- © 2011 American Society of Plant Biologists. All rights reserved.
Abstract
FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1), two transposase-derived transcription factors, are key components in phytochrome A signaling and the circadian clock. Here, we use chromatin immunoprecipitation–based sequencing (ChIP-seq) to identify 1559 and 1009 FHY3 direct target genes in darkness (D) and far-red (FR) light conditions, respectively, in the Arabidopsis thaliana genome. FHY3 preferentially binds to promoters through the FHY3/FAR1 binding motif (CACGCGC). Interestingly, FHY3 also binds to two motifs in the 178-bp Arabidopsis centromeric repeats. Comparison between the ChIP-seq and microarray data indicates that FHY3 quickly regulates the expression of 197 and 86 genes in D and FR, respectively. FHY3 also coregulates a number of common target genes with PHYTOCHROME INTERACTING FACTOR 3-LIKE5 and ELONGATED HYPOCOTYL5. Moreover, we uncover a role for FHY3 in controlling chloroplast development by directly activating the expression of ACCUMULATION AND REPLICATION OF CHLOROPLASTS5, whose product is a structural component of the latter stages of chloroplast division in Arabidopsis. Taken together, our data suggest that FHY3 regulates multiple facets of plant development, thus providing insights into its functions beyond light and circadian pathways.
INTRODUCTION
Light is one of the most important environmental cues that control plant growth and development. As sessile organisms, higher plants have evolved a network of multiple photoreceptors, including phytochromes, cryptochromes, and phototropins, to sense changes in the ambient light environment in order to undergo adaptive growth and development. Among these photoreceptors, the red (R) and far-red (FR) sensing phytochromes (phys) are the best characterized (Neff et al., 2000). In Arabidopsis thaliana, phytochromes are encoded by a five-member gene family, designated PHYA-PHYE. Among these phytochromes, phyA is light labile, whereas phyB-phyE are light stable. phyA is the primary photoreceptor responsible for perceiving and mediating various responses to FR light; whereas phyB is the predominant phytochrome regulating responses to R light (Sharrock and Quail, 1989; Somers et al., 1991; Nagatani et al., 1993; Parks and Quail, 1993; Reed et al., 1993; Whitelam et al., 1993).
In the last two decades, great efforts have been made to extensively screen the signaling intermediates involved in phyA signaling, and several components responsible for the high-irradiance response branch of the phyA pathway have been identified, including FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL), and FHY3 and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) (Whitelam et al., 1993; Hudson et al., 1999; Desnos et al., 2001; Wang and Deng, 2002; Zhou et al., 2005). The roles of these regulators in phyA signaling have been elucidated in recent studies. FHY1 and FHL, two small plant-specific proteins, are required for nuclear accumulation of light-activated phyA since phyA is localized only in the cytosol of fhy1 fhl double mutants (Hiltbrunner et al., 2005; Hiltbrunner et al., 2006). FHY3 and FAR1 are the key transcription factors directly activating the transcription of FHY1 and FHL and thus indirectly control phyA nuclear accumulation and phyA responses (Lin et al., 2007, 2008).
FHY3 and FAR1 encode two homologous proteins that share extensive sequence similarities with MURA, the transposase encoded by the Mutator element of maize (Zea mays; Hudson et al., 2003), and with the predicted transposase of the maize mobile element Jittery (Lisch, 2002; Xu et al., 2004), both of which are members of the Mutator-like element (MULE) superfamily (Lisch, 2002). As a novel transposase-derived transcription factor family, FHY3 and FAR1 genes are stably integrated into the genome and show no detectable sign of terminal inverted repeats or other transposon-like structures (Hudson et al., 2003). Phylogenetic analyses indicate that the FHY3/FAR1-related gene family evolved from one or several related MULE transposases during the evolution of angiosperms through a process termed “molecular domestication” with a concomitant loss of the ability to transpose (Feschotte and Pritham, 2007; Lin et al., 2007). However, why plants have evolved this new type of transcription factors and whether they function in other biological processes remain largely unknown.
Recently, several reports have shed light on the functional diversities and regulatory modes of these transposase-derived transcription factors. First, FHY3 was found to be involved in independently gating phytochrome signaling to the circadian clock (Allen et al., 2006). This finding has been extended by a recent report that FHY3 and FAR1 are involved in maintaining the rhythmic expression of EARLY FLOWERING4 (ELF4), a key player of the central oscillator of the Arabidopsis circadian clock (Li et al., 2011). Second, two recent reports from our group both showed that FHY3 and FAR1 may work in concert with other cofactors to precisely regulate the expression of their target genes. In one study, ELONGATED HYPOCOTYL5 (HY5), a well-characterized bZIP transcription factor involved in promoting photomorphogenesis, was shown to physically interact with FHY3 and FAR1 and act as a repressor in regulating FHY1 and FHL transcription (Li et al., 2010), while in another study, FHY3, FAR1, and HY5 were shown to work together with CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), two MYB-related transcription factors that are the key components of the central oscillator, to generate the rhythmic expression of ELF4 (Li et al., 2011). Therefore, identification of more FHY3 direct target genes and elucidation of new roles of FHY3 in plant development will contribute to a better understanding of these transposase-derived transcription factors.
In this article, we used chromatin immunoprecipitation–based sequencing (ChIP-seq) analysis to identify the global FHY3 direct targets in the Arabidopsis genome. Our data revealed 1559 and 1009 FHY3 direct target genes in darkness (D) and FR light conditions, respectively, including the three previously reported FHY3 direct target genes FHY1, FHL, and ELF4. In addition, we show that FHY3 also directly binds to two motifs in the 178-bp repeats of the Arabidopsis centromeric regions. Moreover, we show that FHY3 plays a role in regulating chloroplast division by directly activating the expression of ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5), a member of the dynamic GTPase family involved in chloroplast division (Gao et al., 2003). Thus, our genome-wide analysis of FHY3 direct target genes provides insights into the functional diversity of this transposase-derived transcription factor family in plants.
RESULTS
Genome-Wide Identification of in Vivo FHY3 Binding Sites
To determine the global in vivo binding sites of FHY3, we performed ChIP-seq analysis using 35S:3FLAG-FHY3-3HA fhy3-4 transgenic lines in which the 3FLAG-FHY3-3HA fusion proteins could largely rescue the long hypocotyl phenotype of the fhy3-4 mutants (Li et al., 2011). The seedlings were grown in D or continuous FR light conditions for 4 d, and then chromatin fragments were prepared using monoclonal anti-FLAG antibodies. As a negative control, anti-FLAG ChIP assays were simultaneously performed using the wild-type (No-0) plants. Before constructing the ChIP DNA library for sequencing, we performed quantitative PCR (qPCR) analysis to check the quality of the precipitated DNA. As FHY1 and ELF4 are two well-characterized direct targets of FHY3 (Lin et al., 2007; Li et al., 2011), we examined whether the promoter fragments of FHY1 and ELF4 bound by FHY3 in vivo were enriched in our ChIP DNA samples. As shown in Supplemental Figure 1 online, the promoter fragments of FHY1 and ELF4 containing FHY3/FAR1 binding sites (FBSs) were specifically enriched in the anti-FLAG ChIP DNAs using 35S:3FLAG-FHY3-3HA fhy3-4 transgenic lines but not in those using the wild-type plants, indicating that our precipitated DNA samples were enriched for the real FHY3 binding fragments in vivo.
We then generated two DNA libraries, one for D and one for FR-grown samples, which were then subjected to ultra-high-throughput Solexa (Illumina) sequencing. A total of 5.5 and 4 million reads (35 bp per read) were obtained for the D and FR libraries, respectively, which were uniquely mapped to the Arabidopsis genome by adopting Model-based Analysis of ChIP-Seq (MACS) software (Zhang et al., 2008). Consequently, 2776 and 1968 uniquely assigned loci were identified by MACS from the D and FR libraries, respectively, as in vivo FHY3 binding peaks (which were called FHY3 binding sites; P value < 10−5) in the Arabidopsis genome (Table 1; see Supplemental Data Sets 1 and 2 online).
Summary of FHY3 Binding Sites in the Arabidopsis Genome
A subsequent detailed analysis revealed that among all these FHY3 binding sites, 63 and 59% (i.e., 1745 and 1171 loci) were assigned to particular Arabidopsis Genome Initiative loci (from −1000 bp of the transcription start site to the 3′ untranslated region) in D and FR, respectively, whereas 37 and 41% (i.e., 1031 and 797 loci) of the binding sites in D and FR, respectively, reside in intergenic regions (Figure 1A, Table 1; see Supplemental Data Sets 1 and 2 online). Of the binding sites assigned to genes, around half of the binding sites in both D and FR conditions are located in the gene promoter regions, whereas the other half are located in genic regions, including exons, introns, 5′ untranslated regions, and 3′ untranslated regions (Figure 1A). These binding sites were assigned to a total of 1559 and 1009 genes in D and FR conditions, respectively, which are hereafter referred to as FHY3 direct target genes (Figure 1B; see Supplemental Data Sets 1 and 2 online). Comparison of the FHY3 direct target genes in the two conditions revealed that a common set of 785 genes are bound by FHY3 in both D and FR light conditions (Figure 1B); therefore a total of 1783 genes are identified in this study as potential FHY3 direct target genes when D and FR data are combined.
Overview of the FHY3 Binding Sites in the Arabidopsis Genome.
(A) Distribution of FHY3 binding sites in the Arabidopsis genome.
(B) Venn diagram showing the number and overlap of FHY3 direct target genes in D and FR light conditions.
(C) Distribution of FHY3 binding sites in the five Arabidopsis chromosomes. The top blue bars and the bottom red bars on each chromosome represent the positions of the FHY3 binding sites in D and FR light conditions, respectively. An oval on each chromosome indicates the location of the centromere.
(D) FHY1, FHL, and ELF4, three experimentally characterized FHY3 direct target genes reported in the previous studies (Lin et al., 2007; Li et al., 2011), were well identified in our genome-wide study of FHY3 direct target genes. For each gene, the mapped ChIP-seq reads in D (black) and FR (red) libraries are shown in the top and middle rows, respectively, and a schematic representation of gene structure is shown in the bottom row.
As expected, the FHY3 binding sites in both D and FR light conditions are distributed more or less evenly across the five chromosomes (Figure 1C). However, unexpectedly, the binding sites are particularly enriched in the centromeric regions (Figure 1C), which will be discussed later. We then checked the FHY3 binding profiles for FHY1, FHL, and ELF4, three well-documented FHY3 direct target genes in previous reports (Lin et al., 2007; Li et al., 2011). As shown in Figure 1D, FHY3 binding signals were specifically enriched in the promoter regions, but rare in the transcribed regions of these three genes in both D and FR light conditions, indicating that our analysis correctly identified the known target genes of FHY3.
Analysis of FHY3 Binding Motifs in Promoter and Genic Regions
For those FHY3 binding sites assigned to genes, we next analyzed their locations in relation to the nearby transcription start sites. Our results revealed that most of the binding sites (52 and 50% in D and FR, respectively) are located in the gene promoter regions (within 1000-bp regions upstream the transcription start sites) (Figure 2A). In the promoter regions, the locations of the FHY3 binding sites are further skewed toward regions immediately upstream of the transcription start sites, with the peak regions being at −300 to +100 bp in both D and FR light conditions (Figure 2A). This distribution pattern is consistent with the determined FHY3 binding sites in the promoters of FHY1, FHL, and ELF4 (Lin et al., 2007; Li et al., 2011) and with the molecular function of FHY3 as a transcription factor.
FHY3 Binds to the FBS Motifs in the Gene Promoters in Vivo.
(A) FHY3 binding sites are highly enriched in the −300 to +100 bp of the promoter regions in both D and FR light conditions.
(B) Percentage of FHY3 binding sites with or without FBS motifs in D and FR light conditions.
(C) Typical FBS motif (CACGCGC) was identified as the only statistically overrepresented motif in the FHY3 binding regions in D and FR light conditions by MEME software.
(D) Distribution of FBS motifs around the FHY3 binding sites (−500 to +500 bp).
Previous studies showed that FHY3 binds to a short motif, CACGCGC, termed FBS present in the promoters of FHY1, FHL, and ELF4 (Lin et al., 2007; Li et al., 2011). Consistent with these reports, a large portion of the identified FHY3 direct target genes (57 and 49% in D and FR, respectively) contain one or more typical FBS motifs (Figure 2B). To investigate whether there is any other potential FHY3 binding motif, we used a motif-searching program, Multiple Em for Motif Elicitation (MEME) (Bailey et al., 2006), to discover statistically overrepresented motifs in the FHY3 binding regions that were assigned to genes. However, MEME failed to identify any other novel motif, and FBS was identified as the only statistically overrepresented motif in these FHY3 binding regions in both D and FR light conditions (Figure 2C). As the remaining FHY3 direct target genes (43 and 51% in D and FR, respectively) do not contain any FBS, FHY3 may bind to these regions through other motifs.
Moreover, analysis of the distribution pattern of FBSs around the FHY3 binding sites (−500 to +500 bp of each predicted FHY3 binding site) revealed that the frequency of a FBS increases to reach a peak roughly at the predicted FHY3 binding sites and decreases to a background level beyond approximately −200 to +200 bp from the FHY3 binding sites in both D and FR light conditions (Figure 2D). These results indicate that the experimentally confirmed FBS may represent a major FHY3 binding motif and that FBS motifs are mainly present within −200 to +200 bp of each predicted FHY3 binding site.
Analysis of FHY3 Binding Motifs in Intergenic Regions
As mentioned above, ~40% of the FHY3 binding sites in both D and FR conditions were not assigned to specific gene loci (which were called the binding sites in intergenic regions; Figure 1A). However, an outstanding feature of this portion of FHY3 binding sites is the particular enrichment in the centromeric regions (Figure 1C). This pattern is in contrast with the previously identified genome-wide binding patterns of several transcription factors, such as HY5, BRASSINAZOLE RESISTANT1 (BZR1), and PHYTOCHROME INTERACTING FACTOR 3-LIKE5 (PIL5; also known as PHYTOCHROME INTERACTING FACTOR1 [PIF1]) (Lee et al., 2007; Oh et al., 2009). Surprisingly, based on the definitions of the centromeric regions (Copenhaver et al., 1999; Kawabe et al., 2006), more than 70% of the FHY3 binding sites in intergenic regions in both D and FR are localized in the centromeric regions of five Arabidopsis chromosomes (Figure 3A; see Supplemental Data Sets 1 and 2 online).
FHY3 Binds to Two Novel Motifs in the 178-bp Centromeric Repeats.
(A) Distribution of the FHY3 binding sites in intergenic regions in D and FR light conditions.
(B) A 12-bp consensus sequence was discovered by MEME among the FHY3 binding sites in the intergenic regions in D and FR light conditions.
(C) Two novel motifs in the 178-bp centromeric repeats in Arabidopsis perfectly match the 12-bp consensus sequence shown in (B) and were thus named FBSC-1 and -2 (shown in red) for FHY3 binding sites in centromeric regions. The boxed nucleotides represent the conserved positions between these two motifs.
(D) Diagram of the wild-type and mutant (mut) centromeric fragments used as EMSA probes. Wild-type FHY3 binding sites in centromeric regions (FBSC-1 and -2) are shown in red, and substituted nucleotides in the mutant probes are shown in blue.
(E) EMSA assays showing that GST-FHY3N protein, but not GST by itself, specifically binds to the wild type (WT) but not the mutated FBSC-1 and FBSC-2 probes. Asterisk indicates nonspecific binding. FP, free probe.
(F) and (G) Distribution of FBSC-1 (F) and FBSC-2 (G) motifs around the FHY3 binding sites in the intergenic regions.
(H) Typical FHY3 binding loci in D (black) and FR (red) conditions in the centromeric regions of the five Arabidopsis chromosomes. The loci were visualized using the Integrative Genomics Viewer genome browser (Robinson et al., 2011).
To investigate whether there are any conserved motifs in these FHY3 binding sites, MEME software was adopted again to search the sequences from −500 bp to +500 bp of the respective FHY3 binding sites in intergenic regions. Interestingly, the FBS motif observed in the gene promoter regions was not found in these binding sites. By contrast, a 12-bp consensus sequence, A[T/A]ACAC[A/T][T/A][G/A][A/C]CA, was discovered among these binding sites in both D and FR light conditions (Figure 3B). An extensive BLAST search of GenBank revealed that two stretches within the 178-bp satellite repeat of Arabidopsis centromeres showed exact identity to this 12-bp consensus sequence (Figure 3C) (Nagaki et al., 2003; Kawabe and Nasuda, 2005). qPCR showed that the fragment of the 178-bp centromeric repeats was specifically enriched in ChIP DNAs using 35S:3FLAG-FHY3-3HA fhy3-4 transgenic lines compared with those using the wild-type plants (see Supplemental Figure 1 online), indicating that FHY3 indeed binds to the 178-bp centromeric repeats in vivo. Electrophoretic mobility shift assays (EMSAs) showed that GST-FHY3N (glutathione S-transferase fused with the N-terminal DNA binding domain of FHY3), but not GST alone, bound to two wild-type probes in vitro, whereas mutating the core sequence of these motifs (CAC → aaa) effectively abolished GST-FHY3N binding to these probes (Figures 3D and 3E), indicating that FHY3 indeed binds to these two motifs in vitro. Thus, these two motifs likely represent a new type of cis-element that confers specific binding for FHY3 to the centromeric repeats and were therefore named FBSC-1 and -2, respectively, for FHY3 binding sites in centromeres. Notably, FBSC-1 and -2 correspond to two of the three highly conserved regions identified in the centromere tandem repeats from 41 Arabidopsis ecotypes (Hall et al., 2003).
We then analyzed the distribution patterns of two FBSC motifs around each putative FHY3 binding site in the centromeric regions (−500 to +500 bp of each predicted FHY3 binding site). Interestingly, both of them display a similar pattern in D and FR light, showing consecutive peaks at intervals of 178 bp (Figures 3F and 3G), consistent with the distribution pattern of the 178-bp repeats in the Arabidopsis centromeric regions. Consequently, FHY3 binding sites showed a consecutive tandem pattern in the centromeric regions of five Arabidopsis chromosomes (Figure 3H).
Determination of FHY3-Regulated Genes in D and FR by Microarray Analysis
We next sought to determine which of the FHY3 direct target genes are transcriptionally regulated by FHY3 in D and FR light conditions. We used the FHY3p:FHY3-GR fhy3-4 transgenic lines expressing FHY3-glucocorticoid receptor (FHY3-GR) fusion proteins under the control of the FHY3 native promoter (Lin et al., 2007), in which, without dexamethasone (DEX), the GR-fusion proteins localize in the cytoplasm, whereas, when treated with DEX, the fusion proteins translocate into the nucleus (Lloyd et al., 1994). The seedlings were grown in D or continuous FR light for 4 d and then treated with DEX or equal volume of ethanol (referred to as MOCK) and grown in the same conditions for a further 2 h before the samples were harvested. RNA samples were extracted from these samples and then hybridized with Affymetrix Arabidopsis ATH1 genome arrays. Four independent biological replicates for each treatment were used for the microarray analysis.
Our microarray data identified 643 and 143 differentially expressed genes in D and FR light conditions, respectively (Figure 4A; see Supplemental Data Sets 3 and 4 online), using the Significance Analysis of Microarrays method (Tusher et al., 2001) with default parameters as well as a manual setting of false discovery threshold <0.027 in D and <0.022 in FR. Notably, 197 genes in D (31% of 643 genes) and 86 genes in FR light (60% of 143 genes) contain FHY3 binding sites (Figure 4B), which were thus defined as FHY3 directly regulated genes (Table 2). Of the 197 FHY3 directly regulated genes in D, 154 (78%) and 43 (22%) genes are activated and repressed by FHY3, respectively (Figure 4B, Table 2). However, of the 86 FHY3 directly regulated genes in FR light, 85 genes (99%) are activated by FHY3, whereas only one gene, At2g20300, encoding a novel receptor-like kinase ALE2 (Tanaka et al., 2007), is repressed by FHY3 (Figure 4B, Table 2).
Identification of FHY3 Directly Regulated Genes by Comparing ChIP-seq and Microarray Data.
(A) Venn diagram showing the number of genes regulated by FHY3 in D, FR, or both conditions based on our microarray analysis.
(B) Venn diagram showing the overlaps of the FHY3 direct target genes (revealed by our ChIP-seq analysis) with FHY3 regulated genes (revealed by our microarray analysis) in D and FR light conditions.
(C) Distribution of FBS motifs in the FHY3 directly regulated genes.
Summary of FHY3 Directly Regulated Genes
Comparison of the FHY3 directly regulated genes in two conditions revealed that FHY3 directly activates the expression of 70 genes regardless of light conditions (Figure 4B). In addition, FHY3 directly represses the expression of 43 genes in D, whereas FR light exposure released the repressive regulation of FHY3 for 42 genes (except for At2g20300 mentioned above; Figure 4B). The constitutive activation of FHY1 and ELF4 and only-in-dark repression of GA2OX6 by FHY3 were confirmed by real-time qRT-PCR analysis (see Supplemental Figure 2 online). These data suggest that the transcriptional activities of FHY3 are regulated by FR light and that FHY3 mainly acts as a transcriptional activator for most of its direct target genes, especially in FR light condition.
Moreover, 144 FHY3 directly regulated genes in D (73% of 197 genes) and 62 genes in FR light (72% of 86 genes) contain typical FBS motifs (Table 2), and most of these FBS motifs are localized in the gene promoter regions (1000 bp upstream the transcription start sites; Figure 4C), indicating that FHY3 may regulate the expression of these genes by directly binding to the FBS motifs in their gene promoters.
Gene Ontology Analysis of FHY3 Direct Target Genes
Gene Ontology (GO) analysis using the Web Gene Ontology Annotation Plot tool (Ye et al., 2006) shows that FHY3 binds to a wide range of genes involved in multiple cellular activities and biological processes (see Supplemental Figure 3 online). For example, genes involved in metabolism, development, and responses to various hormones are statistically enriched in FHY3 direct target genes compared with the whole genome (see Supplemental Figure 3 online). GO analysis was also performed to further analyze the classification of FHY3 directly regulated genes mentioned above. Interestingly, genes involved in transcription, signal transduction, biosynthesis, and development are highly enriched among these FHY3 directly regulated genes (Figure 5A). Notably, the frequency of transcription factors is ~3 times higher than that found in the whole Arabidopsis genome (5.9%; Riechmann et al., 2000) (Figure 5A), suggesting that they may represent early target genes of FHY3 mediating FHY3-regulated transcriptional cascades.
Functional Classification Analysis of FHY3 Direct Target Genes.
(A) Enrichment of selected GO categories in FHY3 directly regulated genes. Numbers on the top are P values (hypergeometric test) by comparing the percentage of the corresponding categories in the FHY3 directly regulated genes in D or FR light with that in the whole genome.
(B) Representative FHY3 direct target genes with known functions in various cellular processes and response/regulatory pathways. FHY3 direct target genes in D, FR, or both conditions are in black, red, or blue, respectively. Genes upregulated and downregulated by FHY3 in our microarray analyses are indicated by the directions of the arrows. ABA, abscisic acid; BR, brassinosteroid; GA, gibberellic acid.
A large portion of FHY3 direct target genes have been studied experimentally; thus, they provide direct evidence and molecular mechanisms on how FHY3 is involved in a wide range of cellular, developmental, and regulatory processes (Figure 5B). Together with our microarray data, FHY3 was shown to directly activate (and occasionally repress) a large number of target genes with known functions in both D and FR light conditions (Figure 5B). In addition to the experimentally characterized direct target genes FHY1/FHL and ELF4, it is interesting to note that other key regulators in light signaling (such as COP1) and in the circadian clock (such as CCA1, LHY, and CHE) (Deng et al., 1992; Schaffer et al., 1998; Wang and Tobin, 1998; Pruneda-Paz et al., 2009) were identified as FHY3 direct target genes in our study. Direct binding of FHY3 to these genes was confirmed by ChIP-qPCR assays (see Supplemental Figure 1 online). It is also intriguing that FHY3 may directly regulate a large number of target genes involved in various hormone responses, such as abscisic acid, gibberellic acid, auxin, brassinosteroid, and ethylene, which awaits further investigation.
FHY3 Coregulates Some Common Target Genes with PIL5 and HY5
To investigate what other transcription factors may be involved in coregulation with FHY3, we systematically analyzed the 1-kb sequences surrounding the FHY3 binding sites (−500 to +500 bp) that were assigned to genes. We looked for enrichment of other known cis-elements for multiple families of transcription factors using the Arabidopsis cis-regulatory element database (Molina and Grotewold, 2005). This analysis revealed that in addition to the FBS motif, which is highly enriched around the FHY3 binding sites, several other cis-elements, including G-box, GCC-box, and ABRE, appeared at a higher frequency surrounding the FHY3 binding sites than in the whole genome (Figure 6A).
FHY3 Coregulates Some Common Target Genes with PIL5 and HY5.
(A) Analyses of the 1-kb sequences surrounding the FHY3 binding sites revealed enrichment of several other cis-elements (in addition to FBS) around the FHY3 binding sites compared with the random genome (permutation test, P < 0.001).
(B) Venn diagram showing the overlap of FHY3 direct target genes in D revealed in our study and PIL5 direct target genes reported in a recent study (Oh et al., 2009).
(C) Distribution of G-box and FBS motifs around the FHY3 binding sites (−500 to +500 bp) among the PIL5 and FHY3 common target genes.
(D) Venn diagram showing the overlap of FHY3 direct target genes in FR light with two sets of HY5 direct target genes reported by Lee et al. (2007) and Zhang et al. (2011).
(E) Distribution of G-box and FBS motifs around the FHY3 binding sites (−500 to +500 bp) among two sets of HY5 and FHY3 common target genes.
G-box is a well-known light-responsive cis-element that could be bound by the basic helix-loop-helix (bHLH) transcription factors (such as PIL5 and PIF3) and bZIP transcription factors (such as HY5) (Chattopadhyay et al., 1998; Oh et al., 2007, 2009; Shin et al., 2007). A recent ChIP-chip analysis identified 748 PIL5-direct target genes in Arabidopsis imbibed seeds (Oh et al., 2009). Comparison of these PIL5 direct target genes with FHY3 direct target genes in D revealed that a total of 136 genes may be coregulated by PIL5 and FHY3 (Figure 6B; see Supplemental Data Set 5 online). Analysis of G-box locations among these PIL5/FHY3 coregulated genes showed that G-box motifs displayed several peaks around the FBS motifs (Figure 6C).
Two recent studies using the ChIP-chip approach identified 3894 and 9606 putative HY5 direct target genes, respectively, in the Arabidopsis genome (Lee et al., 2007; Zhang et al., 2011). To investigate how many genes may be coregulated by FHY3 and HY5, we compared the 1009 FHY3 direct target genes identified in FR light with two sets of HY5 direct target genes identified in two previous studies. Our analysis showed that 572 and 331 genes were coregulated by HY5 and FHY3, respectively, based on two sets of HY5 target genes (Figure 6D). Notably, 283 genes were identified repeatedly in two sets of HY5/FHY3 coregulated genes, including FHY1 and ELF4 (Figure 6D; see Supplemental Data Set 6 online).
Interestingly, HY5 and FHY3 were reported to bind their respective cis-elements in close proximity (<20 bp) to each other in FHY1/FHL and ELF4 promoters (Li et al., 2010, 2011). Therefore, we asked whether this is a general characteristic for HY5 and FHY3 to coregulate their common target genes. Analysis of two sets of HY5/FHY3 common target genes both showed that G-box motifs are preferentially positioned at the locations of, or in close proximity to (within −200 to +200 bp), FBS motifs (Figure 6E), a pattern distinct from that of the G-box locations in the PIL5/FHY3 coregulated genes (Figure 6C). Considering the fact that HY5 and FHY3 physically interact with each other (Li et al., 2010), our data suggest that HY5/FHY3 interaction and cobinding to the promoters may represent a pivotal module to regulate a large number of their common target genes.
FHY3 Is Involved in the Regulation of Chloroplast Division by Directly Activating ARC5 Transcription
It was interesting to notice that FHY3 regulates several genes involved in chloroplast development (Figure 5B). Therefore, we examined whether the fhy3 mutant has any defects in chloroplast development. Interestingly, microscopy observations show that leaf mesophyll cells in fhy3-4 mutants contain fewer and larger chloroplasts than do wild-type cells (Figures 7A and 7B), whereas introduction of 3FLAG-FHY3-3HA (under the 35S constitutive promoter) or FHY3-GR (under the FHY3 native promoter with DEX treatment) fusion proteins into the fhy3-4 mutants rescued the chloroplast defect phenotypes (Figures 7C and 7D), indicating that FHY3 indeed regulates chloroplast development. In addition, chloroplast number was also reduced in the far1-2 mutants and was much further reduced in the fhy3-4 far1-2 double mutants compared with that in the wild-type plants (Figures 7A and 7B), indicating that FHY3 and FAR1 act together in the control of chloroplast development, although FAR1 plays a less predominant role compared with FHY3.
FHY3 and FAR1 Are Involved in the Control of Chloroplast Development.
(A) Comparison of chloroplasts in leaf mesophyll cells of 10-d-old wild-type (No-0 and Landsberg erecta ecotypes), fhy3-4, far1-2, fhy3-4 far1-2, and arc5 mutant plants grown in continuous white light conditions. Bar = 10 μm.
(B) Chloroplast numbers are significantly decreased in the leaf mesophyll cells of fhy3-4, far1-2, and fhy3-4 far1-2 mutants compared with the wild-type plants. More than 20 leaf mesophyll cells were counted for each plant. Student’s t tests were performed to calculate the statistical significance (**P < 0.01 and ***P < 0.001). Error bars represent sd.
(C) and (D) Transgenic expression of FHY3-GR (under the FHY3 native promoter with DEX treatment) or 3FLAG-FHY3-3HA (under the 35S promoter) sufficiently rescued the chloroplast shape (C) and number (D) phenotypes of the fhy3-4 mutants. **P < 0.01 by Student’s t test. Error bars represent sd.
The phenotypes of fhy3, far1, and fhy3 far1 mutants are reminiscent of what was observed in the loss-of-function mutant of ARC5, encoding a cytosolic dynamin-like protein involved in the latter stages of chloroplast division in Arabidopsis (Figure 7A; Robertson et al., 1996; Gao et al., 2003; Yun and Kawagoe, 2009). Interestingly, ARC5 was identified as a FHY3 direct target gene in our study (Figure 5B), and FHY3 specifically binds to the promoter region of ARC5 in both D and FR light conditions (Figure 8A; see Supplemental Figure 1 online). To further confirm whether FHY3 (and possibly FAR1) bind to the ARC5 promoter through the only FBS motif located at approximately −65 bp upstream of the ATG start codon, we performed yeast one-hybrid assays, in which the wild-type and FBS-mutated ARC5 promoter fragments were used to drive LacZ reporter gene expression (Figure 8B). Our results showed that AD-FHY3 and AD-FAR1 bind to the wild type, but not the FBS-mutated ARC5 promoter fragments (Figure 8C), confirming that the typical FBS motif in the ARC5 promoter mediates the binding of FHY3 and FAR1 to the promoter.
FHY3 Directly Binds to the ARC5 Gene Promoter.
(A) ARC5 was identified as a FHY3 direct target gene in both D and FR light conditions. The mapped ChIP-seq reads in D (black) and FR (red) libraries are shown in the top and middle rows, respectively, and a schematic representation of ARC5 gene structure is shown in the bottom row.
(B) Diagram of the wild-type and mutant (m) ARC5 promoter fragments used to drive LacZ reporter gene expression in yeast one-hybrid assays. The adenine residue of the translational start codon (ATG) was assigned position +1, and the numbers above the nucleotide sequence were counted based on this number. FBS motifs are shown in red, and nucleotide substitutions in the mutant fragment are shown in blue.
(C) Yeast one-hybrid assays showing that FHY3 and FAR directly bind to the wild type but not the FBS-mutated ARC5 promoter fragment.
Next, we asked whether the ARC5 transcript level is regulated by FHY3 and FAR1. Real-time qRT-PCR showed that the transcript levels of ARC5, but not those of ARC3 and ARC6, two other genes also involved in controlling chloroplast division (Marrison et al., 1999; Holzinger et al., 2008) but not identified as FHY3 target genes in our study, were severely attenuated in the fhy3, far1, and fhy3 far1 mutants (Figure 9A). However, ARC5 expression was efficiently restored in the 35S:3FLAG-FHY3-3HA fhy3-4 transgenic lines in which 3FLAG-FHY3-3HA fusion proteins were expressed at high levels (Figure 9B; Li et al., 2011). This restoration was further confirmed by examination of ARC5 transcript levels in the FHY3p:FHY3-GR fhy3-4 transgenic seedlings with MOCK or DEX treatment (Figure 9C). Moreover, detailed time-course qPCRs using this transgenic line showed that in both D and FR light conditions, the ARC5 transcript levels were induced rapidly after the DEX, but not the MOCK treatment within 2 h, indicating that FHY3 indeed directly activates ARC5 transcription (Figure 9D).
FHY3 Directly Activates ARC5 Transcription.
(A) Real-time qRT-PCR analysis showing that the transcript levels of ARC5, but not ARC3 or ARC6, were severely attenuated in the 10-d-old continuous white light–grown fhy3-4, far1-2, and fhy3-4 far1-2 mutants compared with the wild-type plants. Error bars represent sd of triplicate experiments.
(B) Real-time qRT-PCR analysis showing that ARC5 transcript level was restored in 10-d-old continuous white light–grown 35S:3FLAG-FHY3-3HA fhy3-4 transgenic plants.
(C) The expression of ARC5 is upregulated by DEX but not MOCK treatment in the FHY3p:FHY3-GR fhy3-4 plants. The expression of ARC3 or ARC6 is not affected by either treatment and served as the negative controls. The wild-type and FHY3p:FHY3-GR fhy3-4 plants were grown under continuous white light for 10 d on the growth medium containing 10 μM DEX or MOCK treatment (equal volume of ethanol added to the medium) before analysis. Error bars represent sd of triplicate experiments.
(D) ARC5 transcript levels were rapidly induced by DEX but not MOCK treatment in FHY3p:FHY3-GR fhy3-4 transgenic plants in both D and FR light conditions. The seedlings were grown in D or FR light conditions for 4 d and then subjected to the indicated treatments and returned to the same conditions for various time periods. Error bars represent sd of triplicate experiments.
Finally, we examined whether restoration of ARC5 in the fhy3-4 mutant could rescue its chloroplast defects by transforming the 35S:FLAG-ARC5 construct into the fhy3-4 mutants. Analysis of the transgenic plants showed that reintroduction of FLAG-ARC5 under the 35S promoter could rescue the chloroplast defects of the fhy3-4 mutant (Figures 10A to 10D). By contrast, ARC5-GFP (green fluorescent protein) fusion proteins under the control of the native ARC5 promoter in the fhy3-4 mutant were not expressed and thus could not rescue its chloroplast defects (Figures 10E and 10F), further confirming that ARC5 is indeed a FHY3 direct target gene in Arabidopsis. Taken together, our data demonstrate that FHY3 controls chloroplast division by directly activating ARC5 transcription.
Overexpression of ARC5 Rescues the Abnormal Chloroplast Phenotypes of the fhy3 Mutants.
(A) and (B) Overexpression of ARC5 rescues the phenotypes of chloroplast shape (A) and number (B) of the fhy3-4 mutants. The plants were grown in continuous WL for 10 d before analysis. Bar = 10 μm in (A). Error bars in (B) represent sd.
(C) ARC5 is overexpressed in the 10-d-old continuous white light–grown 35S:FLAG-ARC5 fhy3-4 transgenic plants. Error bars represent sd of triplicate experiments.
(D) Immunoblots showing that FLAG-ARC5 fusion proteins were expressed in the 10-d-old continuous white light–grown 35S:FLAG-ARC5 fhy3-4 transgenic plants.
(E) GFP-ARC5 fusion proteins driven by the ARC5 native promoter were expressed in the wild type but not in fhy3-4 mutant plants. Note that GFP-ARC5 is localized to the constriction sites of dividing chloroplasts (indicated by arrows) as reported previously (Gao et al., 2003). Bar = 10 μm.
(F) Chloroplast phenotypes of ARC5p:ARC5-GFP and ARC5p:ARC5-GFP fhy3-4 plants grown in continuous WL for 10 d. Bar = 10 μm.
DISCUSSION
Several studies have been reported in recent years profiling genome-wide direct target genes for pivotal plant transcription factors, such as HY5, PIL5, AGAMOUS-LIKE15, and BZR1 (Lee et al., 2007; Oh et al., 2009; Zheng et al., 2009; Sun et al., 2010; Zhang et al., 2011). However, most of these studies were conducted using ChIP-chip, which tends to have low resolution and is often quite noisy in higher organisms (Johnson et al., 2008). In this study, we adopted a newer technique, ChIP-seq, which may effectively surmount the shortcomings of ChIP-chip (Valouev et al., 2008), to identify genome-wide direct target genes of FHY3, another important transcription factor in Arabidopsis.
Genome-Wide Analysis of FHY3 Direct Target Genes
FHY3 and its homolog FAR1 belong to a recently identified transcription factor family, which has been reported to play pivotal roles in phyA signaling and the circadian clock (Whitelam et al., 1993; Hudson et al., 1999; Wang and Deng, 2002; Allen et al., 2006; Lin et al., 2007; Li et al., 2011). Their protein sizes are large (~92 kD), and both of them contain transposase-like domains in the middle of their proteins and were thus proposed to originate from Mutator-like transposases during evolution (Lin et al., 2007). However, whether these transposase-derived transcription factors are also involved in other biological processes in plants remains largely unknown.
FHY3 was shown to associate with phyA in vivo and protect phyA from being recognized by the COP1/SPA protein degradation machinery (Saijo et al., 2008), suggesting that FHY3 may play additional roles in phyA signaling except for activating FHY1/FHL expression, and that its transcriptional activities may be regulated by phyA. In this study, we performed genome-wide analyses to identify global FHY3 direct target genes by ChIP-seq approach and FHY3 regulated genes by microarray analysis. We conducted these analyses in D (phyA plays no role) and FR light (phyA dominates) conditions, to investigate whether the binding and transcriptional regulation activities of FHY3 are regulated by FR light. Several interesting observations were made in our analyses. First, our data clearly showed that FHY3 binds to a large number of genes specifically in D or FR light conditions, respectively (Figure 1B), demonstrating that the binding activities of FHY3 are differently regulated by FR light at least for some target genes. Second, as shown in Figure 4B, the transcriptional regulation activities of FHY3 are also regulated by FR light. An interesting observation is that FHY3 directly represses the expression of 43 genes in D; however, in FR light, the repressive regulation of FHY3 was released for 42 genes (Figure 4B). Finally, it is interesting to notice that FHY3 binds to and regulates the expression of more genes in D than in FR (Figures 1B and 4A). Next, it will be intriguing to investigate how FR light regulates the transcriptional activities of FHY3 (e.g., phyA directly modifies the activities of FHY3 or via other mechanisms not yet identified).
In addition, microarray and GO analyses indicate that FHY3 directly regulates a wide range of genes involved in various developmental processes in plants (Figures 4 and 5). This notion was further supported by the discovery that FHY3 is indeed involved in the control of chloroplast division by activating ARC5 transcription (Figures 7 to 10). Therefore, our genome-wide analysis of FHY3 direct target genes has led to the discovery of novel functions of FHY3 that may be overlooked by traditional genetic or biochemical approaches.
Regulatory Modes of FHY3
A recent study from our group revealed how FHY3 and FAR1 function with their coregulator HY5 to fine-tune the expression of FHY1 and FHL. FHY3 and FAR1 act as the transcriptional activator for FHY1/FHL expression (Lin et al., 2007), whereas HY5 acts as a repressor of FHY1/FHL expression by modulating the transcriptional activities of FHY3 and FAR1 (Li et al., 2010). This is achieved by two distinct mechanisms. The first mechanism involves steric hindrance as the cis-elements of HY5 and FHY3/FAR1 in the FHY1/FHL promoters are very close to each other (<10 bp away; Li et al., 2010). The second mechanism is called “sequestration” through the physical interactions between HY5 and FHY3/FAR1 (Li et al., 2010). This mechanism was also used in the regulation of some plant bHLH transcription factors (de Lucas et al., 2008; Feng et al., 2008; Hornitschek et al., 2009). However, in another report from our group, both FHY3/FAR1 and HY5 were shown to be transcriptional activators for ELF4 expression, and in this case, their respective cis-elements are around 20 bp away in the ELF4 promoter (Li et al., 2011). These interesting observations reflect the complexity of regulatory modes of even same combination of transcription factors on different target gene promoters.
Notably, this study identified a large number of common target genes that may be coregulated by FHY3 and HY5. Most of these genes, especially the 283 genes repeatedly identified in two sets of HY5 target genes, are of high confidence as they always include the well-characterized FHY3/HY5 common target genes FHY1 and ELF4 (Figure 6D). Analysis of G-box locations showed that G-box motifs are preferentially located in close proximity to the FBS motifs in these common target genes (Figure 6E), consistent with their locations in the FHY1/FHL and ELF4 promoters. Thus, for these newly identified FHY3/HY5 common target genes, HY5 and FHY3 might bind to their promoters via their respective cis-elements, which are close to each other. They may modulate the transcriptional activities of each other through their physical interactions or through some other mechanisms, such as steric hindrance. However, more detailed molecular and biochemical studies on more specific target gene promoters need to be conducted in future studies so as to demonstrate how FHY3/FAR1 and HY5 work in concert with each other on their target gene promoters, especially to understand how their roles in gene expression are determined by the relative locations of their cis-elements.
At the same time, it should be noted that other types of transcription factors may be recruited to work together with FHY3 to coregulate the expression of some common target genes. This idea is supported by the fact that several other types of cis-elements are significantly enriched around the FHY3 binding sites (Figure 6A) and by our recent report that CCA1 and LHY, two key transcription factors in the central oscillator of the Arabidopsis circadian clock, also bind to the ELF4 promoter through evening elements located ~100 bp away from the FBS motifs (Li et al., 2011). Comparison of the FHY3 direct target genes with the recently reported PIL5 direct targets revealed a total of 136 genes coregulated by FHY3 and PIL5 (Figure 6B). However, G-box locations in these genes showed a different pattern from those in the FHY3/HY5 common target genes (Figures 6C and 6E), suggesting that the coregulation mechanisms of FHY3/PIL5 may be different from those of FHY3/HY5. Thus, it is necessary to investigate whether FHY3 also physically interacts with PIL5, which may partially account for the different pattern of their binding site localizations.
FHY3 Binds to Two Novel Motifs Present in the 178-bp Centromeric Repeats
One unexpected finding of this study is that most of the FHY3 binding sites in the intergenic regions reside in centromeric regions (Figure 3A). Further analysis revealed that FHY3 directly binds to two motifs present in the 178-bp centromeric repeats (Figures 3C to 3E). To our knowledge, this feature is unique to FHY3, as it was not observed in other genome-wide binding site studies of plant transcription factors, such as HY5, PIL5, and BZR1 (Lee et al., 2007; Oh et al., 2009; Sun et al., 2010; Zhang et al., 2011). It is also interesting to notice that these two motifs are highly conserved in the centromeric tandem repeat sequences of 41 Arabidopsis ecotypes (Hall et al., 2003). As the 178-bp repeats are the dominant sequence elements of Arabidopsis centromeres and centromeric regions govern the transmission of nuclear chromosomes to the next generation of cells (Houben and Schubert, 2003; Nagaki et al., 2003), it will be interesting to investigate the biological significance of FHY3 binding to the centromeric regions in future studies.
Our data, together with those in the previous reports, indicate that FHY3 could bind to at least three various cis-elements (i.e., FBS, FBSC-1, and FBSC-2) in the Arabidopsis genome. Interestingly, all of these motifs share the CAC core sequence. This finding may facilitate the identification of novel FHY3 direct target genes in future studies, as around half of the FHY3 direct target genes do not contain any typical FBS motifs (Figure 2B).
FHY3 Plays a Role in the Control of Chloroplast Development
Chloroplasts are essential for photosynthesis, and light is a major environmental factor determining the biogenesis of chloroplasts (Pogson and Albrecht, 2011). Phytochrome-interacting transcription factors PIFs were shown to be negative regulators of chloroplast development that act by repressing the expression of genes involved in chlorophyll biosynthesis and photosynthesis (Huq et al., 2004; Shin et al., 2009; Stephenson et al., 2009). In this study, we show that FHY3, another transcription factor acting in the phytochrome signaling pathway, also plays a role in chloroplast development. However, in contrast with the roles of PIFs in repressing chlorophyll biosynthesis, FHY3 controls chloroplast division by directly activating ARC5 transcription. Recently, ARC5 was also shown to mediate peroxisome division (Zhang and Hu, 2010); thus, it will be interesting to investigate whether FHY3 is involved in peroxisome division as well. Moreover, as mentioned above, FHY3 associates with phyA in vivo (Saijo et al., 2008); thus, FHY3 may represent another pathway mediating phytochrome-regulated chloroplast development in Arabidopsis.
In summary, our genome-wide identification of FHY3 direct target genes may serve as a new starting point to systematically study the functions of FHY3. Discovery of new roles of FHY3 in plant development and elucidation of more regulatory modes of FHY3 may help in understanding the functional conservation and divergence of these transposase-derived transcription factors during evolution.
METHODS
Plant Materials and Growth Conditions
The wild-type Arabidopsis thaliana used in this study is of the No-0 ecotype, unless otherwise indicated. The arc5 mutant (Gao et al., 2003) is of the Landsberg erecta ecotype, ARC5p:ARC5-GFP (Gao et al., 2003) is of the Columbia ecotype, and the fhy3-4 (Wang and Deng, 2002), far1-2 (Hudson et al., 1999), and fhy3-4 far1-2 (Lin et al., 2007) mutants and FHY3p:FHY3-GR fhy3-4 (Lin et al., 2007) and 35S:3FLAG-FHY3-3HA fhy3-4 (Li et al., 2011) transgenic plants are of the No-0 ecotype and have been described previously. The ARC5p:ARC5-GFP fhy3-4 plants were constructed by crossing ARC5p:ARC5-GFP and fhy3-4 mutant plants. The growth conditions and light sources were as described previously (Shen et al., 2005).
ChIP-seq
The homozygous 35S:3FLAG-FHY3-3HA fhy3-4 (Li et al., 2011) seedlings grown in D or continuous FR light for 4 d were used for ChIP assays following the procedure described previously (Lee et al., 2007). Briefly, 5 g of seedlings were first cross-linked with 1% formaldehyde under vacuum, and then the samples were ground to powder in liquid nitrogen. The chromatin complexes were isolated and sonicated and then incubated with monoclonal anti-FLAG antibodies (Sigma-Aldrich). The precipitated DNA was recovered and used to generate Illumina sequencing libraries according to the manufacturer’s instructions. Sequencing was conducted by Keck DNA sequencing laboratory located at Yale School of Medicine.
ChIP-seq Data Analysis
Sequencing reads from D and FR libraries were mapped to TAIR9 genome release of Arabidopsis using MAQ software (Li et al., 2008; He et al., 2010), allowing for up to two mismatching nucleotides and no gaps. Only uniquely mapped reads were retained for further analyses. The output of the analysis pipeline was converted to browser extensible data files for viewing the data in the Integrative Genomics Viewer genome browser (Robinson et al., 2011).
Numerous peak detection algorithms have been proposed recently for analyzing the ChIP-seq data sets, and they provide more or less similar results (Laajala et al., 2009). In this study, MACS software (Zhang et al., 2008) with default parameters (bandwidth, 300 bp; mfold, 32; P value of 1.00e-05) was used to call peaks representing enriched binding sites as described previously (Wang et al., 2009; He et al., 2010). All binding peaks were sorted based on the following criteria: (1) if a binding site resides in the gene body region, it will be further categorized according to its location in the gene body (i.e., 5′-untranslated region, exon, intron, or 3′-untranslated region); (2) if a binding site is localized in the 1000-bp region upstream of the transcription start site of a gene, it is classified as a binding site in the promoter region in our study; (3) if a binding site is assigned to more than one gene by criterion (1) or (2), then it is regarded as a binding site to each of these genes; and (4) the binding sites not selected by the above three criteria were defined as the binding sites in the intergenic regions.
Microarray Analysis
For microarray analysis, FHY3p:FHY3-GR fhy3-4 transgenic seedlings (Lin et al., 2007) grown in D or continuous FR light for 4 d were treated with 10 μM DEX or MOCK (equal volume of ethanol) for 2 h, and then total RNA was isolated using the RNeasy plant mini kit (Qiagen). RNA quality was assessed with an Agilent 2100 Bioanalyzer, and hybridization to the Affymetrix GeneChip Arabidopsis ATH1 Genome Arrays was performed according to the manufacturer’s instructions. Four independent biological replicates for each treatment were used for microarray analysis. Raw microarray data were normalized using the robust multiarray analysis algorithm (Irizarry et al., 2003) in the BIOCONDUCTOR affy package (http://www.bioconductor.org/packages/2.0/bioc/html/affy.html). Then the microarray data were analyzed using the Significance Analysis of Microarrays method (Tusher et al., 2001) with default parameters as well as a manual setting of false discovery threshold <0.027 in D and <0.022 in FR.
Motif Search and Gene Ontology Analysis
The 1-kb sequences (500 bp upstream and 500 bp downstream) surrounding the FHY3 binding peaks were extracted and then searched for perfect matches to the consensus transcription factor binding motifs using a Perl script. The consensus motif sequences were obtained from the AGRIS and Transfac databases (Wingender et al., 1996; Davuluri et al., 2003). The frequencies of these consensus motifs surrounding the FHY3 binding peaks were compared with those in the whole genome. Gene ontology analyses of the FHY3 direct target genes were performed using the Web Gene Ontology Annotation Plotting tool (http://wego.genomics.org.cn) (Ye et al., 2006).
Real-Time qRT-PCR
Total RNA was isolated from Arabidopsis seedlings using the RNeasy plant mini kit (Qiagen). cDNAs were synthesized from 2 μg total RNA using SuperScript II first-strand cDNA synthesis system (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed using the respective pair of primers and Power SYBR Green PCR Master Mix (Applied Biosystems) with a Bio-Rad CFX96 real-time PCR detection system as described previously (Li et al., 2010). PCR reactions were performed in triplicate for each sample, and the expression levels were normalized to that of an Actin gene. The primers used for qRT-PCR are listed in Supplemental Table 1 online.
Analysis of Chloroplast Phenotypes by Microscopy
Chloroplast size and number were observed as previously described (Osteryoung et al., 1998). Briefly, tips from expanding leaves of 10-d-old seedlings grown in continuous white light were cut and fixed with 3.5% glutaraldehyde and then incubated in 0.1 M Na2-EDTA, pH 9.0, for 15 min at 55°C. The images were recorded with a Carl Zeiss LSM 780 microscope.
EMSA
The GST-FHY3N construct was described previously (Lin et al., 2007). EMSAs were performed using the biotin-labeled probes and the Lightshift Chemiluminescent EMSA kit (Pierce) according to the manufacturer’s instructions. The sequences of the complementary oligonucleotides used to generate the biotin-labeled probes are shown in Supplemental Table 1 online.
Yeast One-Hybrid Assay
The AD-FHY3 and AD-FAR1 constructs were described previously (Wang and Deng, 2002). To generate the ARC5p:LacZ construct, a 191-bp wild-type promoter fragment of ARC5 was amplified by PCR using a pair of primers (see Supplemental Table 1 online) and the genomic DNA of No-0 ecotype as templates and then cloned into the EcoRI-KpnI sites of the pLacZi2μ vector (Lin et al., 2007). To generate the ARC5pm:LacZ construct in which the FBS motif (CACGCGC) was mutated to CACcgcg, the ARC5p:LacZ reporter plasmid was used as the template for mutagenesis reactions using the pair of primers shown in Supplemental Table 1 online and the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.
Yeast one-hybrid assays were performed as described previously (Lin et al., 2007). Briefly, plasmids for AD fusions were cotransformed with the ARC5p:LacZ or ARC5pm:LacZ reporter constructs, respectively. Transformants were grown on SD/-Trp-Ura dropout plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) for blue color development.
Immunoblotting
For immunoblots, 35S:FLAG-ARC5 fhy3-4 homozygous seedlings were homogenized in an extraction buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1% Tween 20, 1 mM PMSF, and 1× complete protease inhibitor cocktail (Roche). Immunoblotting was performed as described previously (Shen et al., 2005) using monoclonal anti-FLAG antibodies (Sigma-Aldrich).
Complementation Analysis
To generate the 35S:FLAG-ARC5 construct, the coding region of ARC5 was first PCR amplified using first-strand cDNAs prepared from 10-d-old Arabidopsis seedlings grown in continuous WL (primers shown in Supplemental Table 1 online) and then inserted into the KpnI-SalI sites of the pF3PZPY122 vector (Feng et al., 2003). Then, a BamHI-EcoRI fragment containing the full-length coding sequence of FLAG-ARC5 was released and inserted into the BamHI-EcoRI sites of the pJim19 (Kan) vector, a binary vector based on pBIN19 with 35S promoter of Cauliflower mosaic virus and kanamycin resistance markers in both bacteria and plants. The 35S:FLAG-ARC5 construct was electroporated into Agrobacterium tumefaciens strain GV3101 and used to transform the fhy3-4 homozygous mutants.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: FHY3 (At3g22170), FAR1 (At2g15090), FHY1 (At2g37678), FHL (At5g02200), ELF4 (At2g40080), HY5 (At5g11260), PIL5/PIF1 (At2g20180), and ARC5 (At3g19720). All raw ChIP-seq data and expression profiling data have been deposited in the Gene Expression Omnibus database under accession numbers GSE30711 and GSE30712.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Validation of Several FHY3 Direct Target Genes and Binding Sites by ChIP-qPCR Analysis.
Supplemental Figure 2. Validation of Expression Changes of ELF4, FHY1, and GA2OX6 in D and FR by Real-Time qRT-PCR Analysis.
Supplemental Figure 3. Functional Classification of the FHY3 Direct Target Genes.
Supplemental Table 1. Summary of Primers Used in This Study.
Supplemental Data Set 1. List of FHY3 Binding Sites in D.
Supplemental Data Set 2. List of FHY3 Binding Sites in FR Light.
Supplemental Data Set 3. List of FHY3 Regulated Genes in D.
Supplemental Data Set 4. List of FHY3 Regulated Genes in FR Light.
Supplemental Data Set 5. List of FHY3 and PIL5 Coregulated Genes.
Supplemental Data Set 6. List of FHY3 and HY5 Coregulated Genes.
Acknowledgments
We thank Katherine W. Osteryoung for providing ARC5p:ARC5-GFP and arc5 mutant seeds. We also thank Guangming He, Lifen Huang, Mingqiu Dai, and Shangwei Zhong for their suggestions on the project. This work was supported by the National Institutes of Health (grant GM47850 to X.W.D.) and the National Science Foundation (Awards IOS-0954313 and IOS-1026630 to H.W.). X.O. was supported by Peking-Yale Joint Center Monsanto Fellowship and a fellowship from the China Scholarship Council.
AUTHOR CONTRIBUTIONS
X.W.D., H.W., and S.L. conceived the project. X.O., G.L., and X.W.D. designed the experiments. J.L. and X.O. performed EMSA assays. J.L. and X.H. performed yeast-one hybrid assays. G.L., X.O., X.M., X.W., and R.L. generated the transgenic plants. B.L., B.C., and H.S. conducted bioinformatics analyses. X.O. performed all other experiments. J.L., X.O., and X.W.D. prepared the manuscript.
Footnotes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Xing Wang Deng (xingwang.deng{at}yale.edu).
↵1 These authors contributed equally to this work.
↵[W] Online version contains Web-only data.
- Received March 10, 2011.
- Revised July 10, 2011.
- Accepted July 17, 2011.
- Published July 29, 2011.
References
