The orientation of transcription factor binding site motifs in gene promoter regions: does it matter?

Abstract

Background: Gene expression is to large degree regulated by the specific binding of protein transcription factors to cis-regulatory transcription factor binding sites in gene promoter regions. Despite the identification of hundreds of binding site sequence motifs, the question as to whether motif orientation matters with regard to the gene expression regulation of the respective downstream genes appears surprisingly underinvestigated.

Results: We pursued a statistical approach by probing 293 reported non-palindromic transcription factor binding site and ten core promoter motifs in Arabidopsis thaliana for evidence of any relevance of motif orientation based on mapping statistics and effects on the co-regulation of gene expression of the respective downstream genes. Although positional intervals closer to the transcription start site (TSS) were found with increased frequencies of motifs exhibiting orientation preference, a corresponding effect with regard to gene expression regulation as evidenced by increased co-expression of genes harboring the favored orientation in their upstream sequence could not be established. Furthermore, we identified an intrinsic orientational asymmetry of sequence regions close to the TSS as the likely source of the identified motif orientation preferences. By contrast, motif presence irrespective of orientation was found associated with pronounced effects on gene expression co-regulation validating the pursued approach. Inspecting motif pairs revealed statistically preferred orientational arrangements, but no consistent effect with regard to arrangement-dependent gene expression regulation was evident.

Conclusions: Our results suggest that for the motifs considered here, either no specific orientation rendering them functional across all their instances exists with orientational requirements instead depending on gene-locus specific additional factors, or that the binding orientation of transcription factors may generally not be relevant, but rather the event of binding itself.

Fig. 1

Schematic illustration of the consequences of the orientation reversal of an hypothetical, non-palindromic cis-regulatory transcription factor binding site motif. As the motif sequence is reversed in sequence direction on the opposing strand creating a reverse-complemented version of the original motif, an identical DNA interaction surface is created, but the associated transcription factor (TF) is required to bind in a 180° rotated orientation. As the TF may be asymmetric and possibly additional protein factors (denoted by light-red circles) associate non-symmetrically, the binding orientation relative to the transcription start site (TSS) of the downstream gene is reversed

Fig. 2

Visualization of the results statistics reported in Table 2 and (Additional file 2: Table S1). Percentages of motifs passing through the various filter criteria (C, D1, D2, and E) as explained in table legend 2 are plotted for the different upstream sequence intervals considered in this study and true motif sets (293 upstream elements, 10 core promoter elements). Results statistics are compared to sets of randomized motifs generated by either assuming base composition as observed in the respective upstream sequence interval (R1) or as observed in true motifs (R2) (see Methods, and Additional file 2: Table S1). Significant differences are annotated as “+”/”#” if percentages obtained for true motifs differed from R1/R2-randomized sets, respectively, with triple-symbols indicating p < 0.001, double-symbols p < 0.01, and single-symbols p < 0.05 after correcting for multiple testing

Fig. 3

Motif orientation preferences as a function of distance from the transcription start site (TSS). Based on mapping statistics alone, the percentage of motifs with significant (binomial test p < 0.05, Benjamini-Hochberg (BH) corrected) to upstream intervals of length 100 nt at different distances from the transcription start site (TSS). Statics were generated for the set of actual motifs (black line) as well as randomized motifs based on the composition of the respective interval (blue line) or based on motif-base compositions (magenta line). For randomized motifs, 10 repeats were performed and the average percentage of motifs with orientation preference computed. P-values indicate significant departure of the respective random expectation from true motifs (binomial test, BH-corrected). Motifs were required to map ten times or more to the respective upstream regional interval or ignored otherwise

Fig. 4

Dinucleotide orientational asymmetries in gene upstream regions. For five upstream regional intervals of length 100 nt (−500..-401, −400..-301,…,-100..-1), logarithmic (base 2) dinucleotide orientation ratios (DORs, see Methods, Eq. 4) of the observed-vs-expected frequency ratios of all possible dinucleotides of its forward relative to the respective reverse-complement version are plotted. Observed-vs-expected frequency ratios measure the departure of actually observed dinucleotide frequencies versus their estimated frequencies based on single base frequencies alone; i.e., treating them as independent events (see Methods for details). Thus, deviations from zero indicate evidence of conditional probability differences between the forward and reverse-complement dinucleotide version and are indicative of orientational preferences. For palindromic dinucleotides (AT,CG,GC,TA), this log-ratio computes as zero. Ratios are plotted for dinucleotide combinations with pairs constituting respective inverse ratios (e.g., TC/GA and GA/TC) necessarily resulting in symmetric graphs

Fig. 5

Preferred motif pair arrangements. For all 18 motif pairs with non-random arrangement distribution, relative frequencies were computed; i.e., counts per arrangement divided by the total count for all eight possible arrangements followed by a summation for each arrangement type across all 18 motifs. Plotted are the resulting summed-up relative frequencies for the two possible motif orders with regard to sequence position and orientation where “+” denotes forward, and “-“reverse-complement orientation. Preferences for co-directionally aligned orientations, i.e., both in forward or both in reverse-complement orientation are evident. The central plot entitled “same motif” shows the relative counts for same-motifs only (Note that the order does not matter and the associated frequencies are identical.), whereas the plot “different motifs” shows the data for two different motifs considered a pair