D3; development Transcription; signal transduction Transcription; CCAAT enhancer binding Table 3. The development related TFs predicted to be enriched in the 4kb to 8kb upstream region of mirna transcription start sites TF ID Ncx FAC1 FOXD3 Msx1 HOXA3 MZF1 SRY Gene Ontology (GO) Function T-cell leukemia homeobox 2; development Fetal Alzheimer s Clone 1; development Forkhead Box D3; development Muscle-Segment Homeobox 1; development Homeobox A3; development Myeloid Zinc Finger 1, differentiation Sex Determining Region Y; differentiation Table 4. The results of expression-based clustering of mirnas Brain Low in ES cells Ubiquitous hsa-mir-10b mmu-mir-139 hsa-let-7b hsa-mir-125a mmu-mir-22 hsa-mir-223 mmu-mir-222 hsa-mir-22 hsa-mir-27a mmu-mir-154 hsa-mir-30a hsa-mir-23a -- hsa-mir

10 466 P. Sethupathy et al. 3.3 Computational Validation of TFBS Predictions Complete validation of the TFBS predictions can be accomplished via chromatin immunoprecipitation (ChIP) assays. However, further credence can be given to the TFBSs via simple computational methods. We have shown that at least nine putative TFBSs are not as frequently found in other appropriate background promoter sets, thereby concluding that they are generally more specific and apparently enriched in promoters of mirnas. Bona fide transcription factors will have binding sites preferentially located proximal to the transcription start site (TSS), rather than distal to the TSS. In other words, although it is certainly possible to have enhancers and other cis-elements significantly upstream of the TSS, most effective TFBSs are found clustered within the first 8kb upstream of the TSS. Only roughly half of the ten predicted TFs have binding sites as far as 8kb-12kb upstream of the putative TSS. Furthermore, even some of the sites that are found 8kb-12kb upstream are less convincing as bona fide cis-elements because their similarity scores based on the TFBS scanning procedures are very low. 4 Discussion In this study we report on a novel algorithm which utilizes EST information to approximate a primary-mirna transcript from a mirna precursor for the purpose of making a reasonable assumption about the promoter location. We also searched these promoter regions for novel motifs and TFBSs regulating mirna transcription. We have predicted at least ten TFs that may be important in the transcriptional regulation of human mirnas. Many of these are TFs that have previously been found to have a role in development. We predict mirna promoter regions to be enriched for TFBSs that recruit TFs known to be involved in development. Several of the predicted TFs are homeobox proteins. These proteins are known to be likely targets of mirnas ([18]). Taken together, these two observations suggest the possibility of a negative feed-back loop whereby mirnas may control their own expression (Figure 2). We have also used recently available mirna expression data to cluster mirna genes according to tissue-specificity. This enabled us to search for tissue-specific motifs and TFBSs. We predict four development related TFs that may be important in the transcriptional regulation of the brain-specific mirnas. Two of these four TFs have specific roles in either neurogenesis or general CNS development. MiRNAs are known to play vital roles in many biological functions, including developmental timing, differentiation, apoptosis, fat metabolism, and growth control ([12]). Additionally, mirnas have specific and varied expression patterns across developmental stages, tissues, and cell types ([12]). We believe that gaining insight in to the transcriptional regulation of mirnas will help to understand the range of mirna expression regulation and to more coherently describe the functional importance of mirnas. We present here a novel approach to locate mirna promoter regions and search for cis-elements that may partially control mirna expression patterns.

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