PIF7‐mediated epigenetic reprogramming promotes the transcriptional response to shade in Arabidopsis

Abstract

For shade‐intolerant plants, changes in light quality through competition from neighbors trigger shade avoidance syndrome (SAS): a series of morphological and physiological adaptations that are ultimately detrimental to plant health and crop yield. Phytochrome‐interacting factor 7 (PIF7) is a major transcriptional regulator of SAS in Arabidopsis; however, how it regulates gene expression is not fully understood. Here, we show that PIF7 directly interacts with the histone chaperone anti‐silencing factor 1 (ASF1). The ASF1‐deprived asf1ab mutant showed defective shade‐induced hypocotyl elongation. Histone regulator homolog A (HIRA), which mediates deposition of the H3.3 variant into chromatin, is also involved in SAS. RNA/ChIP‐sequencing analyses identified the role of ASF1 in the direct regulation of a subset of PIF7 target genes. Furthermore, shade‐elicited gene activation is accompanied by H3.3 enrichment, which is mediated by the PIF7‐ASF1‐HIRA regulatory module. Collectively, our data reveal that PIF7 recruits ASF1‐HIRA to increase H3.3 incorporation into chromatin to promote gene transcription, thus enabling plants to effectively respond to environmental shade.

When an Arabidopsis plant grows in shade, the transcription factor PIF7 triggers the adaptive growth response by guiding H3.3 deposition via the histone chaperone ASF1.

Introduction

Plants are sessile organisms that adapt to various strategies by altering their growth and development in response to environmental changes. As photosynthetic organisms, plants are particularly sensitive to local light environments. Light influences every developmental transition, from seed germination and seedling emergence to flowering, reproduction, and seed formation. Shade‐intolerant plants can detect the proximity and density of neighboring vegetation through perception of a reduced ratio of red (660 nm) to far red (730 nm) light, which triggers the shade avoidance syndrome (SAS) that includes responses, such as an increase in hypocotyl and internode elongation, extended petioles, and changes in leaf hyponasty (Ruberti et al, 2012; Casal, 2013). Prolonged shade exposure can lead to early flowering, reduced branching, and decreased seed yields (Galvao et al, 2019; Zhang et al, 2019; Fernandez‐Milmanda & Ballare, 2021). As an adaptive strategy, SAS allows shaded plants to grow and compete with their neighbors at the cost of grain yield or plant biomass.

In Arabidopsis thaliana, shade‐light filtered through dense planting is perceived by photoreceptors (mainly known as phytochromes). The constitutive SAS phenotype of phytochrome B (phyB) mutant indicates that phyB plays a dominant role in inhibiting SAS (Reed et al, 1993). Shade‐light drives the Pfr‐to‐Pr conversion of phyB, allowing dephosphorylation and accumulation of phytochrome‐interacting factors (PIFs), a family of basic helix–loop–helix (bHLH) transcription factors (Leivar & Quail, 2011; Paik et al, 2017). The activation of PIFs, especially PIF7, promotes shade‐induced gene expression and SAS (Li et al, 2012; Galvao et al, 2019; Zhang et al, 2019). Numerous studies have been reported on transcriptional activity and function (Li et al, 2012; Peng et al, 2018; Jiang et al, 2019; Willige et al, 2021; Yang et al, 2021), translation (Chung et al, 2020), subcellular localization (Huang et al, 2018), and protein stability (Zhou et al, 2021) of PIF7, which have helped on understanding the molecular mechanisms of SAS.

Yeast two‐hybrid (Y2H) assays, conducted in our previous study (Huang et al, 2018), revealed that anti‐silencing function 1 (ASF1) is one of the putative PIF7‐interacting proteins. ASF1 is an evolutionarily conserved histone chaperone of the H3/H4 family (English et al, 2006; Natsume et al, 2007). The molecular functions and biochemical mechanisms of ASF1 have been well studied in animals and yeast. At DNA replication forks, ASF1 plays an important role in regulating the supply of H3.1 and H4 to the chromatin assembly factor 1 (CAF1) in chromatin assembly (English et al, 2006; Eitoku et al, 2008; Otero et al, 2014). ASF1 also provides H3.3‐H4 dimers to histone regulatory homolog A (HIRA) for DNA replication‐independent nucleosome assembly. While the canonical H3.1 is incorporated during DNA replication, the histone variant H3.3 is mainly incorporated upon gene activation and enriched in chromatin at actively transcribed genes (Ahmad & Henikoff, 2002; McKittrick et al, 2004; Stroud et al, 2012; Shu et al, 2014). Yeast cells lacking ASF1 display increased frequency of genome instability (Prado et al, 2004; Ramey et al, 2004). Deficiency of ASF1 in vertebrates and flies leads to embryonic lethality (Moshkin et al, 2002; Schulz & Tyler, 2006).

Two ASF1 homologs (ASF1A and ASF1B) are found in Arabidopsis, which play redundant roles and are shown to participate in S‐phase DNA replication‐dependent chromatin assembly (Zhu et al, 2011). The loss‐of‐function asf1ab double mutant exhibits defective thermotolerance (Weng et al, 2014) and early‐flowering phenotypes (Zhao et al, 2021). Many HIRA‐regulated genes were found belonging to a broad class of responsive genes to the environmental changes (Nie et al, 2014; Duc et al, 2017), however, whether and how ASF1 and HIRA coordinately regulate the transcription of specific genes in plants is still poorly understood.

In this study, we report that ASF1 and HIRA are required for shade‐induced hypocotyl elongation. Our results indicated that ASF1 is recruited by PIF7 to bind to a cluster of shade‐responsive genes. The PIF7‐ASF1‐HIRA complex promotes transcription by depositing H3.3 into chromatin at the target genes. Combined with previous reports (Peng et al, 2018; Willige et al, 2021), our study brings out that PIF7 recruits different chromatin modulators and conducts diverse types of histone variants (H3.3 and H2A.Z) and modifications to reprogram chromatin structure in response to shade.

Results

PIF7 interacts with ASF1A and ASF1B

Before verifying the interaction between PIF7 and ASF1, we first determined the effect of light on ASF1 expression. In Arabidopsis, ASF1A and ASF1B showed high sequence conservation (approximately 77.44% similarity) (Appendix Fig S1A). Both ASF1A and ASF1B mRNA levels were found stable in Col‐0 and pif7 under different light/shade/dark treatments (Appendix Fig S1B), which is consistent with the published RNA‐sequencing (RNA‐seq) data (Appendix Fig S1C, GSE81202). To investigate the effects of light on ASF1A and ASF1B protein abundance, 6‐day‐old white‐light‐grown 35S::GFP‐ASF1A and 35S::ASF1B‐GFP transgenic seedlings were transferred from white light to shade for 2 h. The protein levels of ASF1A and ASF1B (Appendix Fig S1D and E) were found barely changed after shade treatment. Together, these observations indicate that light does not significantly affect the transcription and protein levels of ASF1.

To test the interaction between PIF7 and ASF1, we used a luciferase complementation imaging (LCI) assay. As shown in Fig 1A, cLUC‐tagged PIF7 could interact with nLUC‐tagged ASF1A or ASF1B when they were transiently expressed in Nicotiana benthamiana leaf cells. In an in vitro pull‐down assay, SUMO‐His‐PIF7 was bound by GST‐fused ASF1A or ASF1B (Fig 1B, Appendix Fig S2A). In a semi‐in‐vivo pull‐down assay, GFP‐ASF1A extracted from GFP‐ASF1A seedlings were found to be bound by TF‐His‐PIF7 proteins purified from E. coli (Appendix Fig S2B). Interestingly, the dephosphorylated, but not the phosphorylated, PIF7‐Flash (9 × Myc‐6 × His‐3 × Flag) extracted from 35S:PIF7‐Flash (Li et al, 2012) seedlings was found to be bound by the GST‐fused ASF1 (Fig 1C). Finally, to further examine the interaction between ASF1 and PIF7 in vivo, we crossed the GFP‐ASF1A line with PIF7‐Flash line and performed the co‐immunoprecipitation (Co‐IP) assays. As expected, we found that GFP‐ASF1A formed protein complexes with dephosphorylated PIF7‐Flash (Fig 1D, Appendix Fig S2C). Taken together, these data strongly indicate that PIF7 physically interacts with ASF1, both in vitro and in vivo.

Figure 1. The histone chaperones ASF1A and ASF1B interact with PIF7.

1. Interaction between PIF7 and ASF1A/ASF1B was detected using a luciferase complementation imaging (LCI) assay. N‐terminal and C‐terminal halves of LUC were fused to ASF1A/ASF1B and PIF7, respectively. Luciferin was infiltrated before the LUC activity was monitored.
2. Interaction between PIF7 and ASF1A/ASF1B was detected using a pull‐down assay. SUMO‐His‐fused PIF7 and GST‐ASF1A/ASF1B were purified from E. coli.
3. Interaction between PIF7 and ASF1A/ASF1B was detected using a semi‐in‐vivo pull‐down assay. GST‐ASF1A/ASF1B were purified from E. coli. PIF7‐Flash protein from overexpressing PIF7‐Flash seedlings grown under white light or 1 h of shade treatment was detected using an anti‐MYC antibody.
4. Interaction between PIF7 and ASF1A in Arabidopsis was detected using the Co‐IP assay. Anti‐GFP Sepharose beads were used to precipitate GFP‐ASF1A from overexpressing GFP‐ASF1A * PIF7‐Flash seedlings grown under white light or 1 h of shade treatment.
5. Interaction between HIRA and ASF1A was detected using a pull‐down assay. TF‐His‐HIRA and GST‐ASF1A were purified from E. coli.
6. Interactions between ASF1A and PIF7/HIRA were detected in tobacco leaf cells. The PIF7‐Flash, HIRA‐HA, and GFP‐ASF1A (GFP as control) proteins were co‐expressed in tobacco leaf cells. Anti‐GFP Sepharose beads were used for Co‐IP assay.

Source data are available online for this figure.

Because human ASF1 and HIRA form protein complexes (Tang et al, 2006), we also tested the interactions of Arabidopsis HIRA with ASF1 and PIF7. We first confirmed the interaction between Arabidopsis ASF1A and HIRA by performing an in vitro pull‐down assay using TF‐His‐HIRA and GST‐ASF1A (Fig 1E, Appendix Fig S2A). We further found that TF‐His‐HIRA could also be pulled down by GFP‐PIF7, albeit to a lesser extent (Appendix Fig S2D). Lastly, GFP‐ASF1A simultaneously precipitated both HIRA‐HA and PIF7‐Flash when co‐expressed in tobacco leaves (Fig 1F). Based on these observations, we conclude that ASF1, HIRA, and PIF7 together form a multiple‐protein complex.

The histone chaperone ASF1 and HIRA positively regulate shade‐induced hypocotyl elongation

To investigate the function of ASF1 in SAS, we examined the hypocotyl phenotypes of the asf1a‐2, asf1b‐1, and asf1a‐2asf1b‐1 (asf1ab) mutants (Zhu et al, 2011). Although the single‐mutant asf1a‐2 and asf1b‐1 showed no difference in shade‐induced hypocotyl elongation compared to the wild‐type Col‐0, the double‐mutant asf1ab displayed a shade‐defective phenotype, similar to that of pif7‐1 (Fig 2A). The hira‐1 was lesser shade‐defective than asf1ab. The pif7‐1, asf1ab, and hira‐1 seedlings all showed reduced growth and slower growth rates than Col‐0 seedlings during the first 10 h after shade treatment (Fig 2B and C), indicating that these components are involved in promoting early shade response.

Figure 2. The histone chaperones ASF1A and ASF1B positively regulate shade‐induced hypocotyl elongation.

• A
  Hypocotyl lengths of Col‐0 (wild type), pif7‐1, asf1a, asf1b, asf1ab, and hira‐1 under white light and shade conditions. Seedlings were grown for 4 days under continuous white light and either maintained under white light (WL, with white box) or transferred to shade (SH, with gray box) for 4 days. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one‐way ANOVA with Tukey's honest significant difference (HSD) test. At least 20 seedlings were used for each treatment or genotype.
• B, C
  Growth analysis of Col‐0, pif7‐1, asf1ab, and hira‐1 seedlings treated with shade. New hypocotyl growth and real‐time growth rates were recorded at 15‐min intervals after shade treatment. I to IV representing four phases of growth under shade.
• D
  Hypocotyl lengths of Col‐0, phyB‐9, asf1ab, phyB‐9 * asf1ab, hira‐1, and phyB‐9 * hira‐1 grown under white light. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one‐way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype.
• E
  Hypocotyl lengths of Col‐0, pif7‐1, asf1ab, pif7‐1 * asf1ab, hira‐1, and pif7‐1 * hira‐1 under white light and shade conditions. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one‐way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype.

To determine the relationship of ASF1 and HIRA with phyB, we generated asf1ab * phyB‐9 and hira‐1 * phyB‐9 mutants by crossing. These combined mutants showed shorter hypocotyls than phyB under white light conditions (Fig 2D), indicating that the function of the photoreceptor phyB partially relies on ASF1 and HIRA.

To investigate the functional interactions between PIF7 and ASF1, we crossed asf1ab and hira‐1 with pif7‐1, respectively. As expected, the asf1ab * pif7‐1 and hira‐1 * pif7‐1 mutants showed hypocotyl lengths that were similar to that of pif7‐1 (Fig 2E), suggesting that the function of ASF1 and HIRA in shade‐induced hypocotyl growth depends on PIF7.

In addition to SAS, PIF7 is also important for early responses to elevated temperature in Arabidopsis seedlings (Chung et al, 2020; Fiorucci et al, 2020; Burko et al, 2022). We found defective hypocotyl elongation of asf1ab and hira‐1 at 29°C (Appendix Fig S3), suggesting that the PIF7‐ASF1‐HIRA complex may also play a role in thermomorphogenesis besides shade response. Nonetheless, hereinafter, we focus on shade response to uncover molecular mechanism underlying the PIF7‐ASF1‐HIRA regulation in hypocotyl elongation.

PIF7, ASF1, and HIRA are involved in shade‐induced gene expression

To investigate the roles of ASF1 and HIRA in shade‐induced transcription, we performed RNA‐seq analysis in asf1ab and hira‐1 mutants and compared it with pif7‐1 (Appendix Fig S4A–C). After 1 h shade stimuli, 642 shade‐induced genes were identified in Col‐0 seedlings (fold change > 2 and P‐value < 0.01, Dataset EV1). The expression of these genes was compromised in pif7‐1, as well as in asf1ab and hira‐1 mutants to varying degrees (Fig 3A and B, Dataset EV1). Specifically, there are 374, 222, and 258 genes showing reduced shade induction in pif7‐1, asf1ab, and hira‐1, respectively (Fig 3C, Dataset EV2). Hereinafter we called these genes  PIF7‐regulated, ASF1‐regulated, and HIRA‐regulated shade‐responsive genes, respectively. Furthermore, 132 common genes showed reduced shade induction in all three mutants (Fig 3C). Gene ontology (GO) analysis revealed that these common genes were enriched in functional categories, including auxin‐activated signaling pathway, response to auxin, and regulation of transcription, DNA‐templated (Fig 3D, Table EV1).

Figure 3. The PIF7‐ASF1‐HIRA regulatory module activates the gene expression under shade.

1. Heatmap representing the relative expression levels of shade‐induced genes in Col‐0, pif7‐1, asf1ab, and hira‐1. The red and white rows indicate RNA expression at high and low levels, respectively.
2. Boxplot displays the fold changes in shade‐induced genes in Col‐0, pif7‐1, asf1ab, and hira‐1 by comparing the transcript levels between white light and shade conditions. Different letters indicate significant differences (P < 0.05) as determined by one‐way ANOVA.
3. Venn diagram shows the 132 PIF7‐ASF1‐HIRA co‐regulated shade‐responsive genes.
4. Gene ontology (GO) analysis of 132 PIF7‐ASF1‐HIRA co‐regulated shade‐responsive genes. For each point, the size is proportional to the number of genes, and the colors represent the P‐value.

Remarkably, in response to shade stimuli, 77.2, 66.7, and 70.8% of differentially expressed genes (DEGs) were down‐regulated in pif7‐1, asf1ab, and hira‐1, respectively, indicating a positive effect of PIF7, ASF1, and HIRA on the transcriptional activation of these genes under shade (Appendix Fig S4D, Dataset EV3). Among the identified DEGs, the expression patterns of light‐related genes (ATHB2 and NPY8), hormone‐related genes (PIN7, BIM1, and ARL), and developmental growth‐related genes (CSLC4) were further verified and confirmed using quantitative RT–PCR analysis (Appendix Fig S4E). Based on the RNA‐seq and qRT–PCR data, we conclude that PIF7, ASF1, and HIRA act in transcriptional activation and regulate a substantial common subset of shade‐related genes.

Shade increases ASF1 enrichment in the chromatin of target genes of PIF7

To investigate the chromatin‐binding activity of ASF1 in response to shade exposure, we performed chromatin immunoprecipitation sequencing (ChIP‐seq) analysis using anti‐GFP antibodies on white‐light‐ and shade‐grown GFP‐ASF1A transgenic Arabidopsis plants. Approximately 6,259 peaks (for 5,662 genes) under white light and 9,031 peaks (for 7,950 genes) after 1 h of shade treatment were identified as high‐confidence GFP‐ASF1A‐enriched peaks (Fig 4A, fold change > 1.5, P‐value < 0.01, Dataset EV4). Examination of shade‐induced GFP‐ASF1A‐enrichment distributions revealed that 57.24% of the binding occurred at the coding exon, and 29.91% of them were found at the promoter (Fig 4B, Dataset EV5).

Figure 4. Shade increases ASF1 enrichment at chromatin of target genes of PIF7.

1. Heatmap representing the enriched GFP‐ASF1A‐binding peaks under white light (WL) and shade (SH) conditions. Red and white rows indicate GFP‐ASF1A peaks at high and low levels, respectively.
2. Pie chart represents the genomic distribution of shade‐induced GFP‐ASF1A binding events.
3. Average density plot represents the distribution profile of GFP‐ASF1A for shade‐induced genes (left) and control genes (right). The P‐value was calculated in a window from 1 kb upstream to TES by Welch's t‐test.
4. Integrative genomics viewer (IGV) screenshots showing the distribution of GFP‐ASF1A enrichment at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci.
5. Average density plot representing the distribution profile of GFP‐ASF1A in PIF7‐targeted shade‐responsive genes. The P‐value was calculated in a window from 1 kb upstream to TES by Welch's t‐test.
6. Average density plot representing the distribution profile of GFP‐ASF1A in GFP‐ASF1A‐bound and PIF7‐targeted shade‐responsive genes. The P‐value was calculated in a window from 1 kb upstream to TES by Welch's t‐test.
7. ChIP–PCR analysis of ASF1 enrichment using anti‐ASF1A antibodies at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci. Col‐0 and pif7‐1 seedlings were grown under continuous white light or transferred to the shade for 1 h. Top panels show a schematic representation of the gene structures. The bottom panels represent the effects of shade on ASF1A enrichment. Shade‐increased enrichment of ASF1A was calculated as input% SH minus input% WL. The difference at the P2 locus in Col‐0 was normalized to 1 for each gene. Different letters indicate statistically significant differences (P < 0.05) by one‐way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, where n refers to technical replicates).

The numbers of GFP‐ASF1A‐enriched peaks were slightly increased under shade. Compared to that in white light conditions, the genome‐wide GFP‐ASF1A enrichment was increased in response to shade. Specifically, 2,701 peaks (for 2,566 genes) showed an increase (fold change > 1.5, P‐value < 0.01; Dataset EV5), and these corresponding genes are called hereafter shade‐induced GFP‐ASF1‐bound genes. We found 161 shade‐induced GFP‐ASF1‐bound genes (average density plot shown in Appendix Fig S5A) that overlapped with DEGs in asf1ab (gene list in Datasets EV3 and EV5), thus likely representing genes that are directly regulated by ASF1 (GFP‐ASF1A‐targeted genes). GO analysis revealed that these genes were over‐represented by the “response to light stimulus” and “response to auxin,” which were significantly enriched (Appendix Fig S5B, Table EV2).

Specifically, GFP‐ASF1A enrichment was significantly increased under shade in 642 shade‐induced genes but not in control genes (not regulated by shade; Dataset EV2; Fig 4C). Enhancement in enrichment was more remarkable for 131 GFP‐ASF1A‐bound genes (Appendix Fig S5C). These results indicate that for a subset of shade‐induced genes, shade‐mediated gene activation is accompanied by ASF1 enrichment. The enrichment was illustrated as integrative genomics viewer (IGV) screenshots for the selected genes ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 (Fig 4D).

To examine the possible involvement of PIF7 in shade‐induced ASF1 bindings, we examined shade‐induced GFP‐ASF1A enrichment for the 182 PIF7‐targeted (PIF7‐bound and PIF7‐regulated) shade‐responsive genes (Fig 4E, Dataset EV2). A shade‐enhanced enrichment of GFP‐ASF1A was observed for 43 of these PIF7‐targeted genes (Fig 4F, Dataset EV2), indicating that their activation is associated with PIF7.

Then, we directly investigated the role of PIF7 in the binding of ASF1 to target genes. For this, we used the anti‐ASF1A antibody, which has been proven to be effective in ChIP–PCR analyses (Weng et al, 2014). Shade‐induced enrichment of ASF1A at the selected genes ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 was greatly reduced in pif7‐1 (Fig 4G) and slightly reduced in pifq (Appendix Fig S5D), indicating that the recruitment of ASF1A relies on PIFs, particularly PIF7.

Taken together, our results indicate that PIF7 recruits ASF1 for the transcriptional activation of a subset of genes in response to shade.

Shade induces H3.3 levels at a subset of shade‐responsive genes

Because ASF1 plays a role in the deposition of both H3.1 and H3.3 while HIRA is considered as specific for H3.3 (Tagami et al, 2004), we speculated that PIF7‐ASF1‐HIRA might regulate shade‐induced gene expression by affecting the balance of H3.1 and H3.3 assembly into chromatin. As a primary step, we first performed ChIP‐seq to examine the effect of shade exposure on the genome‐wide landscape distribution of H3.1 and H3.3. We selected HTR13 as the representative for H3.1 and HTR5 for H3.3, both of which have been described previously (Stroud et al, 2012). We generated pHTR13::HTR13‐HA and pHTR5::HTR5‐HA transgenic lines and performed ChIP‐seq analyses using anti‐HA antibodies, and obtained H3.1 and H3.3 density patterns (Fig 5, Appendix Fig S6). Based on a cutoff fold change > 1.5 and P‐value < 0.01, it was found that HTR13 / HTR5 was enriched in up to 10,827 peaks (for 8,746 genes) / 14,623 peaks (for 12,125 genes) under white light, and 10,842 peaks (for 8,527 genes) / 12,718 peaks (for 10,781 genes) after 1 h of shade treatment (Datasets EV6 and EV7). The occupancy of HTR13 / HTR5 detected in this study showed good overlap with previously published datasets obtained in Col‐0 under white light (Wollmann et al, 2012; Appendix Fig S6A and B). Consistent with the previous studies (Stroud et al, 2012; Wollmann et al, 2012), H3.1 was distributed across the gene body and associated with transcriptionally silent regions in the genome (Appendix Fig S6C), whereas the variant H3.3 was predominantly distributed toward the transcriptional end site (TES) and positively correlated with the levels of gene expression (Appendix Fig S6D).

Figure 5. Shade‐enriched H3.3 levels at its target genes.

• A, B
  Heatmaps representing genome‐wide H3.1‐ and H3.3‐enrichment levels from 2 kb upstream of the TSS to 2 kb downstream of the TES under white light (WL) and shade (SH) conditions. Gradient colors represent enrichment levels. The red and white rows indicate H3.1 / H3.3 enrichment at high and low levels, respectively. TSS: transcription start site; TES: transcription end site.
• C
  Average density plots representing the distribution profiles of H3.3 for shade‐induced genes and control genes. The P‐value was calculated in a window from 1 kb upstream to TES by Welch's t‐test.
• D
  Average density plots representing the distribution profiles of H3.1 for shade‐induced genes and control genes. The P‐value was calculated in a window from TSS to TES by Welch's t‐test.
• E
  The box‐plot represents the expression levels of 1,246 protein‐coding genes that displayed increased H3.3 levels in response to shade. The paired t‐test was used for statistical analysis.
• F
  GO analysis of the top 400 genes related to H3.3 enrichment and shade‐induced transcription. For each point, the size is proportional to the number of genes, and the colors represent the P‐value.
• G
  IGV screenshots show the distribution of H3.3 and H3.1 enrichment levels at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci.
• H
  Hypocotyl lengths of Col‐0, pif7‐1, and h3.3kd under white light and shade conditions. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one‐way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype.
• I
  Relative expression levels of ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 in Col‐0, pif7‐1, and h3.3kd seedlings grown under white light or shade conditions. The seedlings were grown under white light for 6 days, and then maintained under white light or transferred to shade for 1 h. Different letters indicate statistically significant differences (P < 0.05) by one‐way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).

Although the peak numbers of HTR13 and HTR5 were slightly reduced under shade, it did not affect the global levels of histone H3.1 and H3.3 (Fig 5A and B, Appendix Fig S6E), which is consistent with the stable mRNA levels of H3 genes (Appendix Fig S6F) and the protein levels of HTR13‐HA and HTR5‐HA detected by western‐blot analysis (Appendix Fig S6G). Interestingly, for the 642 shade‐induced genes, however, we found that shade increased H3.3 level (Fig 5C) and no significant change in the H3.1 level (Fig 5D), indicating that shade‐elicited gene activation is accompanied by H3.3 enrichment. As a control, genes not regulated by shade (Dataset EV2) did not show changes in H3.3 and H3.1 levels under shade (Fig 5C and D). In parallel, we also investigated whether shade‐induced H3.3‐enriched genes were shade responsive. Thus, the 1,323 peaks (1,246 protein‐coding genes) that displayed an increase in H3.3 level in response to shade (fold change > 1.5 and P‐value < 0.01, Dataset EV8) were found to be transcriptionally slightly induced under shade (Fig 5E, Dataset EV9). GO analysis revealed enrichment of “response to auxin,” “negative regulation of transcription, DNA‐templated,” and “brassinosteroid mediated signaling pathway” related terms for the top 400 genes with gaining H3.3 incorporation and increasing expression upon shade in Col‐0 (Fig 5F, Table EV3), with the profiles of ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 shown as examples (Fig 5G).

To further examine the function of H3.3 in shade responses, we obtained an H3.3 knockdown mutant (h3.3kd; Wollmann et al, 2017). h3.3kd displayed a defective phenotype for shade‐induced hypocotyl elongation (Fig 5H), which is largely similar to asf1ab. Moreover, the shade induction of ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 was impaired in h3.3kd (Fig 5I).

Taken together, our data indicate that activation of shade‐induced genes is associated with H3.3 enrichment and that H3.3 is required for a proper subset of shade‐induction gene transcription and hypocotyl elongation.

Shade‐induced H3.3‐enriched levels rely on PIF7‐ASF1‐HIRA regulatory module

Next, we examined the association of PIF7 and ASF1 functions with shade‐induced H3.3 incorporation. We checked the overlap among shade‐induced ASF1‐bound genes, PIF7‐bound genes (GSE156584; Yang et al, 2021), and H3.3‐enriched genes (Appendix Fig S7A). Genome ontology analysis of these genes showed that “regulation of transcription, DNA‐templated,” “regulation of cell size,” and “shade avoidance” were significantly enriched (Appendix Fig S7B, Table EV4). We also found that the enrichments of H3.3 were amplified by shade at PIF7‐regulated shade‐responsive genes, ASF1‐regulated shade‐responsive genes, and HIRA‐regulated shade‐responsive genes (Appendix Fig S7C). Moreover, shade effect on H3.3 enrichment was aggravated at PIF7‐targeted shade‐responsive genes (Appendix Fig S7C). Consistently, the transcriptional levels of the top 400 genes with gaining H3.3 incorporation and increasing expression upon shade in Col‐0 were found compromised in pif7‐1, asf1ab, and hira‐1 (Appendix Fig S7D).

To further test the role of PIF7 in facilitating shade‐induced H3.3 incorporation, we profiled H3.3 occupancy in the pif7‐1 mutant using pHTR5::HTR5‐HA/pif7‐1 transgenic lines under white light and shade conditions (Fig 6A). HTR5 was enriched in up to 16,710 peaks (for 13,418 coding genes) under white light, and 16,363 peaks (for 13,158 coding genes) after 1 h of shade treatment in pif7‐1 (with cutoff fold change > 1.5 and P‐value < 0.01, Dataset EV10). The peaks of H3.3 detected in pif7‐1 largely overlapped with that in Col‐0 (Appendix Fig S7E), suggesting that PIF7 does not affect the distribution pattern of H3.3 in the genome.

Figure 6. Shade‐enriched H3.3 levels at its target genes probably depend on PIF7‐ASF1.

1. Heatmap representing genome‐wide H3.3 enrichment levels from 2 kb upstream of the TSS to 2 kb downstream of the TES in pif7‐1 under white light (WL) and shade (SH) conditions. Gradient colors represent enrichment levels. The red and white rows indicate H3.3 enrichment at high and low levels, respectively.
2. Heatmap representing shade‐increased enrichment of H3.3 at 1246 shade‐induced H3.3 genes under Col‐0 and pif7‐1 background. Shade‐increased enrichment of H3.3 was calculated by enrichment levels of H3.3 under shade minus enrichment levels of H3.3 under white light.
3. Average density plots representing the distribution profile of H3.3 at shade‐induced genes (left) and control genes (right) in Col‐0 and pif7‐1 background. Enhanced H3.3 levels (enrichment levels of H3.3 under shade minus enrichment levels of H3.3 under white light) are visualized around the TES after shade exposure. The P‐value was calculated in a window from 1 kb upstream to TES by Welch's t‐test.
4. Average density plots representing the distribution profile of H3.3 at PIF7‐targeted shade‐responsive genes (left), and GFP‐ASF1A‐bound and PIF7‐targeted shade‐responsive genes (right) in Col‐0 and pif7‐1 background. Enhanced H3.3 levels (enrichment levels of H3.3 under shade minus enrichment levels of H3.3 under white light) are visualized around the TES after shade exposure. The P‐value was calculated in a window from 1 kb upstream to TES by Welch's t‐test.
5. ChIP–PCR analysis of HTR5 (H3.3) levels at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci. Top panels show a schematic representation of the gene structures. The bottom panels represent the effects of shade on H3.3 enrichment. Shade‐increased enrichment of H3.3 was calculated as the input% SH minus input% WL. The difference at the P2 locus in Col‐0 was normalized to one for each gene. Different letters indicate statistically significant differences (P < 0.05) by one‐way ANOVA with Tukey's HSD test. The data shown are the means ± SDs (n = 3, where n refers to biological replicates).
6. Proposed model for shade‐induced H3.3 enrichment and active transcription at the targets of PIF7. Under shade conditions, dephosphorylated PIF7 binds to its targets and recruits the ASF1‐HIRA complex. Consequently, gene‐body‐localized H3.3 is enhanced and target genes of PIF7 are activated, leading to hypocotyl elongation.

The shade‐induced enrichment of H3.3 in Col‐0 was clearly faint in the pif7‐1 mutant (Fig 6B). Then, we calculated the shade‐enhanced H3.3 levels (enrichment levels of HTR5 under shade minus the enrichment levels of HTR5 under white light) in Col‐0 and pif7‐1. For 642 shade‐induced genes, shade‐enhanced H3.3 enrichment was strongly compromised in the pif7‐1 mutant (Fig 6C). Moreover, H3.3 enrichments at the PIF7‐targeted shade‐responsive genes, at the ASF1‐bound and PIF7‐targeted shade‐responsive genes, as well as at the HIRA‐regulated shade‐responsive genes were compromised in pif7‐1 compared to Col‐0 (Fig 6D and Appendix Fig S7F). These results suggest that shade‐enhanced PIF7 activity is a prerequisite for H3.3 deposition and subsequent gene activation.

To further verify whether shade‐enhanced H3.3 levels are dependent on the PIF7‐ASF1‐HIRA regulatory module, we assessed HTR5 occupancy at multiple selected genes in Col‐0, pif7‐1, asf1ab, and hira‐1 by ChIP–PCR using pHTR5::HTR5‐HA transgenic lines. The levels of shade‐induced HTR5 enrichment were reduced in these three mutants at the 3′‐ends of ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 (Fig 6E).

Taken together, these results corroborate the previously established functions of ASF1‐HIRA in H3.3 deposition, thus facilitating nucleosome formation (Horard et al, 2018), and indicate that PIF7 recruits ASF1‐HIRA to increase H3.3 incorporation to activate transcription on a subset of shade‐induced genes.

Discussion

Plant growth and development are predominantly postembryonic and occur in response to environmental cues. This developmental plasticity is thought to be an adaptation to the sessile lifestyle of plants. SAS is an excellent example of phenotypic plasticity in the environment. Genetic and epigenetic factors are intertwined in a complex regulatory network for environmental adaptation (Xiao et al, 2017; Zhao et al, 2020). Transcription factors play an important role in environmentally triggered transcriptional reprogramming events; in addition to acting as transcriptional switches, they can also recruit chromatin modulators to increase the precision of regulation (Zhao et al, 2020).

Phytochrome‐interacting factors serve as regulatory hubs that integrate environmental cues into transcriptional scenario. In this study, we found that PIF7 directly interacts with ASF1 to promote the expression of shade‐responsive genes by mediating H3.3 enrichment at the target genes of PIF7. Under shade conditions, PIF7 can also recruit histone acetylases to promote H4K5ac in some shade‐induced genes, for example, YUC8, YUC9, and PAR1 (Peng et al, 2018). However, YUC8, YUC9, and PAR1 showed stable levels of H3.3 enrichment under shade treatment (Appendix Fig S8A). PIF7 recruits histone acetylase and EIN6 enhancer (EEN) to regulate H3K9ac and H2A.Z profiles at PIF7 core genes, for example, ATHB2 (Willige et al, 2021). The results of our study revealed that there are only a few genes showing more than one epigenetic change in response to shade or low R:FR (Appendix Fig S8B), such as ATHB2 showing enhanced H3.3 and H3K9ac but reduced H2A.Z (Appendix Fig S8C). Therefore, PIF7 may recruit different chromatin modulators (e.g., ASF1, EEN, MRG1/2, and histone acetylase) and conducts diverse types of histone variants (H3.3 and H2A.Z) and modifications (H4K5ac and H3K9ac) in an interactive or non‐interactive manner, likely depending on the target gene context, to reprogram chromatin structure under shade. These epigenetic changes might occur at different speeds in response to shade, and/or in different genes involved in different steps/pathways of regulation of cell proliferation/expansion. The complexity of epigenetic regulatory mechanisms might be advantageous for sedentary plants to precisely control gene transcription in response to dynamic environmental changes.

Most binding peaks of PIF7 occur in the promoter region (Willige et al, 2021; Yang et al, 2021), approximately 25.1 and 6.4% of PIF7‐binding events occur in exons and introns, respectively (Willige et al, 2021). Our results indicated that ASF1‐binding events were mainly enriched in gene bodies (Fig 4B). Shade‐induced binding events of ASF1 and H3.3 enrichment mainly occur at the 3′‐end of the gene bodies (Figs 4 and 5). So how does PIF7 regulate the distribution of H3.3 on the 3′‐end of the gene bodies? One hypothesis is that PIF7 bound in gene bodies can directly recruit the ASF1‐HIRA complex, thereby affecting the enrichment of H3.3 and regulating gene expression. Another hypothesis is that genes can be configured as looped structures (Kadauke & Blobel, 2009). Recent studies have described that active phyB forms a repressive chromatin loop with VIN3‐LIKE1/Vernalization 5 (VIL1/VRN5) in growth‐promoting genes (Kim et al, 2021). The enriched H3.3 at the 3′‐end of flowering locus C (FLC) can enhance active histone modifications (H3K4me3) at the 5′‐end by gene loop formation (Zhao et al, 2021). The three‐dimensional genome chromatin loop structure juxtaposes regulatory elements to regulate transcription with high efficiency (Kadauke & Blobel, 2009). However, whether PIF7‐ASF1‐HIRA affects chromatin loops at shade‐responsive genes remains to be investigated further.

Our results suggest that ASF1 is involved in shade‐induced hypocotyl elongation mainly by activating gene expression, which is correlated with transcriptional activation of PIF7 (Appendix Fig S4D). In this study, we found that ASF1 activates shade‐responsive gene expression mainly by promoting the H3.3 enrichment. The ASF1‐HIRA complex has been reported to promote FLC gene expression by enhancing H3.3 levels during flowering (Zhao et al, 2021). Incorporation of H3.3 into nucleosome likely modulates higher‐order chromatin folding, resulting in an open chromatin conformation (Chen et al, 2013). Nucleosomes are unstable and prone to disassembly, and this instability seems to facilitate the access of transcription factors or other chromatin‐associated factors to these regulatory sites (Jin et al, 2009; Venkatesh & Workman, 2015; Grover et al, 2018). ASF1 can promote H3K56ac in heat stress‐related genes to induce gene expression under heat stress conditions (Weng et al, 2014). H3.3 deposition has been reported to be correlated with H3K4me3 and H3K36me3 levels, which are associated with gene activation (Daury et al, 2006; Loyola et al, 2006; Zhao et al, 2021). However, significant differences were not detected in either H3K4me3 or H3K36me3 levels in YUC8, PRE1, and IAA19 after 1 h of shade treatment (Peng et al, 2018). It will be interesting to further explore the histone modifications that accompany H3.3 under shade conditions.

In addition to the PIF7‐ASF1‐HIRA regulatory module, which plays pivotal roles in SAS, PIF7 together with its homolog PIF4 are important for thermomorphogenesis (Chung et al, 2020; Fiorucci et al, 2020). Shade avoidance responses share phenotypic patterns similar to that of thermomorphogenesis (Ballare & Pierik, 2017; Burko et al, 2022). Both shade and elevated temperature can deactivate phyB, leading to increased PIFs and elongation (Legris et al, 2016; Ballare & Pierik, 2017). Similar to pif4 or pif7‐1, defective hypocotyl elongation was also found in asf1ab and hira‐1 at 29°C, suggesting a role of PIF7‐ASF1‐HIRA complex in thermomorphogenesis (Appendix Fig S3). However, whether ASF1‐HIRA functions together with PIFs in thermomorphogenesis remains to be investigated further.

Based on the results of our study, we propose a working model for how ASF1 activates the transcription on a subset of PIF7 target genes (Fig 6F). Shade induces the dephosphorylation of PIF7 and enhances PIF7 DNA‐binding activity (Li et al, 2012; Willige et al, 2021; Yang et al, 2021). Transcription factor PIF7 mainly binds to promoters and recruits other factors to induce gene expression (Peng et al, 2018; Willige et al, 2021). One of the factors recruited by PIF7 is ASF1, which affects H3.3 enrichment with the help of HIRA. Our results demonstrate that PIF7‐ASF1‐HIRA function on the enrichment of H3.3 and promote gene expression, leading to morphological changes in plants in response to shade.

Materials and Methods

Genetic material and growth conditions

All A. thaliana plants used in this study were of the Col‐0 ecotype. The mutants used in this study have been previously described: pif7‐1, phyB‐9, and PIF7‐Flash (Li et al, 2012); pifq (Yang et al, 2021); asf1a, asf1b, and asf1ab (Zhu et al, 2011); and hira‐1 (Nie et al, 2014). HTR5‐HA transgenic plants were obtained by transformation of Col‐0, pif7‐1, asf1ab with vectors containing the HTR5 genomic DNA. HTR13‐HA transgenic plants were obtained by transformation of Col‐0 with vectors containing the HTR13 genomic DNA. Full‐length ASF1A cDNA was amplified using PCR and ligated into pCambia1300 to generate pCambia1300‐GFP‐ASF1A. These constructs were transferred into Agrobacterium tumefaciens strain GV3101 (WEIDI, Shanghai, China). Transgenic plants were screened on half‐strength Murashige and Skoog (1/2 MS) nutrient medium (Duchefa Biochemie, Haarlem, Netherlands) containing hygromycin and were confirmed using immunoblot analysis. We generated pif7‐1 * asf1ab, pif7‐1 * hira‐1, phyB‐9 * asf1ab, phyB‐9 * hira‐1, and GFP‐ASF1A * PIF7‐Flash plants via crossing. Double mutants were generated by genetic crossing and were verified using phenotypic inspection, PCR genotyping, and/or sequencing.

For phenotypic analysis, seeds were allowed to germinate on plates containing 1/2 MS medium (Duchefa Biochemie, Haarlem, Netherlands) with 1% agar (Sangon, Shanghai, China) and without sucrose. After stratification, the plates were incubated in growth chambers under continuous white light (R, ~20 μmol m⁻² s⁻¹; B, ~20 μmol m⁻² s⁻¹; and FR, ~5 μmol m⁻² s⁻¹) for 4 days at 22°C. The plates were either left in white light or transferred to simulated shade (R, ~20 μmol m⁻² s⁻¹; B, ~20 μmol m⁻² s⁻¹; and FR, ~60 μmol m⁻² s⁻¹) for 4 days before measuring the hypocotyl length. To examine hypocotyl elongation at high temperatures, plants were grown at 22°C under white light (R, ~20 μmol m⁻² s⁻¹; B, ~20 μmol m⁻² s⁻¹; and FR, ~5 μmol m⁻² s⁻¹) for 3–4 days, and were further shifted to 29°C or maintained at 22°C for 4 days. Nicotiana benthamiana plants were grown at 26°C under long‐day conditions with 16 h light.

Hypocotyl measurement

Quantitative measurements of hypocotyls were performed on scanned images of seedlings using ImageJ software. At least 20 seedlings were used per treatment or genotype. The Kinetics of hypocotyl growth was measured by a commercial high‐throughput imaging platform, DynaPlant^® (Microlens Technology, Beijing, http://www.dynaplant.cn/en). Seedlings for kinetics measurement were sown on 1/2 MS medium containing 2% phytagel (Solarbio, P8170) and grown under continuous white light, and then the plates were transferred to simulated shade. The images of hypocotyl growth were captured by the DynaPlant^® platform once every 15 min for each seedling with a physical resolution of 1.2 μm per pixel. The lengths of new hypocotyl growth in the time‐series images were quantified by DynaPlant Analysis software which was provided by the manufacturer. The values shown indicate the means with SEMs.

Firefly luciferase complementation imaging assays

The fragments encoding PIF7 were amplified by PCR and ligated into a pCAMBIA2300‐cLUC vector to produce cLUC‐PIF7. The coding regions of ASF1A or ASF1B were amplified by PCR and ligated into pCAMBIA2300‐nLUC to produce ASF1A‐nLUC or ASF1B‐nLUC. The resulting constructs were transformed into Agrobacterium strain GV3101. Agrobacterium cells harboring different constructs were then infiltrated into N. benthamiana leaves. Three days after infiltration, luciferin (Promega, USA) (2.5 mM, 0.1% Triton X‐100) was spread before LUC activity was monitored by a Tanon 5500 chemical luminescence imaging system (Tanon, China).

Protein pull‐down assay

For Fig 1B, in the GST pull‐down assay, the SUMO‐His‐PIF7 protein was incubated with pretreated GST‐ASF1A or GST‐ASF1B beads for 2 h. GST was used as the negative control. The beads were resuspended in SDS–PAGE‐loading buffer and analyzed by SDS–PAGE and immunoblotting using the anti‐His antibody (GNI4110‐HS).

For Appendix Fig S2B, in a semi‐in‐vivo pull‐down assay, total protein was extracted from GFP‐ASF1A plants grown in duplicate under white light for 8 days using extraction buffer (100 mM Tris–HCl [pH 7.5], 300 mM NaCl, 2 mM EDTA, 1% Trion X‐100, 10% glycerol, and protease inhibitor cocktail). Protein extracts were centrifuged at 16,000 g for 10 min, and the resulting supernatant was incubated with pretreated TF‐His‐PIF7 beads for 2 h. GFP was used as the negative control. Beads were resuspended in SDS–PAGE‐loading buffer, analyzed using SDS–PAGE, and followed by immunoblotting using the anti‐GFP antibody (GNI4110‐GP). For Fig 1C, total protein was extracted from PIF7‐Flash plants grown in duplicate under white light for 8 days, thereafter, one of the duplicates was treated with shade for 1 h, and the other was maintained under white light for an additional 1 h. Protein extracts were centrifuged at 16,000 g for 10 min, and the resulting supernatant was incubated with pretreated GST‐ASF1A or GST‐ASF1B beads for 2 h. GST was used as the negative control. The beads were resuspended in SDS–PAGE‐loading buffer and analyzed by SDS–PAGE and immunoblotting using the anti‐MYC antibody (GNI4110‐MC).

Co‐IP assay

Total protein extracts were prepared from GFP‐ASF1A * PIF7‐Flash seedlings grown in duplicate under white light for 8 days; thereafter, one of the duplicates was treated with shade for 1 h, and the other was maintained under white light for an additional hour. A Co‐IP assay was performed as described previously (Peng et al, 2018). IP was performed using anti‐MYC agarose beads. Input and IP‐resulting fractions were analyzed by western blotting using anti‐MYC and anti‐GFP antibodies.

RNA‐Seq analysis

RNA‐seq was performed as previously described (Yang et al, 2020). In brief, seedlings were grown under white light conditions for 6 days and treated with 1 h shade. Three biological replicates were prepared for each genotype of the plants grown under light and shade conditions. Total RNA was extracted from snap‐frozen tissues using TRIzol reagent according to the manufacturer's instructions (Invitrogen). RNA libraries were constructed and sequenced using Majorbio (http://www.majorbio.com/). Differential expression analysis was performed using DESeq2 with |log2foldchange| > log2(2) and P‐value < 0.01.

Quantitative RT–PCR analysis

Approximately 100 mg of seedlings grown on 1/2 MS media supplemented with 1% agar under different light conditions were collected in Eppendorf tubes, frozen in liquid nitrogen, and ground to a fine powder. Three biological replicates were prepared for each genotype of the plants grown under light and shade conditions. Total RNA was extracted using a TRIzol kit (Promega, USA). Two micrograms of total RNA were reverse transcripted using a First‐Strand cDNA Synthesis Kit (TIANGEN, China) according to the manufacturer's instructions. The cDNAs were then subjected to real‐time qPCR using a CFX Connect Real‐Time System (Bio‐Rad, USA) and SYBR Green qPCR Mix (Mei5 Biochem, China). Three biological replicates per sample were used for the qRT–PCR analysis. The data are presented as means with the SDs of three biological replicates normalized to the expression of the reference gene AT2G39960 (Li et al, 2012). The comparative ΔΔCt method was used to evaluate the relative quantities of each amplified product in the samples. The specificity of the qRT–PCR reactions was determined by melt curve analysis of the amplified products using the standard method. Primers used are listed in Table EV5.

ChIP‐Seq analysis

ChIP‐seq assays were performed as previously described (Yang et al, 2020). For GFP‐ASF1A, HTR13‐HA/Col‐0, HTR5‐HA/Col‐0, and HTR5‐HA/pif7‐1 ChIP‐seq, two biological replicates were prepared for each genotype of plants grown under light and shade conditions. White‐light‐grown seedlings treated with 1 h shade were quickly plucked from plates to perform ChIP assays. Approximately 4 g fresh seedlings were collected and treated with fix buffer [0.4 M sucrose, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 1 mM PMSF, 1% formaldehyde, and 1 × protease inhibitor cocktail tablet (Roche)] under vacuum for about 15 min. Fixation was quenched by adding 0.125 M glycine and infiltrated for 7 min, after which the seedlings were washed three times with cold water and rapidly frozen with liquid nitrogen. The tissue was resuspended in extraction buffer 1 [0.4 M sucrose, 10 mM Tris–HCl (pH 8.0), 10 mM MgCl₂, 5 mM β‐ME, 0.1 mM PMSF, 1 × protease inhibitor cocktail tablet] and mixed at 4°C for 20 min. The lysate was filtered through Miracloth and centrifuged at 1,500 g for 20 min at 4°C. The precipitate was resuspended in extraction buffer 2 [0.25 M sucrose, 1% Triton X‐100, 10 mM Tris–HCl (pH 8.0), 10 mM MgCl₂, 5 mM β‐ME, 0.1 mM PMSF, and 1 × protease inhibitor cocktail tablet] and centrifuged at 12,000 g for 10 min at 4°C. The pellet was then resuspended in 300 μl extraction buffer 3 [1.7 M sucrose, 0.15% Triton X‐100, 10 mM Tris–HCl (pH 8.0), 10 mM MgCl₂, 5 mM β‐ME, 0.1 mM PMSF, and 1 × protease inhibitor cocktail tablet]. Extraction buffer 3 (450 μl) was layered and centrifuged at 16,000 g for 1 h at 4°C. Nuclei were sonicated in 300 μl nuclei lysis buffer [1% SDS, 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, and 1 × protease inhibitor cocktail tablet], and DNA was sheared on a Bioruptor Next Gen UCD‐300 (Diagenode Co., Ltd., Toyama, Japan) for 15 cycles of 30/30 s on/off. The mixture was then centrifuged at 16,000 g for 10 min at 4°C. Four times the volume of ChIP dilution buffer [1.1% Triton X‐100, 1.2 mM EDTA, 167 mM Tris–HCL (pH8.0), 167 mM NaCl, and 1 × protease inhibitor cocktail tablet] was added to the supernatant. The mixture was incubated overnight at 4°C with GFP antibody (ab290, Fig 4) and HA antibody (ab9110, Figs 5 and 6). Fifty microliters of Protein A (Life Technologies) was washed with the ChIP dilution buffer three times and incubated for an additional 1 h. The mixture was then centrifuged at 100 g for 2 min at 4°C. The beads were washed with 1 ml of the following buffers for 5 min while rotating at 4°C: 1 × low salt buffer [150 mM NaCl, 0.1% SDS, 1% Triton X‐100, 2 mM EDTA, and 20 mM Tris–HCl (pH 8)], 1 × high‐salt buffer [500 mM NaCl, 0.1% SDS, 1% Triton X‐100, 2 mM EDTA, and 20 mM Tris–HCl (pH 8.0)], 1 × LiCl wash buffer [250 mM LiCl, 1% NP‐40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris–HCl (pH 8.0)], and 2 × TE buffer [10 mM Tris–HCl (pH 8.0) and 1 mM EDTA]. The immunocomplex was eluted from the beads twice with elution buffer (1% SDS and 0.1 M NaHCO₃) and incubated for 20 min with shaking at 65°C. A total of 20 μl of 5 M NaCl was added, and crosslinking was reversed by incubation at 65°C overnight. Ten microliters of 0.5 M EDTA, 20 μl 1 M Tris–HCl (pH 6.5–8.0), 2 μl 20 mg/ml Proteinase K (Invitrogen), and 3 μl 10 mg/ml RNase A1 was added and the mixture was incubated at 45°C for 1 h. DNA was purified using the HiPure Gel Pure DNA Mini Kit (Magen, D2111‐02). Purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. Libraries were prepared using the Illumina E7630S NEB/NEBNext® Library Quant Kit (Illumina). Amplified libraries were size selected using 2% gel to capture fragments between 100 and 400 bp. The libraries were quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies). Samples without antibodies were used as input controls.

The sample library was sequenced on a NovaSeq6000 PE150 by Shanghai Jiayin Biotechnology Ltd. (http://www.rainbow‐genome.com/). A quality distribution plot and base content distribution were generated using FASTQC. Before read mapping, clean reads were obtained from the raw reads by removing adaptor sequences. The clean reads were aligned to the reference genome sequences using the BWA program. The bam file was generated using the unique mapped reads as an input file, and using macs2 software for call peak with cutoff fold change > 1.5 and P‐value < 0.01. The input file was the peak file and genome fasta file. The DNA sequence was extracted according to the peak file, and the sequence was compared with the motif database to obtain the motif. Read distributions (bigwig file of IP to subtract input) across genes are presented as an average plot. For statistical difference peak analysis, we merged the peak files of each sample using the bedtools software. The counts of the reads over the bed were determined for each sample using bedtools multicov. Differential accessible peak was assessed using DESeq2. Regions were called differentially accessible with cutoff fold change > 1.5 and P‐value < 0.01. DAVID was used to identify gene ontology enrichment in the ChIP–seq data.

ChIP–PCR assay

ChIP was performed as previously described (Yang et al, 2020). For @ASF1 (Weng et al, 2014) ChIP, Col‐0, pif7‐1, and pifq seedlings were grown under white light for 8 days and then treated with shade for 1 h. Two biological replicates were prepared (Fig 4G and Appendix Fig S5D). For @HA (ab9110) ChIP, HTR5‐HA/Col‐0, HTR5‐HA/pif7‐1, HTR5‐HA/asf1ab, and HTR5‐HA/hira‐1 seedlings were grown under white light for 8 days and then treated with shade for 1 h. The seedlings were harvested and cross‐linked for 15 min under vacuum in a cross‐linking buffer (extraction buffer 1 with 1% formaldehyde). Cross‐linking was terminated with 125 mM glycine (pH 8.0) under vacuum for 5 min, and the seedlings were washed three times in double‐distilled water and rapidly frozen. The bioruptor was used at high power with 30 s on/30 s off cycles 15 times until the average chromatin size was approximately 300 bp. An anti‐HA affinity gel was used for IP. qRT–PCR was performed using a kit from Takara to determine the enrichment of immunoprecipitated DNA in the ChIP experiments.

Author contributions

Lin Li: Resources; funding acquisition; writing – original draft; project administration; writing – review and editing. Chuanwei Yang: Resources; data curation; software; formal analysis; funding acquisition; investigation; visualization. Tongdan Zhu: Resources; formal analysis; investigation; visualization. Nana Zhou: Resources. Sha Huang: Resources; formal analysis. Yue Zeng: Resources; formal analysis. Wen Jiang: Resources. Yu Xie: Resources. Wen‐Hui Shen: Funding acquisition; writing – original draft; writing – review and editing.

Disclosure and competing interests statement

The authors declare that they have no conflict of interest.

Supporting information

Appendix

Table EV1

Table EV2

Table EV3

Table EV4

Table EV5

Dataset EV1

Dataset EV2

Dataset EV3

Dataset EV4

Dataset EV5

Dataset EV6

Dataset EV7

Dataset EV8

Dataset EV9

Dataset EV10

Source Data for Appendix

Source Data for Figure 1

The EMBO Journal (2023) 42: e111472

Data availability

All mutants and transgenic lines can be requested from the corresponding authors. RNA‐seq and ChIP‐seq data have been deposited in the NCBI GEO database with accession number GSE189669 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189669) and GSE193955 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193955), respectively.