Arabidopsis chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation

Abstract

Photomorphogenesis is a critical plant developmental process that involves light-mediated transcriptome changes, histone modifications, and inhibition of hypocotyl growth. However, the chromatin-based regulatory mechanism underlying this process remains largely unknown. Here, we identify ENHANCED PHOTOMORPHOGENIC1 (EPP1), previously known as PICKLE (PKL), an ATP-dependent chromatin remodeling factor of the chromodomain/helicase/DNA binding family, as a repressor of photomorphogenesis in Arabidopsis thaliana. We show that PKL/EPP1 expression is repressed by light in the hypocotyls in a photoreceptor-dependent manner. Furthermore, we reveal that the transcription factor ELONGATED HYPOCOTYL5 (HY5) binds to the promoters of cell elongation-related genes and recruits PKL/EPP1 through their physical interaction. PKL/EPP1 in turn negatively regulates HY5 by repressing trimethylation of histone H3 Lys 27 at the target loci, thereby regulating the expression of these genes and, thus, hypocotyl elongation. We also show that HY5 possesses transcriptional repression activity. Our study reveals a crucial role for a chromatin remodeling factor in repressing photomorphogenesis and demonstrates that transcription factor-mediated recruitment of chromatin-remodeling machinery is important for plant development in response to changing light environments.

Figure 1.

Loss of EPP1, Encoding PKL, Triggers a Constitutive Photomorphogenic-Like Response. (A) The epp1-1 mutant is hyposensitive to red (R), far-red (FR), and blue (BL) light compared with the Col wild type. Bar = 2 mm. (B) Hypocotyl length of epp1-1 and wild-type seedlings under various light conditions, as shown in (A). Data are mean ± sd of 30 seedlings. (C) Phenotypes of Col, epp1-1, cop1, and epp1 cop1 seedlings in darkness (Dk). Bar = 2 mm. (D) Hypocotyl length of wild-type and mutant seedlings in darkness. Data represent the mean ± sd of 30 seedlings. (E) Anthocyanin content [(A₅₃₀ − 0.25 × A₆₅₇)/100 seedlings] of wild-type and mutant seedlings in far-red light. Data represent the mean ± sd of triplicate assays. (F) Diagram of PKL/EPP1 and positions of the mutations. Black boxes represent exons, and lines between the boxes indicate introns. Triangles denote T-DNA insertions, and the star indicates a point mutation. (G) Immunoblot analysis showing the presence of PKL in the wild type but not in the epp1 mutants. Stars indicate cross-reacting bands used for a loading control. (H) Multiple epp1/pkl mutant alleles displaying open cotyledons without an apical hook under dark conditions. Bar = 0.5 mm. All seedlings in (A) to (E), (G), and (H) were grown in the light or dark conditions for 5 d. [See online article for color version of this figure.]

Figure 2.

PKL Expression in the Hypocotyls Is Repressed by Light. (A) A qRT-PCR assay showing PKL transcript levels in wild-type seedlings subjected to red (R), far-red (FR), or blue (BL) light or darkness (Dk) for 5 d or in 5-d-old dark-grown seedlings transferred to white light (WL) for the indicated periods of time (0 to 12 h). Data represent the mean ± sd of three biological replicates. (B) qRT-PCR assay showing increased PKL expression, relative to the Col wild type, in phyB, phyA, and cry1 photoreceptor mutants grown in red, far-red, and blue light conditions, respectively, for 5 d. For (A) and (B), relative expression was normalized to that of UBQ1, and data represent the mean ± sd of three biological replicates. (C) GUS staining of ProPKL:GUS transgenic seedlings. Seedlings were grown in white light or darkness for 5 d, 4-d-old dark-grown seedlings were exposed to white light for an additional 1 d (Dk→WL), or 4-d-old light-grown seedlings were transferred to darkness for an additional 1 d (WL→Dk). The inset in the WL panel is an enlargement of the hypocotyl. Bar = 2 mm. (D) GUS staining gradually increased in the hypocotyls of seedlings grown in a decreasing series of light intensities (μmol m⁻² s⁻¹; indicated below) for 5 d. Bar = 2 mm. (E) GUS staining in the hypocotyls was significantly enhanced in the photoreceptor mutants compared with the Col wild type. Seedlings harboring the ProPKL:GUS reporter were grown under the indicated light conditions for 5 d. Bar = 2 mm. (F) Immunoblotting of PKL protein. Seedlings were grown in long-day conditions (16 h day/8 h night) for 3 d and were then transferred to darkness or white light for the indicated period of time at the end of the day. Hypocotyls and cotyledons were detached and harvested for protein isolation. Immunoblotting against the tubulin antibody served as a loading control.

Figure 3.

PKL Directly Promotes the Expression of Cell Elongation Genes. (A) The short hypocotyls of the epp1 mutants correlate with a reduction in cell elongation but not in cell number. Seedlings were grown in white light for 5 d. Data represent the mean ± sd of 30 seedlings. Asterisks indicate significant difference from the wild type at P < 0.01 using Student’s t test. (B) qRT-PCR assay showing reduced expression of various genes involved in cell elongation in 5-d-old white light–grown epp1 mutants relative to the Col wild type. Relative expression was normalized to that of UBQ1. Data represent the mean ± sd of biological triplicates. (C) ChIP-qPCR assay showing enrichment of various cell elongation genes in DNA samples pulled down by PKL antibody or IgG control. Numbers in parentheses indicate regions for amplification, as shown in Supplemental Figure 3A online. The Col wild-type seedlings were grown under white light for 5 d. At4g26900 served as a negative control. Data represent the mean ± sd of triplicates. (D) ChIP-qPCR assay using the H3K27me3 antibody, showing relatively high enrichment of DWF4, EXT3, XTH17, and XTR6 in the epp1-1 mutant compared with the Col wild type. Numbers in parentheses indicate regions for amplification, as shown in Supplemental Figure 3A online. Precipitation by IgG preimmune serum served as a control. IP, immunoprecipitation. Data represent the mean ± sd of triplicates.

Figure 4.

PKL and HY5 Interact with Each Other. (A) Diagram of the domain structures of PKL and various PKL deletions (D1-6). (B) Yeast two-hybrid assay between various fragments or mutant forms of PKL shown in (A) fused to the LexA DNA binding domain and AD-tagged HY5 (AD-HY5) or AD alone. (C) Pull-down assay showing direct interaction between GST-PKL-D5 and His-HY5 fusion proteins in vitro. IB, immunoblotting; IP, immunoprecipitation. (D) BiFC assay showing that YFP^N-PKL and HY5-YFP^C interact to form a functional YFP in the nucleus. Bar = 5 μm. (E) Coimmunoprecipitation assay showing that the PKL antibody could precipitate HY5 in 5-d-old Col wild-type seedlings grown in both white light (1.5 μmol m⁻² s⁻¹) and darkness. (F) Seedling phenotypes of hy5, epp1-1, and epp1 hy5 mutants and the Col wild type after exposure to red (R), far-red (FR), blue light (BL), or darkness (Dk) for 5 d. The seedlings are arranged in identical order in each panel. Bars = 2 mm. (G) Quantification of hypocotyl length of the wild-type and mutant seedlings shown in (F). Data represent the mean ± sd of 30 seedlings.

Figure 5.

PKL Negatively Regulates HY5 by Inhibiting H3K27me3 of Target Loci. (A) Diagram of promoter structures of IAA19 and EXP2. ACE, ACGT-containing cis-element; UTR, untranslated region. E1 to E3 and I1 to I3 indicate fragments for ChIP-qPCR amplification. (B) Yeast one-hybrid assay showing that AD-HY5 binds to the promoter regions of IAA19 and EXP2 via the G-box. wt and m indicate wild-type and mutant forms of the G-box–containing fragments, respectively. “–” means empty AD fusion. (C) ChIP assay showing the relative enrichment of IAA19 (I1 to I3) and EXP2 (E1 to E3) genomic fragments upon precipitation with PKL or HY5 antibodies in light- or dark-grown seedlings. Dk, dark; WL, white light. (D) ChIP assay showing that binding of PKL to the regulatory regions of IAA19 and EXP2 was compromised in the hy5 hyh double mutant, as in the epp1-1 mutant. DNA samples were pulled down with PKL antibody or the IgG sera control. (E) Relative gene expression of EXP2 and IAA19 in hy5, epp1, and epp1 hy5 mutants and the Col wild type following transition from darkness to light for the indicated number of hours. (F) ChIP assay with DNA isolated from Col, epp1-1, hy5, and epp1 hy5 seedlings using anti-H3K27me3 antibody or the IgG control. In (C) to (F), data represent the mean ± sd of triplicates.

Figure 6.

HY5 Has Transcriptional Repression Activity. (A) Diagram of various constructs used in this assay. (B) Plasmid combinations of LUC reporter, GUS internal control, and effectors were cotransformed into Arabidopsis protoplasts. The protoplasts were incubated in weak light for 16 h, and relative activity was expressed as the ratio of LUC to GUS activity. Data represent the mean ± sd of three biological replicates.

Figure 7.

A Proposed Model of the Action of PKL/EPP1 and HY5 in Regulating Arabidopsis Hypocotyl Growth. In darkness, HY5 (and HYH) is largely degraded by the 26S proteasome–mediated pathway, whereas PKL/EPP1 is strongly expressed in the hypocotyl. A small pool of HY5 binds to the proximal promoter of cell elongation–related genes, including IAA19 and EXP2, which enables the recruitment of the chromatin remodeling factor PKL/EPP1 to these target loci through physical interaction. This largely prevents H3K27me3 formation and thereby activates cell elongation genes, leading to the promotion of hypocotyl growth. Other transcription activator(s) (indicated as TF) might also be involved in the activation of gene expression. Light triggers the stabilization of HY5 but reduces the level of PKL/EPP1. This allows the recruitment of more H3K27me3 marks on histones at cell elongation–related loci, leading to their repression and the inhibition of hypocotyl elongation.