Diversity of cis-regulatory elements associated with auxin response in Arabidopsis thaliana

Abstract

The phytohormone auxin regulates virtually every developmental process in land plants. This regulation is mediated via de-repression of DNA-binding auxin response factors (ARFs). ARFs bind TGTC-containing auxin response cis-elements (AuxREs), but there is growing evidence that additional cis-elements occur in auxin-responsive regulatory regions. The repertoire of auxin-related cis-elements and their involvement in different modes of auxin response are not yet known. Here we analyze the enrichment of nucleotide hexamers in upstream regions of auxin-responsive genes associated with auxin up- or down-regulation, with early or late response, ARF-binding domains, and with different chromatin states. Intriguingly, hexamers potentially bound by basic helix-loop-helix (bHLH) and basic leucine zipper (bZIP) factors as well as a family of A/T-rich hexamers are more highly enriched in auxin-responsive regions than canonical TGTC-containing AuxREs. We classify and annotate the whole spectrum of enriched hexamers and discuss their patterns of enrichment related to different modes of auxin response.

Keywords: ARF; AuxRE; Auxin; bHLH; bZIP; bioinformatics; chromatin states; transcriptional regulation.

Fig. 1.

The pipeline for genome-wide association analysis for putative cis-elements associated with auxin response. (A) Three steps of the pipeline: (1) exhaustive hexamer search in the upstream regions; (2) analysis of association between the hexamer’s presence in the upstream region and auxin up- (down-) regulation of the gene; and (3) meta-analysis across all data sets. (B) Permutation test scheme, part of step (3).

Fig. 2.

Scheme of the auxin response network reconstructed on the basis of predicted AuxREs. (A and B) Cis-regulatory elements conferring early auxin response (Table 3). (A) Activation of transcription. (B) Inhibition of transcription. (C–E) Potential coupling hexamers in composite AuxREs out of the whole list of auxin-associated cis-regulatory elements (Supplementary Table S2). The hexamers found significantly enriched within ARF5- (C); ARF2- (D), and ARF6-binding regions (E). The experimental data were taken from DAP-Seq analysis for ARF2 and ARF5 (O’Malley et al., 2016) and ChIP-Seq analysis for ARF6 (Oh et al., 2014). Pins were placed in random positions, as in this analysis we did not study the influence of orientation and relative position of the hexamers towards the TGTC-containing core.

Fig. 3.

Putative AuxREs within chromatin context. (A) The portion of putative AuxREs that were found significantly enriched in the upstream regions associated with nine chromatin states (Sequeira-Mendes et al., 2014). Significance was estimated via one-tailed Fisher’s exact test (see the Materials and methods). One hexamer can be enriched in more than one state. (B) The number of putative AuxREs specifically enriched in the chromatin state islands within the upstream regions of auxin-responsive genes relative to not auxin-responsive genes. Hexamers enriched in both up- and down-regulation are counted twice. A/T-rich hexamers are shown in gray.

Fig. 4.

Putative AuxREs specifically enriched within chromatin states 1, 2, 4, and 5 of auxin-responsive genes. Core promoters tends to possess transcriptionally active chromatin state 1, proximal promoters usually belong to chromatin state 2, and distal promoters to state 4 or state 5 (Sequeira-Mendes et al., 2014). (A) Association with transcriptional activation. (B) Association with transcriptional inhibition. A/T-rich hexamers are shown in gray. For details see Supplementary Table S4.