Comparison of structural patterns of different features

Different from traditional promoter prediction methods that are mostly based on sequence compositional features, in this paper we first carry out an extensive comparison between promoter and non-promoter sequences in order to explore whether promoters possess specific structures. Here, our investigation focuses on thirteen different kinds of structural features, including duplex free energy, stacking energy, DNA denaturation, duplex disrupt energy, protein deformation, Z-DNA, DNA-bending stiffness, A-philicity, nucleosome position, propeller twist, protein-DNA twist, B-DNA twist and bendability. For these features, different structure models have been derived from various biochemical experiments. According to these models, DNA sequences can be transformed into distinct structural profiles, as illustrated in Figure 1. Although these structural models are based on dinucleotides or trinucleotides, several studies have proven that these structural features are different in terms of the nucleotide information, and offer additional thermo-physical information [20, 32, 33]. Based on the conversion schemas, we transform the promoter and non-promoter sequences into numerical vectors and examine their similarities and differences with regard to these structural features on the DBTSS human TSS dataset (see Methods). For all these thirteen kinds of structural features, we plot and compare the structural profiles of promoters and non-promoters (Figure 2).

Through a comprehensive analysis, we obtain some interesting observations. First, for each feature, the structural values along promoter sequences are different from those along non-promoter sequences. Second, promoters have specific patterns of structural profiles, with overall stable values and two sharp troughs or peaks. The first clear trough (peak) locates at the position -30 bp upstream of TSS, where the TATA-binding protein (TBP) binds. The second trough (peak) is around the TSS. These two troughs (peaks) may guide transcription apparatus to locate the appropriate initiation site, which is in agreement with the fact that promoters bear a special structure that is essential to the assembly of the transcription machinery. On the contrary, the structural profiles of non-promoters do not exhibit such patterns. The different patterns may originate from the strongly conserved sequences, such as the TATA box around the -30 bp position and the Inr element at the TSS position. However, recent studies indicate that the presence of these elements is not enough to explain the specific structural patterns of promoters. Promoter sequences without the TATA element still have a clear trough in the structural profile of DNA denaturation, although the trough is not as deep as that with TATA box [20]. This finding suggests that promoter sequences can denature more easily than non-promoter sequences, no matter whether they contain those elements or not. In our previous study [34], the analysis on CpG-related and non-CpG related promoters shows that the two different kinds of promoter sequences exhibit similar structural patterns. As a result, we may conclude that the structural features are not only affected by the specific elements such as TATA box, Inr element and CpG island, but also by long-range combinations of nucleotides. Third, as shown in Figure 2, there are two types of structural features according to the profile patterns: either a large trough stretching over the TSS region is visible, such as the plots in the first two rows, or a peak is visible, such as the features in the last three rows. These distinctive peak or trough regions are possibly useful for identifying TSSs. Furthermore, compared with the non-promoter sequences, the structural patterns of promoters with regard to some features are distinctive and clear, such as energy-related features, DNA-bending stiffness and DNA denaturation. By analyzing the structural models, we observe that these features are closely related with the stability of promoter sequences. In contrast, the profiles of B-DNA twist, protein deformation and protein-DNA twist are ambiguous and noisy. These structural features have different capabilities of differentiating promoters and non-promoters. These findings suggest that a variety of possible mechanisms may lead to promoter recognition by RNA polymerase. The binding of RNA polymerase could result from the DNA sequence preferences. Alternatively, promoter recognition may also be guided by the boundaries of the specific structural peaks or troughs around the TSSs.

Comparison of classification performance of different structural features

The above analysis implies that the structures of DNA sequences are important signals in promoter recognition of RNA polymerase, and integrating structural features may be helpful for promoter prediction. In order to identify the most promising features, we move forward to test how well each individual feature can differentiate promoters and non-promoters. The experiments are conducted on the DBTSS human promoter dataset. For each feature, we respectively convert the positive promoter and negative non-promoter sequences into corresponding numerical feature vectors. Because of the outstanding classification performance of the support vector machine (SVM) classifier, we apply it to discriminate promoters from non-promoters. We train thirteen SVM classifiers corresponding to all these different structural features and test their classification performance, which is evaluated by three measures, including sensitivity, specificity and F-measure.

Figure 3 shows the five-fold cross-validation performance of all these thirteen structural features on the human dataset. The experimental results indicate that these structural properties are indeed different in predictive power for promoters. Generally, protein deformation, DNA-bending stiffness and protein-DNA twist have fairly high sensitivity values, but specificity values are slightly lower than those of the other features. On the contrary, duplex free energy, DNA denaturation, Z-DNA and DNA-bending stiffness strike a balance between sensitivity and specificity, leading to much higher values of F-measure.

Ranking structural features by filter methods

To identify the discriminative structural features for promoter prediction, we adopt different filter feature selection methods to select feature subsets from the thirteen kinds of structural features. First, each sequence (length 251 bp) is converted to thirteen numerical vectors corresponding to the thirteen different structural features. Since the values of these features are at different levels, we respectively normalize these vectors into the range 0[1]. Second, we combine these feature vectors into an integrated vector whose dimension is 13*251, and add a Boolean dimension as class label with 'true' for promoters and 'false' for non-promoters. Third, various filter selection methods based on four evaluation metrics, including information gain (IG), Chi Square (CHI), ReliefF and Correlation-based Feature Selection (CFS), are used to select the most discriminative dimension subsets (see Methods). Then, all these dimensions are ranked based on the scores that are assigned by the feature selection methods. For the selected dimension subset, we calculate the ratio of each feature in the subset, that is, the ratio of the number of dimensions related to this feature over the total number of dimensions in the selected dimension subset. Only the top-ranked features are used for further classification purpose. In order to obtain a general trend, for each evaluation criterion, we select several dimension subsets with growing dimensionality, including 100, 200, 300, 400 and 500. Furthermore, we use the SVM classifier to evaluate the predictive ability of each selected subset.

Ranking structural features by wrapper methods

Wrapper methods provide alternatives to perform multivariate feature selection, which are dependent on the target classifiers whose accuracies are utilized to evaluate the candidate feature subsets. Thus, the selected feature subsets are closely related to specific classifiers, and the classification performance is usually better than that of filter methods. As it is prohibitively costly to evaluate all possible subsets, we adopt the genetic search strategy to search for the optimal subsets, in combination with SVM and kNN classifiers respectively.

Table 3 summarizes the performance of the wrapper feature selection methods. The first two rows report the classification results with the wrapper feature selection, while the third and fourth rows are the classification results without feature selection. From the table, we observe that the classifiers with the wrapper feature selection achieve much better classification performance than those without feature selection. Compared with the results of the filter feature selection methods, the wrapper methods are better in most cases, except ReliefF. For the wrapper methods, we also compute the ratio of each feature in the selected subset as mentioned above. From the distribution plots in Figure 4, the subsets selected by the wrapper methods indicate the similar trend as the filter methods. The ratios of duplex free energy, DNA-bending stiffness and DNA denaturation in the selected subsets are much higher than those of the other features, indicating that they are more predictive for promoter prediction.

Classifier	Wrapper	Specificity	Sensitivity	F-measure	ROC score	Dimensionality
SVM	SVM	0.761	0.723	0.742	0.786	320
KNN	KNN	0.705	0.731	0.718	0.745	300
SVM	NULL	0.733	0.72	0.731	0.732	All
KNN	NULL	0.637	0.753	0.69	0.723	All

The performance comparison of two classifiers (SVM and kNN) for promoter classification: with the wrapper feature selection (the first two rows) vs. without wrapper feature selection (the last two rows).

Classification performance comparison with other existing methods

In this section, we evaluate the effectiveness of the selected structural feature subsets in promoter prediction (termed as FSPP in this paper), and compare them with nine existing promoter prediction methods. We select five classical methods that perform well in previous comparative studies [12, 35], including FirstEF [36], Eponine [37], DragonGSF [7], McPromoter [8] and ARTS [38]. These methods are all traditional, in the sense that they were mainly built on sequence compositional features, such as CpG island, TATA box and a number of motifs. FirstEF is based on quadratic discriminant analysis of promoters, the first exon and the first donor site. Eponine constructs a relevance vector machine based on TATA box in a G+C rich domain. DragonGSF utilizes artificial neural network. McPromoter also adopts neural network to predict promoters, but uses combined features from different segments. By using support vector machine, ARTS integrates several compositional features and two structural features. Meanwhile, we include four newly proposed methods that depend on structural features to predict promoters. EP3 directly uses a deviation from the average structural value to locate the promoter regions [20]. Observing the distinct structural patterns of promoter regions, PNNP develops a pattern-based promoter prediction method [34]. Based on DNA melting temperature, Profisi predicts promoters using simple thresholding [39]. ProSOM applies self-organizing maps to distinguish promoters from non-pomoters [40].

Core promoter datasets

To extensively analyze the structural features of promoter sequences, we retrieve a collection of promoter sequences from DBTSS (version 7.0) [41]. DBTSS is built on experimentally validated TSSs, which is mainly based on full-length cDNA transcripts, and the 5'-ends of oligo-cap selected cDNAs are experimentally determined [42]. This dataset provides the best training data to recent computational studies in genomic annotation and promoter analysis. DBTSS includes the promoter sequences of several species. Here, our analysis is based on one of the largest dataset, the human dataset. We extract the sequences from 200 bp upstream to 50 bp downstream flanking TSSs as the promoter sequences. Through filtering, we obtain 11,682 experimentally validated human TSSs. Correspondingly, we retrieve the same number of non-promoter sequences. In order to avoid the position bias in extracting sequences from the human genome, we build a randomized non-promoter dataset by shuffling the real promoter sequences. From the promoter dataset, we obtain a first-order Markov model that preserves the dinucleotide frequencies. Furthermore, based on the Markov model, the randomized non-promoter sequences are generated.

To unbiasedly compare the performance of different promoter prediction methods, the comparative evaluation is conducted on another dataset - the CAGE dataset, which is different from our training dataset and has a wider coverage of human genome. The CAGE dataset is based on the cap analysis gene expression (CAGE) technique and is retrieved from the Riken institute website [3]. As previous study, only tag clusters identified by two or more tags are included in our analysis [35]. Mapping these tags to human genome allows us to obtain 181,046 unique human TSSs. The whole genome (hg19) is retrieved via the UCSC Genome Browser [43].

Calculating structural profile

Because of important roles in different key biological processes, many studies have focused on biophysical understanding of the intrinsic structural properties of DNA sequences. Recent experimental analyses have shown that, in some manner, eukaryotic core promoters are indeed marked by specific structures comparing with coding or non-regulatory sequences. For instance, the duplex free energy content at TSS positions is much higher than that at the other positions. As a region with high free energy is more active than a region with low thermodynamic energy content [21], the stable regions may provide a contrasting background for the highly unstable peaks, and make the peaks prominent for guiding transcription apparatus to locate the appropriate transcription sites [34]. Also, the propeller twist capacity measures the rigidity of a sequence [44]. A region with high negative propeller twist is rigid, which makes this region hard to wrap around a nucleosome and facilitates RNA polymerase to bind in this area [45]. Besides these two features, we also investigate other eleven structural properties, including stacking energy [26], DNA denaturation [22], duplex disrupt energy [24], protein deformation [25], Z-DNA [23], bending stiffness [27], A-philicity [28], nucleosome position [29], protein-DNA twist [25], B-DNA twist [30], and bendability [31].

In order to quantitatively analyze structural properties of promoters, we convert the retrieved promoter and non-promoter sequences into numerical structural profiles corresponding to different structural features. For each feature, the calculation of structural profile is divided into two steps, as illustrated in Figure 1. We first transform each DNA sequence into a numerical vector. Each dinucleotide (or trinucleotide) is replaced by its corresponding structural value [18, 20]. The transformation models for different structural features are obtained from various bio-chemical experiments, and are summarized as the validated conversion schemas by Florquin et al. [18]. Specifically, since genome-wide nucleosome maps for different species have been recently published, we use the new in vivo human nucleosome positioning data from Schones et al.'s study to calculate the structural profiles related to nucleosome positioning [46]. We take all the nucleosome-bound sequences from their collection, and then align these sequences with regard to their centers. By using the same method as in the previous study [47], we obtain the new nucleosome parameters. Accordingly, the new structural profiles related to nucleosome positioning are calculated. Second, we use a sliding window approach to smooth the raw profiles, with a window-size of 3 nt and a step-size of 1 bp. When the window slides along a sequence, a vector of structural values is output. We plot the average value on each position to get the structural profile of the sequence with regard to each feature. At last, every sequence has thirteen structural profiles corresponding to all these structural features.

Feature selection framework

Feature selection has been an important issue in machine learning, data mining and statistics fields, and has drawn much attention in diverse bioinformatics applications [48]. Here, to gain a deeper insight into the structural properties of promoters, to avoid the overfitting problem and to improve the performance of promoter prediction models, we adopt different feature selection techniques to analyze these structural features we studied. We identify features that are highly correlated with promoter sequences and effective in promoter recognition. According to the combination mode between feature selection and classification process, feature selection techniques generally fall into the following two categories: wrapper and filter methods [49].

A wrapper method depends on a target classifier to identify the optimal feature subset that provides the target classifier with the best classification performance. The feature selection process consists of a search in the feature space guided by the performance of the target classifier. While bringing good accuracy for the final classifier, wrapper methods are computationally expensive. Filter methods are independent of any target classifier. These methods select features by looking only at the intrinsic properties of the data, such as class separability or correlation between features and classes. First, a feature relevance score is calculated, and the low-scoring features are filtered. Afterwards, selected features can be presented as input to a classification algorithm. Hence, the filter feature selection methods are computationally efficient and can easily scale to high-dimensional datasets.

Generally, feature selection techniques differ from each other with respect to two main aspects: evaluation criterion of features and search strategy in the feature space. To gain a systematical and unbiased analysis, here we take advantage of various feature selection techniques based on different evaluation functions in conjunction with search strategies, as shown in Figure 5. For filter methods, four different evaluation criteria are adopted to evaluate features, assigning a score to each feature that suggests how valuable the feature is for classification. The four evaluation criteria include two univariate criteria: information gain (IG) and Chi Square (CHI), and two multivariate criteria: ReliefF and Correlation-based Feature Selection (CFS) [48]. Then, all features are ranked by their scores and the highly relevant features are obtained according to a given threshold. At last, these features subsets selected by different filter methods are evaluated by classification performance. As the calculation of correlation coefficients among features is costly and it is prohibitively expensive to rank each feature subset, we adopt the genetic search strategy for CFS, which is different from the other criteria. For wrapper models, the target classifier and search strategy are critical elements. We respectively use SVM and kNN as target classifiers, utilizing the accuracy of classification to measure the quality of selected features. Meanwhile, the genetic search algorithm is applied to search for optimal feature subsets in the feature space. This combination is due to the following reasons. In literature, SVM classifier is widely used because of its high classification accuracy [50], and the instance-based classifier kNN is quite efficient [51]. Genetic search is effective in high-dimensional search space. All our feature selection methods are developed based on an open-source data-mining tool, Weka [52]. Finally, we report the 5-fold cross validation estimate of classification accuracy.

Performance measures

TP, FP and FN represent the numbers of true positives, false positives and false negatives respectively. Generally, sensitivity is the proportion of correct predictions of TSSs over all experimental TSSs. Specificity is the proportion of correct prediction of TSSs out of all counted positive predictions. The higher the value of Se is, the more false positives may be reported, and the lower the value of Sp is. It is a trade-off to balance sensitivity and specificity. F-measure is a single measure that can compare prediction methods with different sensitivity and specificity. Meanwhile, the quality of a classifier can be evaluated by ROC score, which computes the area under the ROC curve [53]. The value of the ROC score ranges from zero to one, with a score of 0.5 corresponding to random guess and a score of 1.0 indicating perfect separation. The ROC score indicates the accuracy with which a classifier separates promoters from non-promoters.