Combinatorial Complexity in a Transcriptionally Centered Signaling Hub in Arabidopsis

Abstract

A subfamily of four Phytochrome (phy)-Interacting bHLH transcription Factors (PIFs) collectively promote skotomorphogenic development in dark-grown seedlings. This activity is reversed upon exposure to light, by photoactivated phy molecules that induce degradation of the PIFs, thereby triggering the transcriptional changes that drive a transition to photomorphogenesis. The PIFs function both redundantly and partially differentially at the morphogenic level in this process. To identify the direct targets of PIF transcriptional regulation genome-wide, we analyzed the DNA-binding sites for all four PIFs by ChIP-seq analysis, and defined the genes transcriptionally regulated by each PIF, using RNA-seq analysis of pif mutants. Despite the absence of detectable differences in DNA-binding-motif recognition between the PIFs, the data show a spectrum of regulatory patterns, ranging from single PIF dominance to equal contributions by all four. Similarly, a broad array of promoter architectures was found, ranging from single PIF-binding sites, containing single sequence motifs, through multiple PIF-binding sites, each containing one or more motifs, with each site occupied preferentially by one to multiple PIFs. Quantitative analysis of the promoter occupancy and expression level induced by each PIF revealed an intriguing pattern. Although there is no robust correlation broadly across the target-gene population, examination of individual genes that are shared targets of multiple PIFs shows a gradation in correlation from strongly positive, through uncorrelated, to negative. This finding suggests a dual-layered mechanism of transcriptional regulation, comprising both a continuum of binding-site occupancy by each PIF and a superimposed layer of local regulation that acts differentially on each PIF, to modulate its intrinsic transcriptional activation capacity at each site, in a quantitative pattern that varies between the individual PIFs from gene to gene. These findings provide a framework for probing the mechanisms by which transcription factors with overlapping direct-target genes integrate and selectively transduce signals to their target networks.

Arabidopsis SAURs are critical for differential light regulation of the development of various organs

“…Because previous studies have shown that PIFs positively regulated auxin biosynthesis in response to high temperature or shade (36)(37)(38), the degradation of PIFs should cause some of the decrease in auxin levels after light illumination in hypocotyls. The differential regulation of target genes by PIFs may be achieved by their cofactors, and this speculation is supported by previous ChIP-seq and RNA-seq data, which showed that PIFs may both positively and negatively regulate their target genes (16)(17)(18). Based on our transcriptomic analysis, thousands of genes are differentially expressed in cotyledons and hypocotyls (subgroup 7 in Dataset S6), and some may work as PIF cofactors.…”

“…In Arabidopsis, single or multiple loss-of-function mutants of PIF genes display short hypocotyls in red light and in the dark, indicating that PIFs are repressors of photomorphogenesis (13). Chromatin immunoprecipitation (ChIP)-chip and ChIP-Seq studies have shown that PIFs regulate thousands of downstream genes (14)(15)(16)(17)(18). These genes are released from the control of the PIFs upon illumination because light suppresses both the amounts and the activities of the PIFs in various ways (19).…”

Neighbor Detection Induces Organ-Specific Transcriptomes, Revealing Patterns Underlying Hypocotyl-Specific Growth

“…We confirmed the importance of PIFs beyond shadeinduced auxin production in cotyledons by comparing the auxin responsiveness of yuc2 yuc5 yuc8 yuc9 and pif4 pif5 pif7 mutants (Supplemental Figure 4F) (Nozue et al, 2011;Hornitschek et al, 2012;Hersch et al, 2014). Consistent with the additive function of PIF4 and PIF5 during shade avoidance (Lorrain et al, 2008;Hornitschek et al, 2012;Pfeiffer et al, 2014), both bound to a highly overlapping set of shade-induced genes (Figure 3). The rapid and strong overrepresentation of auxin-regulated genes among our shade-regulated gene list (Figures 2 and 4) prompted us to determine whether many of these genes are also bound by ARF transcription factors (Guilfoyle, 2015).…”

“…Moreover, given that many PIF target genes are themselves transcriptional regulators (Hornitschek et al, 2012;Oh et al, 2012;Pfeiffer et al, 2014), this finding suggests the involvement of other transcription factors later in the response. PIFs are known to share many target genes; for example, PIF4, PIF5, and PIF7 bind to the YUC8 promoter (Hornitschek et al, 2012;Li et al, 2012;Pfeiffer et al, 2014). We tested the degree of PIF binding overlap by reanalyzing genome-wide PIF4 ChIP (chromatin immunoprecipitation) data in etiolated seedlings .…”

A Negative Feedback Loop between PHYTOCHROME INTERACTING FACTORs and HECATE Proteins Fine-Tunes Photomorphogenesis in Arabidopsis

“…RNA was isolated from 3-d-old dark-grown wild-type Col-0, pif1, pifQ, and hec1 hec2 seedlings. RT-qPCR was performed for PIL1, PIL2, HFR1, FHL, SDR, XRT7, and AtHB-2, which are the direct targets of PIFs as previously shown (Zhang et al, 2013;Pfeiffer et al, 2014). Results show that the expression of all those PIF target genes is stimulated in the hec1 hec2 mutant compared with the wild type in the dark ( Figure 5B).…”