Selected NASCAR candidates.

<p>The first 8 entries correspond to the candidates that showed expression in medaka.</p><p>* = partial motif co-linearity, DF = “double flanked”, SF = “single flanked”</p><p>Selected NASCAR candidates.</p

NASCAR workflow.

<p>(1) Seeds perfectly matching between query (i.e. enhancer) and target (e.g. genomic window) sequence (small black segments) are extended up- and downstream (red segments) using a match/mismatch scoring scheme to generate a raw motif profile. Motifs that overlap the predefined window boundaries are also taken into account and virtually extend the window (grey areas). (2) As a next step, overlapping regions of the extracted raw motifs in the target sequence are determined (grey areas) and the smaller motif truncated whenever it overlaps a larger one (2 to 3). Motifs smaller than the initial seed size after truncation are discarded in this step. (3) Same filtering procedure is repeated in the query sequence for the processed profile (3 to 4). (4) Motifs below the noise threshold (bright blue segment) are discarded and the basic similarity (“PURE”) score calculated from the fully filtered motif profile (dark blue). (5) In addition, a pattern detection method searches for co-linear arrangements in the profile (grey area). Panel shows the same motif composition as (4) but in a co-linear configuration. This time, the motif below the noise threshold (bright pink) is kept as it is contained in a pattern. The score of the full pattern (all pink motifs) is subsequently added to the previously calculated basic score, resulting in the “COMB” score. For a given enhancer, the whole process is repeated window by window until the last window in the target sequence is reached.</p

Deletion of conserved motifs (grey area) from the predicted fish regions results in change of enhancer activity in both tested constructs.

<p><b>Schematic on the right shows the motif configuration in the human and medaka locus for hs1344 and hs865, respectively. The full grey area is deleted from the medaka enhancer and the remaining sequence tested for reporter expression. Images on the left show the reporter activity of the medaka constructs prior to and after the deletion.</b> Hs1344 ol2-1delta gains two symmetrical domains in the midbrain (red arrowheads), while hs865 ol2-1delta shows a loss of expression in the central part of the original domain.</p

Handling Permutation in Sequence Comparison: Genome-Wide Enhancer Prediction in Vertebrates by a Novel Non-Linear Alignment Scoring Principle

<div><p>Enhancers have been described to evolve by permutation without changing function. This has posed the problem of how to predict enhancer elements that are hidden from alignment-based approaches due to the loss of co-linearity. Alignment-free algorithms have been proposed as one possible solution. However, this approach is hampered by several problems inherent to its underlying working principle. Here we present a new approach, which combines the power of alignment and alignment-free techniques into one algorithm. It allows the prediction of enhancers based on the query and target sequence only, no matter whether the regulatory logic is co-linear or reshuffled. To test our novel approach, we employ it for the prediction of enhancers across the evolutionary distance of ~450Myr between human and medaka. We demonstrate its efficacy by subsequent <i>in vivo</i> validation resulting in 82% (9/11) of the predicted medaka regions showing reporter activity. These include five candidates with partially co-linear and four with reshuffled motif patterns. Orthology in flanking genes and conservation of the detected co-linear motifs indicates that those candidates are likely functionally equivalent enhancers. In sum, our results demonstrate that the proposed principle successfully predicts mutated as well as permuted enhancer regions at an encouragingly high rate.</p></div

Predicted candidates do not only differ in their motif configuration but also their conservation levels.

<p>Hs1344 shows significant motif specific conservation among teleosts, hs1049 and hs882 also among placental mammals. Together with the co-linear configuration and the orthologous gene(s) in flanking position this indicates that the motifs shared between human and medaka are likely to be orthologous counterparts. The random motifs (light colours) show very diverse conservation levels, which are generally lower within both clades.</p

Motif composition and reporter gene activity of selected human VISTA enhancers.

<p>For all enhancers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141487#pone.0141487.s005" target="_blank">S5 Fig</a>. (A) Comparison of the known human enhancer sequence and the predicted enhancer in medaka. The coloured boxes represent the motifs identified by NASCAR to assess the similarity of each pair. Upper track always displays the motif positions in the human sequence (colour coded by position), lower track shows the configuration in the medaka region. All Motifs are draw in size relative to the used window size. Motif heights in the lower track represent the motif score, orientation (up/down) indicates the relative orientation (forward/reverse) compared to the query sequence. (B) Expression pattern of the human (hg19) or medaka (ol2-1) enhancers. Lens activity is part of the reporter construct and allows distinguishing between successful and negative injections. All pictures are taken at 10 days post injection (10dpi). In all cases both enhancers show strikingly similar pattern.</p

TFBS motif profile search (EEL) misses the enhancers found by NASCAR.

<p>Bar plot showing the score assigned by EEL to the enhancer identified by NASCAR and the top 25 ranked regions genome wide with respect to each human enhancer. Error bars show standard deviation.</p

Secondary constructs also show enhancer activity.

<p>For each enhancer, the two motif tracks in the middle show the motifs in the human sequence used for prediction of either of the two medaka regions. Grey bars between the tracks are motif projections to the other sequence, black bars indicate motifs shared between both predictions. (A) The secondary construct for hs1344 shows additional expression in more posterior regions of the brain but partially overlaps the domains of the primary construct. (B) The secondary construct for hs865 does not show significant overlap with the primary construct but the combined expression domain of both resembles much better the reported expression domain in mouse (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141487#pone.0141487.s005" target="_blank">S5 Fig</a>). (C) For hs882, one additional candidate was found in close proximity to the initial prediction but sharing almost no motifs. Nonetheless, hs882:ol2-2 shows enhancer activity in the brain like hs882:ol2-1.</p

P-values for Wilcoxon rank-sum-test of motif conservation in placental mammals and teleosts.

<p>One asterisk (*) denotes p-value ≤ 0.05</p><p>two asterisks (**) means p-value ≤ 0.01.</p><p>P-values for Wilcoxon rank-sum-test of motif conservation in placental mammals and teleosts.</p