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PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants

Abstract

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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Fig. 1: Role of PIF7 and phyB in low R:FR–induced hypocotyl growth.
Fig. 2: Low R:FR exposure induces genome-wide binding of PIF7.
Fig. 3: Low R:FR light exposure controls genome-wide H2A.Z occupancy.
Fig. 4: DNA binding of PIF7 initiates low R:FR–induced H2A.Z removal at its target genes.
Fig. 5: A PIF7–INO80 regulatory module facilitates low R:FR–induced H2A.Z removal.

Data availability

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.

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

B.C.W., M.Z., M.C., J.R.E. and J.C. designed the research. B.C.W. and M.Z. performed RNA-seq and ChIP–seq experiments. M.Z., Y.H., J.R.N. and H.C. analyzed the sequencing data and performed bioinformatics analyses. Plasmid cloning was done by B.C.W., M.Z. and A.P. Generation of genetic material, phenotyping, western blotting, pull-down and co-IP experiments were conducted by B.C.W and by A.P. under B.C.W.’s supervision. C.Y.Y. performed immunolocalization and confocal imaging analysis. R.M.G. and S.A.T. shared plasmid clones and protein interaction data. B.C.W., M.Z., C.Y.Y., M.C., J.R.E. and J.C. prepared the figures and wrote the manuscript.

Corresponding authors

Correspondence to Joseph R. Ecker or Joanne Chory.

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Competing interests

The authors declare no competing interests.

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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.

Extended data

Extended Data Fig. 1 PIF7 activity is regulated by low R:FR light exposure.

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.

Source data

Extended Data Fig. 2 Low R:FR light manipulates H2A.Z dynamics.

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.

Extended Data Fig. 3 Low R:FR light exposure induces global PIF7 DNA binding.

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).

Extended Data Fig. 4 Low R:FR induced H3K9 hyperacetylation depends on PIFs.

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.

Extended Data Fig. 5 PIF-EEN/INO80C interaction and een mutant complementation.

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).

Source data

Extended Data Fig. 6 PIF7 construct and light conditions.

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 information

Reporting Summary

Supplementary Tables 1–7

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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Source Data Fig. 5

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Source Data Extended Data Fig. 1

Unprocessed blots of Extended Data Fig. 1.

Source Data Extended Data Fig. 5

Unprocessed blots of Extended Data Fig. 5.

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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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