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Direct Targeting of Light Signals to a Promoter Element-Bound Transcription Factor

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Science  05 May 2000:
Vol. 288, Issue 5467, pp. 859-863
DOI: 10.1126/science.288.5467.859

Abstract

Light signals perceived by the phytochrome family of sensory photoreceptors are transduced to photoresponsive genes by an unknown mechanism. Here, we show that the basic helix-loop-helix transcription factor PIF3 binds specifically to a G-box DNA-sequence motif present in various light-regulated gene promoters, and that phytochrome B binds reversibly to G-box–bound PIF3 specifically upon light-triggered conversion of the photoreceptor to its biologically active conformer. We suggest that the phytochromes may function as integral light-switchable components of transcriptional regulator complexes, permitting continuous and immediate sensing of changes in this environmental signal directly at target gene promoters.

Plants use a set of sensory photoreceptors to monitor the environment for informational light signals (1). The phytochrome (phy) family, comprising five members (phyA to phyE) inArabidopsis, track the red (R) and far red (FR) light wavelengths by virtue of their capacity for photoinduced, reversible switching between two conformers: the R-absorbing, biologically inactive Pr form and the FR-absorbing, biologically active Pfr form. Each phy molecule is a dimer of subunits that consist of a ≈125-kD polypeptide with a covalently bound tetrapyrrole chromophore that is autocatalytically attached by the apoprotein (2). Light-driven Pfr formation induces changes in the expression of numerous genes underlying various aspects of plant photomorphogenesis, and promoter analysis has identified a number of cis-acting light-responsive elements (LREs) and some cognate DNA-binding proteins involved in regulating expression (3).

Considerable progress has been made in recent times toward identifying molecular components potentially involved in early steps in the signaling pathways linking the phytochromes to photoresponsive genes. Evidence from photoreceptor mutants inArabidopsis indicates that individual members of the phy family have differential photosensory and/or physiological functions in controlling development (4–7), and genetic screens have identified several loci specific to either phyA or phyB signaling pathway segments (8–13). Molecular cloning of two of these loci, FAR1 andSPA1, specific to phyA signaling, has revealed that they encode nuclear proteins (13, 14). Yeast two-hybrid screening for phytochrome-interacting proteins has identified PKS1, a cytoplasmic protein (15), NDPK2, a nucleoside diphosphate kinase (16), and PIF3, a nuclear-localized basic helix-loop-helix (bHLH) protein (17). The functions of PKS1 and NDPK2 in phytochrome signaling remain to be determined. However, because PIF3 belongs to the bHLH superfamily of transcription factors (18,19), the possibility of a direct signaling pathway from the photoreceptor to target genes is suggested. This suggestion is consistent with recent evidence that phyA and phyB are induced to translocate from the cytoplasm to the nucleus upon Pfr formation (20, 21). To explore this possibility, we examined whether PIF3 has sequence-specific DNA binding activity and, if so, whether phyB would interact with DNA-bound PIF3.

Using a random binding site selection (RBSS) procedure, we identified a palindromic hexanucleotide DNA sequence—CACGTG, known as a G-box motif (3,22–26)—as the core PIF3 target element (Fig. 1A). The specificity of this interaction was verified by electrophoretic mobility shift assay (EMSA), with the use of a G-box containing probe (G-wt) representative of those selected by RBSS (Fig. 1B), and recombinant PIF3 synthesized in the TnT in vitro transcription-translation system. Figure 1C shows that the low-mobility complex formed in the presence of thePIF3 template–programmed TnT reaction (lane 3) was effectively competed by unlabeled G-wt probe (lanes 4 to 6), but not an unlabeled mutant probe (lanes 7 to 9) containing a single T to G substitution in the G-box (G-mut, Fig. 1B). By contrast, the higher mobility complex formed by an endogenous TnT reaction component (Fig. 1C, lane 2) was competed equally well with G-wt and G-mut unlabeled probes. The data show that PIF3 does indeed bind DNA, with target sequence specificity characteristic of the bHLH family, and probably as a homodimer, on the basis of the known structure of DNA-bHLH protein complexes (27). The G-box motif is found in a variety of light-regulated genes and has been implicated in the regulation of some by functional assay (3,22–26). This motif is a representative of the more general E-box motif, CANNTG, considered to be the core consensus sequence for bHLH proteins in nonplant systems (18,19). The PIF3 bHLH domain alone is sufficient for sequence-specific binding to the G-box, similar to other bHLH proteins (Fig. 1D) (19, 27).

Figure 1

PIF3 is a sequence-specific DNA-binding protein that targets G-box motifs through its bHLH domain. (A) Summary of DNA sequences selected by random binding site selection (RBSS) (45) with either E. coli–produced GST:PIF3:flag protein (top) or TnT-expressed His6:PIF3 protein (bottom) (42). The consensus-selected G-box is indicated in bold type. (B) Upper-strand nucleotide sequences of the probes used for the experiments in (C) and (D). The G-box sequence is highlighted in bold type. G-wt was selected by RBSS. G-mut contains a point mutation in the G-box sequence (underlined). The nucleotides added for probe labeling are indicated in lowercase. (C and D) Sequence specificity of PIF3 DNA-binding activity determined by competitive EMSA. (C) PIF3 binding to labeled G-wt probe is competed specifically by cold G-wt but not by cold G-mut. A nonspecific complex formed by a TnT component in the absence of PIF3 (*) is competed equally by G-wt and G-mut probes. Lane 1, no protein; lane 2, mock-translated TnT; lanes 3 to 9, PIF3. The binding complexes were competed by the addition of none (lane 3), 5× (lanes 4 and 7), 25× (lanes 5 and 8), and 125× (lanes 6 and 9) molar excess of unlabeled G-wt (lanes 4 to 6) or G-mut (lanes 7 to 9). (D) A truncated fragment of PIF3 containing only the bHLH domain fused to GST (G:bhPIF3) binds to the labeled G-wt probe, and this binding is also competed by cold G-wt, but not by cold G-mut. Lane 1, PIF3; lanes 2 to 8, G:bhPIF3. The binding complexes were competed by the addition of none (lane 2), 5× (lanes 3 and 6), 25× (lanes 4 and 7), and 125× (lanes 5 and 8) molar excess of unlabeled G-wt (lanes 3 to 5) or G-mut (lanes 6 to 8). Proteins responsible for the binding complexes are indicated schematically on the right. Cross-hatching indicates bHLH domain. Gray box indicates GST protein. FP, free probe; (*) nonspecific binding complex.

Full-length, chromophore-conjugated phyB interacts with PIF3 that is not bound to DNA only upon conversion to the Pfr form (28). To determine whether phyB would bind to PIF3 that had formed a complex with its DNA target site, we performed EMSA with PIF3 and phyB together (Fig. 2A). Neither the PHYB apoprotein nor photoactive phyB in either conformer interacted directly with the DNA probe (Fig. 2B, lanes 3 to 5). Similarly, neither PHYB nor the phyB Pr form (PrB) altered the mobility or abundance of the PIF3-DNA complex when added to that complex, indicating the absence of any interaction (Fig. 2B, lanes 7 and 8). By contrast, R irradiation of chromophore-conjugated phyB induced formation of a discrete, lower mobility complex, presumably corresponding to a ternary complex between PIF3, phyB, and the DNA probe (Fig. 2B, lane 9). The data indicate, therefore, that phyB does indeed bind specifically to DNA-bound PIF3, but only upon R light–induced conversion to the Pfr form (PfrB).Figure 2B also shows that phyB does not interact with the bHLH domain of PIF3 (G:bhPIF3) when this isolated domain is bound to its target sequence (lanes 10 to 13). The data indicate, therefore, that the conformer-specific recognition of PIF3 by phyB requires molecular determinants outside the DNA-binding domain.

Figure 2

PIF3 simultaneously binds G-box DNA and the active form of phyB (PfrB). (A) Design of experiments in (B). PHYB refers to full-length phytochrome B apoprotein. phyB refers to photoactive phytochrome B, after chromophore attachment to PHYB, depicted by the small black rectangle (42). After coincubation of proteins with labeled G-wt probe, the samples were given a pulse of FR or R (46) and incubated on ice in the dark (Dk) for 2 additional hours before EMSA. (B) The binding complex formed between PIF3 and the G-wt probe is shifted in the presence of R-irradiated photoactive phyB, and this supershifted complex is dependent on full-length PIF3. Lane 1, no protein; lane 2, mock-translated TnT; lanes 3, 7, and 11, 2 μl of PHYB; lanes 4, 5, 8, 9, 12, and 13, 2 μl of phyB; lanes 6 to 9, PIF3; lanes 10 to 13, G:bhPIF3. (C) Experimental design for (D). Either phyB alone (lanes 3 and 4) or PIF3 and phyB together (lanes 5 to 12) were incubated for 3 hours in the dark (Dk) after being given a R and/or FR pulse, before G-box probe addition and EMSA. After an initial R pulse (R) (lanes 3 and 5 to 8) a FR pulse was given either immediately [R + FR(0)], after 1 hour [R + FR(1)], or 2 hours [R + FR(2)] (lanes 6, 7, and 8, respectively). Conversely, after an initial FR pulse (FR) (lanes 4 and 9 to 12), a R pulse was given immediately [FR + R(0)], after 1 hour [FR + R(1)], or 2 hours [FR + R(2)] (lanes 10, 11, and 12, respectively). (D) The R-induced shift in the PIF3:G-wt probe binding complex caused by the presence of phyB is photoreversible. Lane 1, mock-translated TnT; lanes 2 and 5 to 12, PIF3; lanes 3 to 12, 2 μl of phyB. Proteins responsible for the binding complexes are indicated to the right. FP, free probe; (*) nonspecific binding complex; PfrB, biologically active form of phyB, formed by R pulse.

To determine whether the R light–induced binding of phyB to DNA-bound PIF3 was reversible, we examined the effects of FR pulses given after an initial R pulse on the ternary complex detected by EMSA. The amount of R-induced complex was rapidly reduced by subsequent exposure to FR (Fig. 2D), indicating that the interaction triggered by Pfr formation was rapidly reversed by reconversion to Pr. These data indicate that phyB recognition of DNA-bound PIF3 requires maintenance of the photoreceptor in the biologically active (Pfr) form.

The G-box motif is neither present in all light-regulated promoters, nor is it confined to light-regulated genes. On the contrary, it is found in a broad range of plant gene promoters responsive to a diversity of nonlight-related stimuli (3,22–26, 29, 30). Moreover, most studies aimed at identifying plant DNA-binding proteins that recognize this motif report the cloning of bZIP class factors rather than bHLH proteins (22, 30). To address this apparent complexity in relation to PIF3, we examined whether PIF3 was capable of recognizing the G-boxes in photoresponsive genes in the context of their native flanking sequences. This is pertinent because the nucleotides flanking the core hexamer E-box motif have been shown to influence the specificity of bHLH family-member recognition of binding sites containing this core motif (31, 32). Figure 3B shows that the G-box–containing sequences from the promoters of four light-regulated genes,RBCS-1A, CCA1, LHY, andSPA1, all interacted effectively with PIF3, despite deviations from the consensus sequence of the PIF3 binding site (Fig. 1A) at the positions flanking the CACGTG hexanucleotide core in some cases (Fig. 3A). To determine whether PIF3 might recognize non–G-box motifs in other functionally defined LREs in photoresponsive genes, we examined PIF3 binding to the GT1, Z, and GATA motifs representing consensus sequences from several light-regulated promoters (33). PIF3 exhibited no detectable interaction with these motifs (Fig. 3C), further verifying the sequence-specific nature of the G-box recognition. Together the data indicate that PIF3 is indeed capable of sequence-specific binding to the G-box–containing promoters of a variety of light-regulated genes.

Figure 3

PIF3 binds to G-box–containing promoter sequences from various light-regulated genes. (A) Upper-strand nucleotide sequences of the probes used in the EMSA experiments in (B) and (C). The probes contain G-box and surrounding sequences fromRBCS-1A (25), CCA1 (38,39), LHY (40), and SPA1(14) promoters. The G-box sequence is highlighted in bold and the coordinates are in parentheses. (B) Competition of PIF3 binding to G-wt by the different G-box sequences from the four light-regulated genes. Lanes 1 and 6, mock-translated TnT; lanes 2 to 5, 7 to 16, PIF3; lanes 3, 8, 11, and 14, 5× molar excess of cold probe; lanes 4, 9, 12, and 15, 25× molar excess of cold probe; lanes 5, 10, 13, and 16, 125× molar excess of cold probe; lanes 3 to 5,RBCS-1A/G-box cold probe; lanes 8 to 10,CCA1/G-box cold probe; lanes 11 to 13, LHY/G-box cold probe; lanes 14 to 16, SPA1/G-box cold probe. (C) PIF3 binding to labeled G-wt probe is competed only by the LRE that contains a G-box (47). Lanes 1 to 9, PIF3; lanes 2, 4, 6, and 8, 25× molar excess of cold probe; lanes 3, 5, 7 and 9, 125× molar excess of cold probe; lanes 2 and 3, GT1 cold probe; lanes 4 and 5, Z cold probe; lanes 6 and 7, G cold probe; lanes 8 and 9, GATA cold probe (47).

To determine whether PIF3 is necessary for the phytochrome-regulated expression of these genes, we examined the effect of continuous R light (Rc) on their mRNA levels in wild-type and PIF3-antisense (17) seedlings. The rapid (within 1 hour) Rc-induced increase in expression of CCA1 andLHY was reduced in the PIF3-antisense seedlings (Fig. 4, A and B). By contrast, the similarly rapid increase in SPA1 expression was unaffected in the antisense plants. Two more slowly induced genes also showed no difference in expression between wild-type and antisense plants. These were the G-box–containing gene RBCS-1A(34) and CHS (Fig. 4, A and B) for which there is no evidence of a functionally active, fully palindromic G-box in Arabidopsis (22, 35,36). On the other hand, the absence of the HY5 bZIP protein in the hy5 null mutant (37) caused no reduction in the photoresponsiveness of CCA1,LHY, or SPA1, but markedly reduced the induction of CHS (Fig. 4, C and D). Together these data suggest that there are multiple classes of promoters in phytochrome-responsive genes: G-box–containing promoters that require PIF3 for responsiveness; G-box–containing promoters that do not require PIF3 for responsiveness, despite their capacity to bind PIF3 in vitro; and promoters lacking evidence of functionally active G-boxes that do not require PIF3 for responsiveness, but nevertheless do require the bZIP factor HY5, considered to be a G-box–binding protein, for responsiveness.

Figure 4

The light-induced expression ofCCA1 and LHY is reduced in the PIF3Arabidopsis antisense line A22. (A) RNA blot analysis of CCA1, LHY, SPA1, andCHS mRNA levels in the A22 line (17) and its corresponding wild type (No-0) in Rc for increasing periods (48). (B) Quantitative determination of the relative levels of the transcripts shown in (A) (49). (C) RNA blot analysis of CCA1, LHY,SPA1, and CHS mRNA levels in a hy5mutant line [hy5-1 allele (37)] and its corresponding wild-type (Ler) (48). (D) Quantitative determination of the relative levels of the transcripts shown in (C) (49).

On the basis of this pattern of expression profiles, we suggest that a subclass of rapidly induced genes, represented byCCA1 and LHY, may be direct targets of phytochrome regulation through binding of the photoreceptor to PIF3, which is in turn bound to G-box promoter elements. Other subclasses of phytochrome-responsive genes apparently have alternative response pathways independent of PIF3. It is intriguing that CCA1 andLHY encode similar MYB-class proteins that have been implicated in phytochrome-regulated CAB gene expression and/or circadian clock regulation (38–40). It is possible, therefore, that PIF3 represents the entry point for phytochrome regulation of the plant circadian clock, as well as initiating one branch of the phytochrome-induced gene expression cascade (41).

The data presented here and elsewhere (17, 20, 21, 28) suggest that the phytochromes may integrate into, and function as photoswitchable components of, transcription-regulator complexes directly at target promoter sites after light-induced translocation from cytoplasm to nucleus (Fig. 5). The function of PIF3 in this scheme would be to recruit phyB specifically to the designated promoters. Regardless of the biochemical basis of the ensuing signaling transactions between phyB and the transcriptional machinery, the data suggest that plants have evolved a mechanism whereby an extracellular signal can be monitored continuously and directly by the control elements of target genes, thereby potentially permitting almost instantaneous modulation of transcription rates in response to changes in signal content.

Figure 5

Model depicting the proposed mechanism of phyB regulation of gene expression. R-induced conversion of phyB from its cytoplasmically localized, biologically inactive Pr form (PrB) to its active Pfr form (PfrB) triggers translocation to the nucleus (20, 21), where it binds to PIF3 that is constitutively nuclear (17) and bound as a presumptive dimer to G-box motifs in target promoters. Bound PfrB then activates (or represses) transcription either directly, by functioning as a coregulator in recruiting and/or biochemically or allosterically modifying components of the preinitiation complex (PIC) or associated factors (solid arrowhead), or indirectly, by biochemically or allosterically modifying the presumptive transcriptional regulatory activity of PIF3, which then in turn recruits coregulator or PIC components (open arrowheads). Subsequent reconversion by FR of bound PfrB to PrB causes rapid dissociation of the photoreceptor from DNA-bound PIF3, disrupting the enhanced (or reduced) transcriptional activity of target genes. In the short term, subsequent reconversion by R of PrB to PfrB, either before dissociation or nearby in the nucleoplasm, would rapidly reestablish the previous enhanced (or reduced) transcriptional state.

  • * To whom correspondence should be addressed. E-mail: quail{at}nature.berkeley.edu

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