The interplay between light receptors and PHYTOCHROME-INTERACTING FACTORs (PIFs) serves as a regulatory hub that perceives and integrates environmental cues into transcriptional networks of plants1,2. Although occupancy of the histone variant H2A.Z and acetylation of histone H3 have emerged as regulators of environmentally responsive gene networks, how these epigenomic features interface with PIF activity is poorly understood3,4,5,6,7. By taking advantage of rapid and reversible light-mediated manipulation of PIF7 subnuclear localization and phosphorylation, we simultaneously assayed the DNA-binding properties of PIF7, as well as its impact on chromatin dynamics genome wide. We found that PIFs act rapidly to reshape the H2A.Z and H3K9ac epigenetic landscape in response to a change in light quality. Furthermore, we discovered that PIFs achieve H2A.Z removal through direct interaction with EIN6 ENHANCER (EEN), the Arabidopsis thaliana homolog of the chromatin remodeling complex subunit INO80 Subunit 6 (Ies6). Thus, we describe a PIF–INO80 regulatory module that is an intermediate step for allowing plants to change their growth trajectory in response to environmental changes.
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All mutants and transgenic lines can be requested from the corresponding authors. All sequence data can be accessed at GEO (accession GSE139296). ChIP–seq and RNA-seq data can be browsed at http://neomorph.salk.edu/aj2/pages/hchen/PIF7-INO80-H2AZ.php. Source data are provided with this paper.
Leivar, P. & Monte, E. PIFs: systems integrators in plant development. Plant Cell 26, 56–78 (2014).
Paik, I., Kathare, P. K., Kim, J. I. & Huq, E. Expanding roles of PIFs in signal integration from multiple processes. Mol. Plant 10, 1035–1046 (2017).
Kumar, S. V. et al. Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 484, 242–245 (2012).
van der Woude, L. C. et al. HISTONE DEACETYLASE 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion. Proc. Natl Acad. Sci. USA 116, 25343–25354 (2019).
Coleman-Derr, D. & Zilberman, D. Deposition of histone variant H2A.Z within gene bodies regulates responsive genes. PLoS Genet. 8, e1002988 (2012).
Sura, W. et al. Dual role of the histone variant H2A.Z in transcriptional regulation of stress-response genes. Plant Cell 29, 791–807 (2017).
Zander, M. et al. Epigenetic silencing of a multifunctional plant stress regulator. eLife 8, e47835 (2019).
Burgie, E. S. & Vierstra, R. D. Phytochromes: an atomic perspective on photoactivation and signaling. Plant Cell 26, 4568–4583 (2014).
Rockwell, N. C. & Lagarias, J. C. Phytochrome evolution in 3D: deletion, duplication, and diversification. New Phytol. 225, 2283–2300 (2020).
Franklin, K. A. Shade avoidance. New Phytol. 179, 930–944 (2008).
Casal, J. J. Shade avoidance. Arabidopsis Book 10, e0157 (2012).
Morgan, D. C. & Smith, H. Linear relationship between phytochrome photoequilibrium and growth in plants under natural radiation. Nature 262, 210–212 (1976).
Li, L. et al. Linking photoreceptor excitation to changes in plant architecture. Genes Dev. 26, 785–790 (2012).
Chung, B. Y. W. et al. An RNA thermoswitch regulates daytime growth in Arabidopsis. Nat. Plants 6, 522–532 (2020).
Hornitschek, P. et al. Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling. Plant J. 71, 699–711 (2012).
Oh, E. et al. Genome-wide analysis of genes targeted by PHYTOCHROME INTERACTING FACTOR 3-LIKE5 during seed germination in Arabidopsis. Plant Cell 21, 403–419 (2009).
Oh, E., Zhu, J. Y. & Wang, Z. Y. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat. Cell Biol. 14, 802–809 (2012).
Zhang, Y. et al. A quartet of PIF bHLH factors provides a transcriptionally centered signaling hub that regulates seedling morphogenesis through differential expression-patterning of shared target genes in Arabidopsis. PLoS Genet. 9, e1003244 (2013).
Steindler, C. et al. Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression. Development 126, 4235–4245 (1999).
Sessa, G. et al. A dynamic balance between gene activation and repression regulates the shade avoidance response in Arabidopsis. Genes Dev. 19, 2811–2815 (2005).
Hassidim, M., Harir, Y., Yakir, E., Kron, I. & Green, R. M. Over-expression of CONSTANS-LIKE 5 can induce flowering in short-day grown Arabidopsis. Planta 230, 481–491 (2009).
Barnes, C. E., English, D. M. & Cowley, S. M. Acetylation & Co: an expanding repertoire of histone acylations regulates chromatin and transcription. Essays Biochem. 63, 97–107 (2019).
Boycheva, I., Vassileva, V. & Iantcheva, A. Histone acetyltransferases in plant development and plasticity. Curr. Genomics 15, 28–37 (2014).
Alatwi, H. E. & Downs, J. A. Removal of H2A.Z by INO80 promotes homologous recombination. EMBO Rep. 16, 986–994 (2015).
Brahma, S. et al. INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers. Nat. Commun. 8, 15616 (2017).
Papamichos-Chronakis, M., Watanabe, S., Rando, O. J. & Peterson, C. L. Global regulation of H2A.Z localization by the INO80 chromatin-remodeling enzyme is essential for genome integrity. Cell 144, 200–213 (2011).
Watanabe, S. et al. Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C. Nat. Commun. 6, 7108 (2015).
Mao, Z. et al. Anp32e, a higher eukaryotic histone chaperone directs preferential recognition for H2A.Z. Cell Res. 24, 389–399 (2014).
Obri, A. et al. ANP32E is a histone chaperone that removes H2A.Z from chromatin. Nature 505, 648–653 (2014).
Zilberman, D., Coleman-Derr, D., Ballinger, T. & Henikoff, S. Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature 456, 125–129 (2008).
Fiorucci, A. S. et al. PHYTOCHROME INTERACTING FACTOR 7 is important for early responses to elevated temperature in Arabidopsis seedlings. New Phytol. 226, 50–58 (2019).
Jung, J. H. et al. Phytochromes function as thermosensors in Arabidopsis. Science 354, 886–889 (2016).
Legris, M. et al. Phytochrome B integrates light and temperature signals in Arabidopsis. Science 354, 897–900 (2016).
Lorrain, S., Allen, T., Duek, P. D., Whitelam, G. C. & Fankhauser, C. Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. Plant J. 53, 312–323 (2008).
Khanna, R. et al. The basic helix-loop-helix transcription factor PIF5 acts on ethylene biosynthesis and phytochrome signaling by distinct mechanisms. Plant Cell 19, 3915–3929 (2007).
Leivar, P. et al. The Arabidopsis phytochrome-interacting factor PIF7, together with PIF3 and PIF4, regulates responses to prolonged red light by modulating phyB levels. Plant Cell 20, 337–352 (2008).
Leivar, P. et al. Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness. Curr. Biol. 18, 1815–1823 (2008).
Reed, J. W., Nagpal, P., Poole, D. S., Furuya, M. & Chory, J. Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell 5, 147–157 (1993).
Kandasamy, M. K., McKinney, E. C., Deal, R. B., Smith, A. P. & Meagher, R. B. Arabidopsis actin-related protein ARP5 in multicellular development and DNA repair. Dev. Biol. 335, 22–32 (2009).
Nakagawa, T. et al. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 104, 34–41 (2007).
Jaillais, Y. et al. Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor. Genes Dev. 25, 232–237 (2011).
Wu, X. et al. Modes of intercellular transcription factor movement in the Arabidopsis apex. Development 130, 3735–3745 (2003).
Yoo, S. Y. et al. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221, 523–530 (2005).
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Nito, K., Wong, C. C., Yates, J. R. 3rd & Chory, J. Tyrosine phosphorylation regulates the activity of phytochrome photoreceptors. Cell Rep. 3, 1970–1979 (2013).
Galvão, V. C. et al. PIF transcription factors link a neighbor threat cue to accelerated reproduction in Arabidopsis. Nat. Commun. 10, 4005 (2019).
Feng, C. M., Qiu, Y., Van Buskirk, E. K., Yang, E. J. & Chen, M. Light-regulated gene repositioning in Arabidopsis. Nat. Commun. 5, 3027 (2014).
Sauer, M., Paciorek, T., Benková, E. & Friml, J. Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat. Protoc. 1, 98–103 (2006).
Kaufmann, K. et al. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat. Protoc. 5, 457–472 (2010).
Langmead, B. Aligning short sequencing reads with Bowtie. Curr. Protoc. Bioinform. 32, 11.7.1–11.7.14 (2010).
Song, L. et al. A transcription factor hierarchy defines an environmental stress response network. Science 354, aag1550 (2016).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Zang, C. et al. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 25, 1952–1958 (2009).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Guo, Y., Mahony, S. & Gifford, D. K. High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints. PLoS Comput. Biol. 8, e1002638 (2012).
Machanick, P. & Bailey, T. L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Dennis, G.Jr. et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, R60 (2003).
Shin, H., Liu, T., Manrai, A. K. & Liu, X. S. CEAS: cis-regulatory element annotation system. Bioinformatics 25, 2605–2606 (2009).
Ramírez, F., Dündar, F., Diehl, S., Grüning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).
Kent, W. J. et al. The human genome browser at UCSC. Genome Res 12, 996–1006 (2002).
Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).
Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).
Eustermann, S. et al. Structural basis for ATP-dependent chromatin remodelling by the INO80 complex. Nature 556, 386–390 (2018).
We thank A. Nagatani for kindly providing the anti-phyB antibody, X. Wu for materials and advice regarding Gibson cloning, J. Swift and H. Liu for critical comments on our manuscript and T. Haque for help with genotyping. B.C.W. was supported by an EMBO Long-Term Fellowship (ALTF 1514-2012), the Human Frontier Science Program (LT000222/2013-L) and the Salk Pioneer Postdoctoral Endowment Fund. M.Z. was supported by the Salk Pioneer Postdoctoral Endowment Fund as well as by a Deutsche Forschungsgemeinschaft (DFG) research fellowship (Za-730/1-1). This work was supported by grants from the National Science Foundation (NSF) (MCB-1024999, to J.R.E.), the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (DE-FG02-04ER15517, to J.R.E.), the Gordon and Betty Moore Foundation (GBMF3034, to J.R.E.) and the National Institutes of Health (NIH) (2R01GM087388, to M.C., and 5R35GM122604, to J.C.). J.C. and J.R.E. are investigators of the Howard Hughes Medical Institute.
The authors declare no competing interests.
Peer review information Nature Genetics thanks Frederic Berger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Hypocotyl length measurements of WT (n = 30/30), pif457 (n = 30/32, P = 0.271/P < 0.001) and pif457 expressing PIF7:PIF7:4xMYC (n = 36/36, P = 0.823/P < 0.001) in white light or in responses to low R:FR. Stars denote statistically significant differences between WT and the other genotypes for the respective light condition (two-way ANOVA, Tukey’s multiple comparisons test, n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). b, Hypocotyl length measurements of WT (n = 26, P < 0.001), phyB (n = 33), phyB pif457 (n = 23, P < 0.001) and phyB pif457 expressing PIF7:PIF7:4xMYC (n = 34, P = 0,027) grown in WL. Stars denote statistically significant differences between phyB and the other genotypes (one-way ANOVA, Tukey’s multiple comparisons test, n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). c, Immunoblot with anti-MYC of immunoprecipitated PIF7:4xMYC that had been treated with boiled (inactive) or native (active) λ-phosphatase (λ-PPase). PIF7:4xMYC was immunoprecipitated from 6 days old seedlings, grown in WL and harvested at ZT4. Mark next to cropped blot represent 50 kDa. d, Immunoblot analysis of pif457 PIF7:PIF7:4xMYC in LD. 6-day-old seedlings continued to grow in WL or where exposed to low R:FR at ZT0. Marks next to cropped blots represent 50 kDa (PIF7:4xMYC) or 37 kDa (ACTIN), respectively. e, Colocalization of PIF7:4xMYC and phyB containing nuclear speckles per nucleus. The number of phyB nuclear bodies (n = 105), PIF7 nuclear bodies (n = 98), and co-localized PHYB/PIF7 nuclear bodies were scored and used to calculate the percentage of co-localization of PHYB and PIF7. f, Aggregated profile shows the low R:FR dependent difference between PIF7 binding at ZT4. PIF7 binding was determined in WL and low R:FR-exposed pif457 PIF7:PIF7:4xMYC seedlings by ChIP-seq. PIF7 occupancy is shown from 1 kb upstream to 1 kb downstream of the 500 strongest PIF7 binding events. In a, b and e, boxes extend from the 25th to 75th percentiles. Middle lines represent medians. Whiskers extend to the smallest and largest values, respectively.
a, Heatmap visualizes absolute H2A.Z of all Arabidopsis thaliana protein-coding genes (TAIR10) at the indicated time points and light treatments. H2A.Z occupancy was determined by ChIP-seq in WT seedlings and calculated as the log2 fold change between H2A.Z ChIP and IgG control sample. b, AnnoJ genome browser screenshot visualizes the light quality-dependent H2A.Z occupancy at the COL5 gene at ZT0, ZT8 and ZT16. The WT IgG track serves as a control and all tracks were normalized to their sequencing depth. c, Quantification of H2A.Z levels at the gene body of COL5 is shown. Occupancy of H2A.Z was determined by ChIP-seq in one experiment and calculated as the ratio between H2A.Z and IgG control. d, Schematic overview illustrates the experimental setup that was used to investigate chromatin dynamics in low R:FR light responses for experiments shown in Figure 3c to e. e,f, Alternative presentation of results shown in Figure 3c and 3d. Aggregated profiles visualize low R:FR-induced H2A.Z loss and incorporation after two hours of low R:FR exposure (e), and after an additional two-hour-long WL recovery phase (f). Profiles are shown for genes that are differentially expressed after two hours of low R:FR exposure.
a, Levels of H2A.Z at ATHB2 in WT and pif457 seedlings at the indicated time points are shown. Occupancy of H2A.Z was determined by ChIP-seq (n = 1) and calculated as the ratio between H2A.Z and IgG. b, Aggregated profiles visualize the low R:FR-mediated activation of PIF7 after short low R:FR exposures (5, 10 and 30 min). PIF7 binding was determined in WL and low R:FR-exposed pif457 PIF7:PIF7:4xMYC seedlings by ChIP-seq and was calculated as the ratio between H2A.Z ChIP-seq samples and IgG control sample. PIF7 occupancy is shown from 1 kb upstream to 1 kb downstream of the 500 strongest PIF7 binding events. c, Bar plot illustrates increase of low R:FR-induced PIF7 DNA binding events. PIF7 binding events were determined by GEM through the direct comparison of the respective low R:FR-exposed and WL-exposed PIF7 ChIP-seq replicates (n = 3).
a, Aggregated profiles visualize the increase of H3K9ac at the most dynamic 200 genes after short low R:FR exposures (5, 10 and 30 min). H3K9ac occupancy was determined in WL and low R:FR-exposed pif457 PIF7:PIF7:4xMYC seedlings by ChIP-seq and was calculated as the ratio between WL and low R:FR-treated H3K9ac ChIP-seq samples. b, AnnoJ genome browser screenshot visualizes PIF7 binding and H3K9 acetylation at the ATHB2 gene and its closest relatives (ATHB4, HAT2, HAT3). Genome-wide occupancy of PIF7 and H3K9ac under constant light conditions was determined in the same pif457 PIF7:PIF7:4xMYC chromatin by ChIP-seq whereas under LD conditions at ZT4, WT (H3K9ac), pif457 (H3K9ac) and pif457 PIF7:PIF7:4xMYC (PIF7) chromatin was used. All tracks were normalized to the respective sequencing depth. The areas marked in red indicate PIF7 binding and H3K9 hyperacetylation. c, Quantification of relative H3K9ac levels at the promoters of ATHB2, ATHB4, HAT2 and HAT3 in low R:FR-exposed pif457 PIF7:PIF7:4xMYC seedlings. H3K9ac occupancy was calculated as the ratio between the respective ChIP-seq sample from one experiment and the WT IgG control. d, Aggregated profiles visualize the increase of H3K9ac at the most dynamic 200 genes after 4 hours of low R:FR exposure at ZT4. H3K9ac occupancy was determined in WL and low R:FR-exposed WT and pif457 seedlings by ChIP-seq and was calculated as the ratio between WL and low R:FR-treated H3K9ac ChIP-seq samples. e, Quantification of relative H3K9ac levels at the promoters of ATHB2, ATHB4, HAT2 and HAT3 in WL and low R:FR-exposed WT and pif457 seedlings. H3K9ac occupancy was calculated from one experiment as the ratio between the H2A.Z ChIP-seq sample and the WT IgG control.
a, Hypocotyl length of WT (n = 15/17, P < 0.001), pif457 (n = 17/13, P = 0.996), een (n=14/14, P < 0.001) and pif457 een (n = 12/15, P = 0.994) seedlings grown in WL or in response to low R:FR. b, Pull-down assay with in vitro translated proteins. ARP4, ARP5, ARP6, EEN, INO80 insertion domain (INO80Insert)64, and RVB2 were tagged with FLAG and PIF4 with HA. FLAG:GFP served as negative control. c, Hypocotyl length of WT (n = 16/18), een (n = 19/18, P = 0.406/P < 0.001) and een UBQ10:GFP:EEN line #3 (n = 15/17, P > 0.999/P = 0.49), #7 (n = 15/17, P > 0.999/P = 0.319) and #11 (n = 16/17, P > 0.999/P = 0.977). d, Aggregated H2A.Z profiles of all Arabidopsis genes (TAIR10) in WL and low R:FR-treated WT and een seedlings show H2A.Z occupancy around the TSS. e, Spearman’s correlation plot shows the correlation of read coverages between WL and low R:FR-treated WT and een H2A.Z ChIP-seq samples. Clustering was determined by the degree of correlation. f, Box plots show level of H2A.Z loss at the 20 most dynamic genes in WT, pif457 and een seedlings at ZT4 for three independent experiments. Boxes extend from the 25th to 75th percentiles. Middle lines represent the median. Stars denote statistically significant differences in comparison to WT (one-way ANOVA, Tukey’s multiple comparisons test, n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). g, Hypocotyl length measurements of WT (n = 37/35), een (n = 30/31, P = 0.806/P < 0.001) and een UBQ10:GFP:INO80C line #5 (n = 40/38, P = 0.877/P = 0.145), #7 (n = 37/37, P > 0.999/ P > 0.999) and #17 (n = 38/37, P > 0.999/ P > 0.999). h, Pull-down assay with in vitro translated proteins. INO80C was tagged with FLAG and PIF4 with HA. FLAG:GFP served as a negative control. In a, c and g, boxes extend from the 25th to 75th percentiles. Middle lines represent medians. Whiskers extend to the smallest and largest values, respectively. Stars denote statistically significant differences between light conditions (a) or versus WT for the respective light condition (c and g) (two-way ANOVA, Tukey’s multiple comparisons test, n.s. P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001).
a, The PIF7:PIF7:4xMYC construct consists of a 4064 bp genomic PIF7 fragment starting at 2500 bp upstream of the PIF7 start codon and is fused to a 4xMYC tag. b, Light spectra and fluence rate for white light and low R:FR conditions. c, Light intensities in μmols m-2 s-1 and the ratio between red and far-red light for the two light conditions used in this study.
Supplementary Table 1: List of PIF7 DNA binding events at ZT4 under low R:FR light exposure; Supplementary Table 2 Overview of Spearman’s correlations of genomic datasets; Supplementary Table 3: List of genes with significant H2A.Z enrichment and low R:FR-induced H2A.Z reduction; Supplementary Table 4: List of genes with differential expression and H2A.Z reduction after 2 hours of low R:FR light exposure; Supplementary Table 5: List of significant PIF7 DNA-binding events after short low R:FR light exposure times; Supplementary Table 6: List of genes that show an H3K9ac increase and R:FR-induced H2A.Z reduction in WT seedlings; Supplementary Table 7: List of used primers.
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Willige, B.C., Zander, M., Yoo, C.Y. et al. PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants. Nat Genet 53, 955–961 (2021). https://doi.org/10.1038/s41588-021-00882-3
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