PHYTOCHROME INTERACTING FACTORs (PIFs) are bHLH transcription factors that bind to G-box elements in the target genes to regulate their expressions downstream of phytochromes in Arabidopsis. PIF1 is a key negative regulator of light-dependent seed germination. A previous genome-wide PIF1 targeting analysis indicated that PIF1 binds 748 sites in imbibed seeds, only 59% of which possess G-box elements. This suggests the G-box is not the sole determinant of in vivo PIF1 targeting. It is possible that the targeting of PIF1 to specific sites is stabilized by PIF1-interacting Transcription Factors (PTFs) that bind other nearby sequence elements.
This study reports that PIF1 targeting sites are enriched with not only G-boxes but also with other hexameric sequence elements named G-box Coupling Elements (GCEs). One of these GCEs, GCE2 possesses an ACGT core and serves as a binding site for group A bZIP transcription factors which are known to inhibit seed germination under ABA signaling. PIF1 interacts with ABI5 and other group A bZIP transcription factors, and together they target a subset of PIF1 binding sites in vivo. Decoupling of elements by T-DNA insertion in co-targeted locus leads reduction of PIF1 binding to the target and subsequent reduction of the target gene expression. In vitro single molecule fluorescence imaging confirms that ABI5 facilitates PIF1 binding to DNA fragments possessing either multiple G-boxes or the GCE2 alone. Thus, this study demonstrates that in vivo PIF1 targeting to specific binding sites is determined by its interaction with PTFs and their binding to GCEs. Further, this study suggests that in seedlings, the DNA binding specificity of PIFs also might be determined by these mechanisms. Taken together, this study strongly suggests that the interaction between PIFs that bind to G-boxes and PTFs that bind to GCEs determines in vivo binding sites of PIFs.