# VRN2-PRC2 facilitates light-triggered repression of PIF signaling to coordinate growth in Arabidopsis

Rory Osborne, Anne-marie Labandera, Alex J Ryder, Anastasia Kanali, Tianyuan Xu, Oluwatunmise Akintewe, Maximillian A Schwarze, Christian D Morgan, Sjon Hartman, Eirini Kaiserli, et al.

► To cite this version:

Rory Osborne, Anne-marie Labandera, Alex J Ryder, Anastasia Kanali, Tianyuan Xu, et al. VRN2-PRC2 facilitates light-triggered repression of PIF signaling to coordinate growth in Arabidopsis. Developmental Cell, 2025, 60, pp.2046 - 2060.e5. 10.1016/j.devcel.2025.03.001. hal-05273809

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Submitted on 23 Sep 2025

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# Developmental Cell

## VRN2-PRC2 facilitates light-triggered repression of PIF signaling to coordinate growth in *Arabidopsis*

### Graphical abstract

<figure><img src="image_2.png" /></figure>

### Authors

Rory Osborne, Anne-Marie Labandera, Alex J. Ryder, ..., Sjon Hartman, Eirini Kaiserli, Daniel J. Gibbs

### Correspondence

d.gibbs@bham.ac.uk

### In brief

Osborne et al. show that the angiosperm-specific PRC2 subunit VRN2 is enriched in hypoxic regions of the *Arabidopsis* shoot, where it is required for stable deposition of H3K27me3 in key PIF4 target genes. This keeps these genes epigenetically poised for light-triggered repression to coordinate cell-expansion-mediated growth as leaves mature.

### Highlights

* VRN2 is enriched in hypoxic shoot tissues and negatively regulates cell expansion

* VRN2-PRC2 promotes the stable deposition of H3K27me3 in PIF4 target genes

* H3K27me3 facilitates light-triggered repression of PIF4 signaling to inhibit growth

* VRN2 may connect hypoxia gradients to the epigenetic control of development

<figure><img src="image_3.png" /></figure>
## Article

# VRN2-PRC2 facilitates light-triggered repression of PIF signaling to coordinate growth in *Arabidopsis*

Rory Osborne,<fnref n="1" /> Anne-Marie Labandera,<fnref n="1" />,<fnref n="5" /> Alex J. Ryder,<fnref n="1" /> Anastasia Kanali,<fnref n="1" /> Tianyuan Xu,<fnref n="2" /> Oluwatunmise Akintewe,<fnref n="1" /> Maximillian A. Schwarze,<fnref n="1" /> Christian D. Morgan,<fnref n="1" /> Sjon Hartman,<fnref n="1" />,<fnref n="3" />,<fnref n="4" /> Eirini Kaiserli,<fnref n="2" /> and Daniel J. Gibbs<fnref n="1" />,<fnref n="6" />,<fnref n="7" />,*

## SUMMARY

VERNALIZATION2 (VRN2) is a flowering plant-specific subunit of the polycomb-repressive complex 2 (PRC2), a conserved eukaryotic holoenzyme that represses gene expression by depositing the histone H3 lysine 27 trimethylation (H3K27me3) mark in chromatin. Previous work established VRN2 as an oxygen-regulated target of the N-degron pathway that may function as a sensor subunit connecting PRC2 activity to the perception of endogenous and environmental cues. Here, we show that VRN2 is enriched in the hypoxic shoot apex and emerging leaves of *Arabidopsis*, where it negatively regulates growth by establishing a stable and conditionally repressed chromatin state in key PHYTOCHROME INTERACTING FACTOR (PIF)-regulated genes that promote cell expansion. This function is required to keep these genes poised for repression via a light-responsive signaling cascade later in leaf development. Thus, we identify VRN2-PRC2 as a core component of a developmentally and spatially encoded epigenetic mechanism that coordinates plant growth through facilitating the signal-dependent suppression of PIF signaling.

## INTRODUCTION

Changes in chromatin structure play an important role in regulating gene expression in eukaryotes. For example, a diverse range of modifications on histone tails can influence gene expression by altering histone compaction, DNA accessibility, and transcription factor occupancy.<fnref n="1" /> One well-characterized histone mark is histone H3 lysine 27 trimethylation (H3K27me3), a repressive modification that is deposited by the polycomb-repressive complex 2 (PRC2) in most animals and land plants.<fnref n="2" />,<fnref n="3" /> H3K27me3 is often mitotically stable, potentiated by a PRC2-mediated “read-and-write” mechanism that transmits the modification as DNA replicates to trigger long-term gene repression.<fnref n="4" /> However, H3K27me3 can also be erased by specific demethylases<fnref n="5" /> or co-exist with activating marks in a bivalent state that facilitates dynamic changes in gene expression.<fnref n="6" />⁻¹² Thus, PRC2-mediated H3K27me3 typically functions as part of a broader set of chemical modifications that control the local chromatin state to fine-tune gene expression.

In *Arabidopsis thaliana* (hereafter *Arabidopsis*), PRC2s are classified according to which of three homologs of the

*Drosophila* SUPPRESSOR OF ZESTE 12 (Suz12) subunit they recruit (Figure 1A).¹³,¹⁴ This includes the sporophyte-specific Suz12 orthologs EMBRYONIC FLOWER 2 (EMF2) and VERNALIZATION 2 (VRN2), which can interchangeably associate with the two sporophyte methyltransferases CURLY LEAF (CLF) and SWINGER (SWN), as well as gametophyte-specific FERTILIZATION INDEPENDENT SEED 2 (FIS2) and its cognate methyltransferase MEDEA (MEA). Several discrete and overlapping functions for each of these different complexes have been ascribed.<fnref n="2" />,¹³⁻¹⁶ VRN2-PRC2 has been linked to the regulation of vernalization and flowering time,¹⁷,¹⁸ seed development and dormancy,¹⁹,²⁰ preventing somatic cell de-differentiation,²¹ and repressing root development.¹⁸ Apart from vernalization, where VRN2-PRC2 is part of the mechanism that epigenetically silences *FLOWERING LOCUS C* (FLC),¹⁷,²² the specific targets of VRN2 involved in many of these responses are unknown or only partially characterized.

VRN2 protein stability is controlled by the PROTEOLYSIS6 (PRT6) N-degron pathway.²³ This proteolytic pathway regulates hypoxia-stress responses and nitric oxide (NO) signaling in plants,²⁴,²⁵ and it has recently been shown to transduce

<footnote n="1">¹School of Biosciences, University of Birmingham, Edgbaston B152TT, UK</footnote>

<footnote n="2">²School of Molecular Biosciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK</footnote>

<footnote n="3">³Present address: Plant Environmental Signalling and Development, Faculty of Biology, University of Freiburg, Freiburg 79104, Germany</footnote>

<footnote n="4">⁴Present address: CIBSS-Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg 79104, Germany</footnote>

<footnote n="5">⁵Present address: Université de Bordeaux, UMR Ecophysiologie et Génomique Fonctionnelle de la Vigne, 210 Chemin de Leysotte, Villenave d'Ornon, 33140 Bordeaux France</footnote>

<footnote n="6">⁶X (formerly Twitter): @DJ_Gibbs</footnote>

<footnote n="7">⁷Lead contact</footnote>

<footnote>*Correspondence: d.gibbs@bham.ac.uk
https://doi.org/10.1016/j.devcel.2025.03.001</footnote>
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endogenous hypoxia into the regulation of leaf and lateral root development via LITTLE ZIPPER 2 (ZPR2) and GROUP VII ETHYLENE RESPONSE FACTOR (ERFVII) substrates, respectively.²⁶,²⁷ As a target of this pathway, VRN2 is highly labile but relatively enriched in the shoot apical meristem (SAM), root meristem (RM), and lateral root primordia (LRP), known hypoxic niches.¹⁸,²³,²⁸,²⁹ We previously showed that VRN2 negatively regulates root growth in *Arabidopsis*.¹⁸ Moreover, VRN2 is stabilized throughout plant tissues in response to environmental signals that inhibit its turnover, such as cold-exposure- and flooding-induced hypoxia, and *vrn2* mutants are sensitive to low oxygen (O₂) stress and root waterlogging.²³ This suggests that VRN2 may act as a “sensor” subunit of the PRC2 that coordinates H3K27me3 deposition in response to positional and environmental cues that influence O₂ availability or otherwise inhibit the N-degron pathway.

An important environmental signal controlling plant growth and development is light, which is transduced by a suite of

<caption>**Figure 1. VRN2 represses rosette growth**</caption>

(A) Schematic showing the four subunits of the PRC2 in *Arabidopsis thaliana*.

(B) Histochemical staining of 3-week-old Col-0 and *prt6-1* rosettes expressing *pVRN2::VRN2-GUS*, and Col-0 expressing *p5xHRPE::GUS*. Scale bar, 1 cm. Inset: magnified SAM region.

(C) Rosettes of 3-week-old lines grown under long-day photoperiodic conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹). Images were digitally extracted for comparison. Scale bar, 1 cm.

(D) Leaf series from individual 3-week-old plants grown as in (C). Cotyledon (ct) and leaf number (1–9) are shown under *vrn2-5*.

(E) Total rosette area of denoted lines. Black and white squares and circles correspond to 4 independent biological repeats. Statistically significant differences were calculated by one-way ANOVA followed by Tukey's test (*p* < 0.05), presented in compact letter display (CLD) format.

(F) Confocal images of (i) SAM, (ii) RM, and (iii) stage VIII LRP in 7-day-old *vrn2-5/VRN2-YFP* seedling. Scale bars: (i) 75 µm, (ii and iii) 150 µm.

receptors that perceive different wavelengths.³⁰ In *Arabidopsis*, the phytochrome A (phyA) and phyB photoreceptors, respectively, transduce far-red (FR) and red light into transcriptional changes through degrading PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors.³¹,³² PIFs are positive regulators of growth-promoting gene expression, though recent reports also indicate some repressive roles.³³ While phy-mediated degradation of PIFs is key for redirecting transcription in response to light and temperature,³⁴,³⁵ other signals are also required to dampen PIF signaling.³⁶ Moreover, there is increasing evidence that phy/PIF signaling is under DNA and chromatin-associated epigenetic control.³⁷ For example, phyB was shown to interact with the VIN3-LIKE 1(VIL1)/VERNALIZATION5(VRN5) PRC2 accessory protein to form a repressive chromatin loop at a PIF4 target gene,³⁸ and the RELATIVE OF EARLY FLOWERING 6 (REF6) H3K27me3 demethylase contributes to PIF-regulated thermomorphogenesis and shade memory responses.³⁹,⁴⁰

Here, we sought to investigate broader roles for VRN2 in the control of plant development. We found that VRN2 is enriched in the hypoxic SAM and emerging leaves of wild-type (WT) plants and that *vrn2* mutants have larger rosettes due to increased cell size. We show that VRN2-PRC2 negatively regulates plant growth by repressing the PIF4-regulated transcriptome specifically in the light. A spatiotemporal analysis of VRN2 protein abundance and localization suggests that VRN2-PRC2 functions early on in leaf organogenesis, facilitating the stable deposition of H3K27me3 in specific growth-promoting PIF4 target genes. We propose that this function is important for setting a stable and conditionally repressed epigenetic state at these loci,
<figure><img src="image_10.png" /><figcaption>Figure 2. Upregulation of growth-promoting genes in vm2 seedlings and rosettes</figcaption></figure>

(A) Schematic and Venn diagram describing the RNA-seq strategy and results in seedlings (top) and rosettes (bottom). A total of 491 vm2-5 upregulated genes were identified across the three comparisons, of which 45 (red triangle) were observed in at least two comparisons. Histochemically stained plants indicate localization of VRN2-GUS in respective mutants for descriptive purposes.

(B) Dot plot showing the GO term enrichment of respective subsets of genes described in (A). y axis indicates the term, x axis indicates the percentage of positive genes in term per query, dot size indicates number of genes enriched in term, and color indicates the -log₁₀ of the adjusted p value.

(legend continued on next page)
allowing for subsequent light-mediated inhibition of PIF signaling
in the cell expansion phase of leaf development. As such, we
identify the VRN2-PRC2 module as an important regulator of
light-responsive gene expression that may also act to transduce
natural hypoxia gradients into the epigenetic control of plant
growth.

RESULTS

VRN2 represses leaf growth

In WT (Col-0) *Arabidopsis* rosettes, VRN2 protein fused to β-glucoronidase (VRN2-GUS) is enriched in the SAM and emerging leaf primordia, with further weak signal in the petioles and parts of the vasculature (Figure 1B), a pattern similar to that seen for the hypoxia reporter *pHRPEx5::GUS*.²⁶ This localization pattern is the result of post-translational degradation of VRN2 via the N-degron pathway, since VRN2-GUS accumulates to a high level in all tissues in *prt6-1* in the absence of changes in mRNA expression (Figure 1B; Labandera et al.¹⁸ and Gibbs et al.²³). We observed that *vm2-5* and *prt6-1 vm2-5* mutant rosettes were larger than Col-0 and *prt6-1*, respectively (Figure 1C). To quantify this, we dissected and measured the area⁴¹ of individual cotyledons and leaves from 3-week-old rosettes, which showed that the increased size is due to consistently larger leaves across their developmental chronology (Figures 1D, 1E, and S1A). Correlating with this, *vm2-5* rosettes had significantly increased aerial fresh weight compared with WT (Figure S1B). We generated a transgenic *vm2-5* line expressing a VRN2-YFP (yellow fluorescent protein) fusion driven by ~2 kb of the native VRN2 promoter (hereafter: *vm2-5/VRN2-YFP*) and confirmed its nuclear localization and characteristic post-translational enrichment in hypoxic SAM, RM, and LRP regions (Figures 1F, S1C, and S1D).¹⁸,²³ This transgene reverted leaf size and fresh weight to WT levels (Figures 1E, S1B, and S1E-S1G), indicating that the VRN2-YFP fusion protein is functional and sufficient to complement the ectopic growth phenotype of *vm2-5*.

Upregulation of growth-promoting genes in *vrn2*
seedlings and rosettes

To identify VRN2 target genes responsible for the larger pheno-
types in vm2 mutants (Figure 1; Labandera et al.¹⁸), we conducted
an RNA sequencing (RNA-seq) analysis on several mutants at
different developmental stages: (1) Col-0, vm2-5, prt6-1, and
prt6-1 vm2-5 at the 11-day-old seedling stage and (2) Col-0 and
vm2-5 at the 3-week-old rosette stage (Figure 2A). Given that

VRN2-PRC2 represses gene expression, we specifically looked for genes significantly upregulated (>1.5-fold, *p* < 0.05) in *vm2-5* lines vs. respective controls. This revealed 139 upregulated genes in *vm2-5* vs. Col-0 seedlings, 104 upregulated genes in *prt6-1* *vm2-5* vs. *prt6-1* seedlings, and 300 upregulated genes in *vm2-5* vs. Col-0 rosettes (Figure 2A; Table S1). By combining these datasets, we identified a core set of 45 “*vm2-5* UP” differentially expressed genes (DEGs). This included 11 SMALL AUXIN UPREGULATED RNA (SAUR) genes, several transcription factors of the HAT/ARABIDOPSIS THALIANA HOMEOBOX PROTEIN (ATHB) family, various auxin signaling genes (SHORT HYPOCOTYL 1 [SHY1], SHY2, and IAA19), and genes linked to brassinosteroid signaling and cell-wall expansion (e.g., PACLOBUTRAZOL RESISTANCE 6 [PRE6] and EXPANSIN 8 [EXPA8], respectively) (Figure 2C). Additionally, many other related genes—including 7 further SAURs, 3 more IAAs, 3 more PREs, and the auxin biosynthesis gene YUC9—were present in individual comparisons (Figure S2B). By contrast, there was almost no overlap between the downregulated gene sets, where VRN2 was the only common downregulated gene in all three comparisons (Figure S2A).

Gene Ontology (GO) term enrichment of the 45 vm2-5 UP genes revealed an overrepresentation of terms linked to “response to auxin,” “response to red or far-red light,” “shade avoidance,” and “growth” (Figures 2A and 2B; Table S2), which prompted us to investigate the impact of VRN2 on growth under specific light conditions. We observed elongated hypocotyl phenotypes in vm2-5 vs. WT seedlings when grown in continuous white, red, and blue light but not in FR light, where the hypocotyl length was slightly reduced in vm2-5 (Figures 2D, 2E, and S2C). Furthermore, we observed an increase in epithelial pavement cell area in vm2-5 compared with WT and vm2-5/VRN2-YFP in continuous white light (Figures 2F–2H and S2D). Together, these RNA-seq and phenotypic data indicate that the larger size of plants lacking VRN2 can be attributed to increased expression of genes driving cell expansion and that VRN2 likely functions downstream of phyB (red light receptor) and potentially cryptochrome (blue light receptor) signaling.

VRN2 is required for spatiotemporal and light-triggered repression of SAURS

SAURs are a large group of plant-specific proteins that promote cell expansion.⁴²,⁴³ Given the large number of *SAURs* present in *vm2-5* UP DEGs (11/45; Figure 2C), we investigated the connection between VRN2 and *SAUR* expression further. We crossed a *pSAUR19::GUS* reporter line⁴⁴ to *vm2-5*, *prt6-1*, and *prt6-1*

(C) Heatmap showing the log₂ fold change in respective comparisons of the 45 vm2-5 UP genes. Asterisks indicate the 7 genes that were upregulated in all 3 comparisons, and gray boxes indicate genes associated with the GO terms "response to auxin" and "response to red or far-red light" as described in (B).

(D) Hypocotyl lengths of 7-day-old seedlings grown under white light (16 h L:8 h D; 100 µmol m⁻²·s⁻¹). Data comprise 3 biological replicates. Boxplots indicate minimum to maximum values, the 25ᵗʰ to 75ᵗʰ percentile with lines indicating the median of the data.

(E) Hypocotyl lengths of 7-day-old seedlings grown under continuous red (1 µmol m⁻²·s⁻¹), far red (1 µmol m⁻²·s⁻¹), or blue (1 µmol m⁻²·s⁻¹) light. Data comprise 2 biological replicates. Boxplots indicate minimum to maximum values, the 25ᵗʰ to 75ᵗʰ percentile with lines indicating the median of the data. Statistically significant differences in (D) and (E) were calculated by one-way ANOVA followed by Tukey's test (*p* < 0.05), presented in CLD format.

(F) Representative images of epidermal cells from Col-0 and vm2-5. Cells are colored according to area; red = 85–300 µm², light green = 300–1,600 µm², mid green = 1,600–3,200 µm², dark green >3,200 µm². Gray cells are guard cells/stomata.

(G) Average area of epidermal cells from leaf 3 of 2-week-old Col-0 and vm2-5 plants. Statistically significant differences were calculated by Student's t test. Boxplots indicate minimum to maximum values, the 25th to 75th percentile with lines indicating the median of the data.

(H) Number of epidermal pavement cells per 0.24 mm² from leaf 3 of 2-week-old Col-0 and vm2-5 plants. Statistically significant differences were calculated using a Student's t test. Data in (G) and (H) are derived from 2 images from 6 independent plants for each genotype. Boxplots indicate minimum to maximum values, the 25th to 75th percentile with lines indicating the median of the data.
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vm2-5 and monitored promoter activity by anti-GUS western blot-
ting (Figures 3A and S3A). When comparing GUS signal in skoto-
morphogenic (i.e., dark-grown) seedlings, no difference in pro-
moter activity was observed. However, in photomorphogenic
(i.e., light-grown) seedlings and rosette leaves harvested in the
light, GUS levels were much higher in vm2-5 and prt6-1 vm2-5
compared with Col-0 and prt6-1. SAUR19 is highly expressed in
dark-grown seedlings and rapidly downregulated during de-eti-
olation.⁴⁵ Directly comparing pSAUR19::GUS activity in etiolated
vs. photomorphogenic seedlings on the same blot confirmed
these dynamics in Col-0 and showed that this repression is
compromised in vm2-5 (Figure S3B). Furthermore, an RT-qPCR
analysis of SAUR19 and SAUR24 expression over a diel time
course revealed similar mRNA levels in Col-0 and vm2-5 at zeit-
geber time (ZT) 22 and ZT 0 (i.e., in the dark) but much higher
expression levels in light at ZT 4 and ZT 8 in vm2-5 (Figures 3B
and S3C). In line with this, we found that the larger rosette of
vm2-5 is also evident in plants grown under a 24-h white light
regime, suggesting that the growth phenotype is light dependent
rather than dark dependent (Figure S3D).

We also examined the spatial pattern of ectopic pSAUR19::
GUS expression in 7- and 10-day-old seedlings and 4-week-
old rosettes (Figures 3C and S3E). In Col-0, pSAUR19::GUS
expression is largely restricted to hypocotyl, petiole, and vascu-
lature regions, but in vm2-5, it is expressed throughout the whole
plant, including the expansion zone of leaves. To investigate the
temporal dynamics of this misexpression further, we stained
seedlings at sequential days after germination and found that
spatial differences in GUS expression were only observed from
day 5, indicating that ectopic pSAUR19 activity in vm2-5 is initi-
ated during seedling development (Figure S3F). Thus, our data

<caption>Figure 3. VRN2 is required for spatiotemporal and light-triggered repression of SAURs</caption>

(A) Anti-GUS western blot of pSAUR19::GUS in dark (top) and light (bottom; 16 h L:8 h D; 100 µmol m⁻²·s⁻¹) grown seedlings. Coomassie brilliant blue (CBB) staining shows equal loading.

(B) RT-qPCR quantifying SAUR19 and SAUR24 expression in Col-0 and vm2-5 at the indicated zeitgeber (ZT) time points. Data are presented relative to Col-0 at ZT 0. Each point represents the mean of 2 technical repeats from one biological replicate. Error bars = SEM. A similar trend was observed across 3 independent biological replicates (Figure S3).

(C) Histochemical staining of representative Col-0 and vm2-5 plants expressing pSAUR19::GUS at 7 days (top) and 4 weeks (bottom) of growth. Scale bars, 1 mm (top) and 1 cm (bottom), respectively.

reveal that VRN2 is required for light-triggered and spatiotemporal repression of *SAURs*.

The VRN2-PRC2 module is epistatic
to PIF4

The findings presented in Figures 2 and 3
prompted us to investigate VRN2 func-

tion within the context of phytochrome/PIF signaling. We
compared the vm2-5 UP DEGs with several published PIF chro-
matin immunoprecipitation (ChIP) datasets and found that 71%
(32/45, *p* < 0.001), 53% (24/45, *p* < 0.001), and 58% (26/45,
*p* < 0.001) are direct binding targets of PIF4, PIF5, and PIF7,
respectively (Figure S4A; Table S1).⁴⁶⁻⁴⁸ Among the 32 reported
PIF4 targets, 25 also had significantly reduced mRNA abun-
dance in the *pif4* mutant vs. WT⁴⁹, including all 6 members of
the *SAUR19-24* subfamily, *HAT2*, *HAT4*, and several *IAAs* (Fig-
ure 4A; Table S1). We chose to further explore the genetic and
functional interactions between VRN2 and PIF4 by generating
and phenotypically characterizing a *pif4-2 vm2-5* double mutant.
The large leaf, elongated hypocotyl, and elevated *SAUR19*
expression phenotypes of *vm2-5* were abolished in *pif4-2*
*vm2-5*, instead resembling the *pif4-2* single mutant (Figures
4B-4E). Furthermore, an RNA-seq analysis revealed that a ma-
jority of the *vm2-5* UP DEGs (in particular the 32 that are direct
PIF4-binding targets; Figure 4A) are downregulated in *pif4-2*
*vm2-5* vs. *vm2-5* (Figure 4F; Table S1).

PIF4 is also a major regulator of thermomorphogenesis, where
it promotes hypocotyl elongation in response to warm tempera-
ture via induction of auxin and brassinosteroid synthesis that
ultimately converge on *SAUR* expression.⁴⁶,⁵⁰,⁵¹ Hypocotyl
extension in photomorphogenic seedlings grown at 28°C was
significantly induced in both Col-0 and *vm2-5* relative to 22°C,
but the response was severely dampened in *pif4-2* and *pif4-2*
*vm2-5* double mutants (Figure 4D). This was corroborated by
*SAUR19* mRNA levels, which were similarly induced after 6 h
transfer to 28°C in Col-0 and *vm2-5* (Figure 4E).

Since PIF4 is necessary for the ectopic growth phenotypes
observed in vm2-5 mutants, we next tested if PIF4 itself is a
<figure><img src="image_12.png" /><figcaption>**Figure 4.** The VRN2-PRC2 module is epistatic to PIF4</figcaption></figure>

(A) Venn diagram showing the overlap between the 45 *vrn2-5* UP genes (Figure 2A), *pif4* down genes,⁴⁹ and genes described as HC PIF4-binding targets.⁴⁶

(B) Representative images of 3-week-old rosettes grown under long-day conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹). Scale bar, 1 cm.

(C) Quantification of total rosette area after leaf dissection of 3-week-old plants grown as in (B). Statistically significant differences calculated by one-way ANOVA followed by Tukey's test (*p* < 0.05) and are presented in CLD format. Boxplots indicate minimum to maximum values, the 25th to 75th percentile with lines indicating the median of the data.

(D) Hypocotyl lengths of seedlings grown under long-day photoperiodic conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹) at 22°C or 28°C. Each point represents a single seedling. Data shown are pooled from 3 independent biological replicates. Boxplots indicate minimum to maximum values, the 25th to 75th percentile with lines indicating the median of the data.

(E) RT-qPCR quantifying *SAUR19* expression in 7-day-old seedlings grown under long-day photoperiodic conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹) at 22°C or following transfer to 28°C for 6 h. Each bar represents the average of data from 2 biological replicates; error bars = SEM. Data presented relative to Col-0 at 22°C. Statistically significant differences in (D) and (E) were calculated by one-way ANOVA followed by Tukey's test (*p* < 0.05) and are presented in CLD format.

(F) Heatmap showing log₂ fold changes of the 45 *vrn2-5* UP genes and *YUC9* in respective RNA-seq dataset comparisons. Asterisks indicate the 32 genes that were shown to be direct PIF4 binding targets (see Figure 4A).

(G) RT-qPCR quantifying *PIF4* expression in Col-0 and *vrn2-5* at the indicated ZT time points. Data are presented relative to Col-0 at ZT 0. Each bar represents the average of data from 3 biological replicates; error bars = SEM.
target of VRN2. PIF4 mRNA abundance was unaltered in *vrn2-5* relative to Col-0 (Figure 4G), and steady-state levels of PIF4 protein were also equivalent in both genotypes (Figure 4H), ruling out the hypothesis that VRN2 directly regulates PIF4 expression. Furthermore, we could not detect interaction between VRN2-YFP and PIF4 in dark or red light conditions by co-immunoprecipitation, despite effectively capturing interaction with the PRC2 component FIE (Figure 4I). We also did not detect an interaction between VRN2-YFP and phyB, and, unlike phyB-CFP, VRN2-YFP did not form nuclear photobodies *in vivo* in response to red light exposure (Figure S4D). We therefore concluded that VRN2-PRC2 does not associate with the same multi-protein complex as PIF4 or phyB to regulate the expression of growth-promoting genes and instead hypothesized that it co-targets some or all of PIF4's target genes.

It was recently shown that the PRC2 accessory protein VIL1/VRN5 interacts and cooperates with phyB to regulate PIF-dependent expression dynamics at *HAT4* by promoting the formation of a chromatin loop.³⁸ Chromatin loop formation was abolished in both *vil1* and *vrn2* mutants, corroborating the fact that VIL1 is a specific interactor of VRN2. In our study, *HAT4* was identified as one of only 7 genes that was ubiquitously upregulated across all three RNA-seq comparisons (Figure 2C). To understand the relative contributions of VRN2 and VIL1 to light-triggered regulation of *SAUR* expression, we crossed *vrn2-5/pSAUR19::GUS* to a *vil1-1* mutant and monitored promoter activity. Here, we observed similar de-repression of the *pSAUR19::GUS* in the light, which was not amplified in the *vrn2-5 vil1-1* double mutant, indicating an epistatic rather than additive effect and implicating the two proteins in the same pathway for controlling *SAUR19* expression (Figures 4J and S4C). Interestingly, however, when we compared our *vrn2-5* UP DEGs with two previously published *vil1* RNA-seq datasets sampled at different developmental stages (1-day-old germinated seeds⁵² and 14-day-old light-grown seedlings harvested at ZT 6⁵³), we saw very little overlap (only 3 genes, a complete absence of *SAURs* and *HAT4*, etc.) as well as an absence of GO terms linked to “red/far red” or “shade avoidance” (Figure S4B). This suggests that control of gene expression by the VRN2-VIL1 module is dynamic, likely age and developmental stage regulated, and that there is possible redundancy of function with other factors.

## VRN2-PRC2 stably methylates key hub genes in the PIF4 signaling cascade

Our data indicate that VRN2 represses the PIF4-regulated transcriptome. However, while the RNA-seq analysis (Figure 2) provides insight into the broader gene expression changes driving the growth phenotypes in *vrn2-5*, it cannot differentiate between direct and indirect (i.e., downstream) targets of VRN2. To resolve

this, we performed protein chromatin sequencing (ChIP-seq) with VRN2-YFP-expressing plants to identify VRN2-bound loci, as well as H3K27me3 ChIP-seq to identify genes that are hypomethylated in *vrn2-5* vs. Col-0. After peak calling, 1,474 regions of the mappable *Arabidopsis* genome were found to interact with VRN2-YFP, while 703 regions exhibited reduced histone methylation in *vrn2-5*. We observed enrichment of the previously reported VRN2 targets *WIND1*, *WIND2*, and *WIND3* in our VRN2-YFP ChIP.²¹ and a depletion of H3K27me3 in *vrn2-5* at several known PRC2 targets (*ULT1*, *LEC1*, *NIT2*, and *AGL15*) (Figures S5A and S5B). Furthermore, over 80% of identified loci in our WT H3K27me3 dataset are found in other published datasets⁵⁴⁻⁵⁶ (Figure S5C).

Comparison of the VRN2-YFP peaks and the H3K27me3 alignment data revealed a broad reduction in H3K27me3 signal for these specific regions in *vrn2-5* relative to Col-0 (Figure 5A), despite total levels of H3K27me3 being equivalent between genotypes that either lack (i.e., *vrn2-5*) or over accumulate (i.e., *prt6-1*) VRN2 (Figure 5B). The 1,474 VRN2-YFP peaks and 703 regions with reduced H3K27me3 levels corresponded to 1,265 and 699 genes, respectively (Table S3). Of these, 118 genes were common to the two datasets, revealing that approximately 10% of VRN2-bound genes are also methylated by VRN2-PRC2 (Figure 5C; Table S3). These 118 genes were designated as “high-confidence (HC) VRN2 targets” ($p \le 0.001$; Figure S5E). Among them were the auxin biosynthesis genes *YUC8* and *YUC9*, the auxin efflux transporter *PIN-FORMED 1* (*PIN1*), and the abscisic acid (ABA) receptors *PYROBACTIN RESISTANCE 1 LIKE 2* (*PYL2*) and *PYL3* (Figure 5D). Interestingly, *YUC8* is a key intermediate in the PIF4 signaling cascade that drives *SAUR* expression,⁵¹ while *YUC9* acts synergistically with *YUC8* during the shade avoidance response⁵⁷ and is also upregulated in *vrn2-5* vs. Col-0 (Figure S2B). Annotated peaks identified in only one of the ChIP datasets were designated “VRN2-YFP” or “*vrn2*-hypomethylated” targets, respectively (Table S3). We posited that many of these could also represent bona fide VRN2-YFP targets. Among the latter was *HAT4* (Figure 5E), which was hypomethylated at defined promoter response elements (REs) and upregulated in the *vrn2-5* transcriptome (Figure 2B). Interestingly, these REs were previously reported as sites for phyB, PIF4, and VIL1 association,³⁴,³⁸ highlighting them as critical sites for the convergence of multiple transcriptional, light sensing, and chromatin remodeling factors.

We designed primers overlapping the identified peaks and using ChIP-PCR confirmed *YUC8*, *YUC9*, *PIN1*, and *HAT4 RE1* and *RE2* as direct VRN2-YFP-binding targets (Figure 5F). Given that VRN2 specifically represses growth in the light, we also assessed whether H3K27me3 deposition at these genes is dynamically regulated during the photoperiod. While our analysis confirmed that all four genes possess reduced levels of

(H) Western blot of PIF4 protein levels at ZT 22, ZT 0, and ZT 4 in 7-day-old seedlings of Col-0 and *vrn2-5* grown in long day photoperiodic conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹). Ponceau S staining of RbcS (small subunit of rubisco) shows equal loading.

(I) Western blot of VRN2-YFP, FIE, PIF4, and phyB following anti-GFP immunoprecipitation of VRN2-YFP from 14-day-old seedlings grown under long day photoperiodic conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹) and transferred at ZT 0 to red light (RL; 10 µmol m⁻²·s⁻¹) or dark for 4 h. CBB staining shows equal loading and bead washing.

(J) Western blot showing *pSAUR19::GUS* expression in 4-day-old seedlings in the dark or in the light. *vrn2-5 vil1-1* without *pSAUR19::GUS* was used as a negative control. CBB staining shows equal loading. Numbers beneath the light blot are band intensities normalized to CBB relative to Col-0. Seedling images are for descriptive purposes only.
A

<figure><img src="image_13.png" /></figure>

B

<figure><img src="image_14.png" /></figure>

C

<figure><img src="image_15.png" /></figure>

(p=<0.001)

High confidence VRN2 targets

D

<figure><img src="image_16.png" /></figure>

YUC8

YUC9

<figure><img src="image_17.png" /></figure>

PIN1

PYL2

PYL3

E

<figure><img src="image_18.png" /></figure>

HAT4

F

<figure><img src="image_19.png" /></figure>

H

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HAT4

G

<figure><img src="image_21.png" /></figure>

(legend on next page)
H3K27me3 in *vrn2-5* relative to Col-0, we did not observe any statistically significant variation in H3K27me3 levels between ZT 0 and ZT 8 in Col-0 (Figure 5G). This finding corroborates other studies, which suggest that the H3K27me3 repressive mark is often stably present in genes with environment-responsive functions.⁷⁻¹⁰,³⁸ Despite the stable presence of H2K27me3, several of these genes showed elevated expression at ZT 8 in *vrn2-5* compared with Col-0 (Figure 5H), similarly to *SAUR19* and *SAUR24* (Figure 3B).

Although they are prevalent *vrn2-5* UP DEGs, we observed neither H3K27me3 nor VRN2-YFP enrichment in *SAUR19-24* and *SAUR60-65* (Figure S5D; Table S1), indicating that their ectopic upregulation in *vrn2-5* mutants is a downstream consequence of loss of VRN2 function. Indeed, while the overlap between *vrn2-5* hypomethylated genes and total *vrn2-5* UP DEGs was significant ($p \le 0.001$), the overlap between VRN2-YFP targets and *vrn2* UP genes was not ($p = 0.38$) (Figure S5E). This is likely due to limitations in resolution with respect to protein vs. histone ChIP sequencing, giving rise to a lower overlap (3.8%, Figure S5E). Nonetheless, this low number is similar to that seen when looking at PIF4 targets vs. downregulated genes in *pif4* vs. Col-0 (4.7%; Figure 4A) and suggests that many DEGs are downstream of VRN2-PRC2 function.

Overall, our combined ChIP analyses revealed a range of HC VRN2 targets linked to diverse cellular processes, including flowering time, meristem identity and maintenance, and auxin signaling, and suggest that VRN2's role in repressing PIF signaling is a consequence of it targeting a small set of “hub” genes (*HAT4* and *YUC8/9*) that then trigger broader transcriptional changes to repress growth in the light.

## Spatiotemporal and light-responsive analysis of VRN2 stability

VRN2-mediated repression of PIF4 target genes is light dependent (Figures 3 and 4), which prompted us to explore whether this could be due to changes in VRN2 mRNA expression and/or VRN2-YFP protein abundance across a diel time course (Figures 6A and 6B). We saw no obvious differences in transcript

levels or protein stability, suggesting that VRN2 itself is not regulated by light or time of day. We therefore considered that there might be a developmentally encoded mechanism at play, particularly because (1) target gene H3K27me3 levels appear to be stable (Figure 5G), (2) the larger rosette size of *vrn2-5* mutants corresponds to an increase in cell size, a process that occurs later in leaf development (Figures 2F–2H), and (3) there is a spatial disconnect between VRN2 accumulation in the SAM and emerging leaves and where *SAUR19* is upregulated in *vrn2-5*—namely, the cell expansion zone of older leaves (Figures 1B, 3C, and 6C). To explore this spatiotemporal disconnect further, we conducted an RT-qPCR analysis of direct VRN2-PRC2 target genes *HAT4*, *YUC8*, and *YUC9* (Figure 5) in dissected leaves of 3-week-old rosettes, which confirmed that they also show elevated expression in the older leaves of *vrn2-5* compared with WT (Figure 6D).

Rosettes displayed a very specific pattern of VRN2-GUS localization across leaf development (Figures 6E, 6F, and S6). Signal was strong throughout the meristem, leaf primordia, and youngest leaf 9 but receded in a basal direction (i.e., toward the petiole) as leaves aged. This was restricted to the petiole in leaf 6 and was completely absent in older leaves where VRN2-target genes are ectopically expressed (Figures 6D and S6). This wave of VRN2-GUS depletion appears to correspond to the cell division arrest front, where cells exit mitosis and subsequently contribute to leaf growth through expansion alone.⁵⁸,⁵⁹ Moreover, it is post-translationally regulated, since VRN2-GUS is strongly stabilized throughout all leaves in the *prt6-1* mutant (Figure 1B).¹⁸,²³ Thus, VRN2 abundance is tightly regulated across leaf development, which perhaps points to a gradient of O₂ availability as organs emerge from the hypoxic shoot apical meristem,²⁶ divide, and then expand. Indeed, hypoxia-responsive genes have previously been shown to be active in the SAM, leaf primordia, and young leaves but not in older leaves.⁶⁰ As such, we propose a developmentally and spatially encoded mechanism whereby VRN2-PRC2 stably deposits H3K27me3 in target genes early in leaf organogenesis, establishing a conditionally repressed epigenetic state that keeps them poised for

### Figure 5. VRN2-PRC2 stably methylates key hub genes in the PIF4 signaling cascade

(A) Coverage plots (top) and associated heatmaps (bottom) showing the distribution of H3K27me3 in Col-0 and *vrn2-5* across regions targeted by VRN2-YFP. Data show coverage across the combination of 3 biological replicates, which were normalized by reads per genome coverage to account for the differences in the sequencing depth of the protein vs. histone ChIP assays.

(B) Western blot showing abundance of H3K27me3 in denoted lines. αH3 was used to quantify equal loading.

(C) Overlap of genes (HC VRN2 targets; $p \le 0.001$ see Figure S5E) depleted for H3K27me3 in *vrn2-5* (*vrn2-5* hypomethylated; blue) and targeted by VRN2-YFP (yellow).

(D) IGV plots showing alignment of HC VRN2 targets. Black and gray tracks represent Col-0 and *vrn2-5* H3K27me3 coverage, respectively, and “overlay” represents these same tracks mapped over one another. Black dotted boxes indicate regions identified as significantly depleted for H3K27me3 in *vrn2-5*. Yellow track represents VRN2-YFP coverage across the same loci, and orange boxes indicate regions significantly enriched by VRN2-YFP. Data show coverage across the combination of 3 biological replicates, normalized by reads per genome coverage. The position/direction of the genomic sequence for each gene is shown in blue beneath the tracks.

(E) As in (D) for the *vrn2-5* hypomethylated gene *HAT4*.

(F) ChIP-PCR of VRN2-YFP expressing plants showing enrichment for genomic regions using primers denoted as red lines in d and e, calculated by normalizing % input values to the enrichment of 5SrDNA. Bars represent the average value from 2 biological replicates. Error bars = SEM; statistically significant differences in enrichment between Col-0 and VRN2-YFP were calculated by Student's t test ($p < 0.05$ *, $p < 0.01$ **, $p < 0.0001$ ****).

(H) RT-qPCR quantifying gene expression in Col-0 and *vrn2-5* at the indicated ZT time points. Data are presented relative to Col-0 at ZT 0. Bars represent the mean of 2 biological replicates; error bars = SEM.

(G) ChIP-PCR to evaluate H3K27me3 levels in Col-0 and *vrn2-5* at defined genomic regions using primers denoted as red lines in (D) and (E). H3K27me3 levels at ZT 0 and ZT 8 were calculated by normalizing Ct values according to histone H3 enrichment and are presented as relative enrichment compared with the 5SrDNA negative control. Data represent the average value from at least 2 biological replicates. Error bars = SEM. Statistically significant differences in (G) and (H) were calculated by two-way ANOVA followed by Tukey's test ($p < 0.05$) and are presented in CLD format.
A

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B

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E

pVRN2::VRN2-GUS

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F

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C

Col-0/pVRN2::VRN2-GUS

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vrn2-5/pSAUR19::GUS

D

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G

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light-triggered repression by the phyB-VIL1 module³⁸,⁵³ once
cells enter the expansion phase (Figure 6G).

DISCUSSION

We have shown that VRN2-PRC2 epigenetically suppresses growth in *Arabidopsis* through light-specific attenuation of PIF signaling, actioned through histone methylation of specific hub genes in the PIF4 transcriptional network. RNA-seq revealed that many *vrn2-5* upregulated genes promote cell-expansion-mediated growth (Figures 2C and S2) and have been previously reported as PIF4 signaling targets, including *HAT2*, *HAT4*, *YUC9*, and the *SAUR19-24* family.⁵⁰,⁵¹,⁶¹ Using a *pSAUR19::GUS* reporter, we demonstrated that this gene is both upregulated and expressed ectopically in *vrn2-5* (Figures 3A, 3B, S3A, and S3B). Furthermore, we found that this regulation was light dependent and only observable from 5 days after germination (Figure S3E), suggesting that the regulatory role of VRN2-PRC2 is initiated during seedling establishment and not during embryogenesis. The enhanced growth phenotype in *vrn2-5* was completely abolished in the *pif4-2 vm2-5* mutant, as was the upregulation of many *vrn2-5* UP DEGs (Figures 4D and 4E). Our data suggest that the epigenetic regulation of only a small number of genes by VRN2-PRC2 has a significant impact on the PIF-regulated transcriptional landscape. Of particular interest are *HAT4*, *YUC8*, and *YUC9*, which have previously been shown to function downstream of PIF4 and upstream of *SAURs*.⁵⁰,⁵¹,⁵⁷

It was previously reported that phyB associates with the PRC2 accessory protein VIL1 to trigger the formation of a repressive chromatin loop in *HAT4* and that this is negatively regulated through COP1-mediated degradation of VIL1 in the dark.³⁸,⁶² VIL1 is a specific interactor of VRN2,⁶³,⁶⁴ and in accordance with this, chromatin loop formation was abolished in a *vrn2* mutant.³⁸ We observed increased *SAUR19* promoter activity in *vil1-1* and showed that this effect was not additive in *vrn2-5* *vil1-1*, suggesting that these proteins do indeed function in the same pathway to repress growth-promoting gene expression. This is further supported by our observation that VRN2-YFP physically associates with two regulatory elements critical for loop formation in the *HAT4* promoter, which also exhibit reduced H3K27me3 deposition in *vrn2-5* relative to WT (Figures 5E and

5F). Whether other VRN2 targets are also regulated by phyB/
VIL1-dependent chromatin loop formation will require further
investigation.

Our data suggest that VRN2 negatively regulates leaf growth through restricting cell expansion (Figures 1C–1E and 2F–2H). Interestingly, another N-degron pathway target, the transcription factor ZPR2, was previously shown to coordinate leaf emergence from the hypoxic SAM in *Arabidopsis*,²⁶ suggesting that multiple O₂-regulated substrates of this proteolytic pathway influence leaf development. The spatial disconnect between VRN2 localization in the SAM and the ectopic expression of *pSAUR19::GUS* and other VRN2-PRC2 target gene mRNAs in the mature leaves of *vrn2-5* alludes to a meristem-associated or early developmental function for VRN2 that either persists through leaf maturation or is transduced across the leaf tissue. One possible mechanism for the latter is that targeting of *YUC8*, *YUC9*, and *PIN1* by VRN2-PRC2 influences the synthesis and/or transport of bio-active auxins, which potentiate PIF4 signaling (Figure 5D).⁵⁰,⁵¹ A closer examination of auxin production, abundance, signaling, and transport in the *vrn2-5* mutant is therefore warranted.

Our data indicate that VRN2 is stable in the dividing regions of
developing leaves but is destabilized via the N-degron pathway
in the expansion region of mature leaves (Figures 1B, 6E, 6F, and
S6). This suggests that there may be a gradient of O₂ availability
across the developing leaf, or that other signals impeding VRN2
turnover exist in the meristem and emerging leaves. For
example, NO levels also feed into the control of N-degron sub-
strates, including VRN2.²³,⁶⁵⁻⁶⁷ Although VRN2-signal appears
to be absent in mature leaves, it is possible that a small pool of
stable VRN2 remains in these tissues as part of the PRC2, for
example, due to shielding of its N terminus. Under this mecha-
nism, VRN2 might be maintained at PRC2 target genes to
directly facilitate the binding of secondary regulators while also
maintaining H3K27me3 levels. Interestingly, it was recently re-
ported that emerging leaves are subjected to a time-of-day-
dependent hypoxic cycle linked to starch availability during the
night.⁶⁰ which might also lead to transient stabilization of VRN2
in areas of the plant that normally appear normoxic. It is also
plausible that EMF2-PRC2, which has some functional overlap
with VRN2 in certain processes,¹⁵,¹⁶ could help to maintain this
mark in older leaves after its initial deposition by VRN2-PRC2.

Figure 6. Spatiotemporal and light-responsive analysis of VRN2 stability

(A) RT-qPCR quantifying VRN2 expression in Col-0 grown under long day photoperiodic conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹) at the indicated ZT timepoints. Data are presented relative to Col-0 at ZT 0. Each point represents the average of data from 3 biological replicates. Error bars = SEM. One-way ANOVA revealed no significant differences.

(B) Western blot showing VRN2-YFP levels in 7-day-old seedlings at the indicated ZT time points. CBB staining was used to quantify equal loading.

(C) Histochemical staining of rosettes expressing Col-0/*pVRN2::VRN2-GUS* and *vrn2-5/pSAUR19::GUS*, illustrating the spatial disconnect between VRN2 localization in Col-0 and ectopic *pSAUR19* promoter activity in *vrn2-5*.

(D) RT-qPCR of *HAT4*, *YUC8*, and *YUC9* in the dissected SAM and leaves 1–8 of 3-week-old *Arabidopsis* rosettes grown under long day conditions (16 h L:8 h D; 100 µmol m⁻²·s⁻¹). Statistically significant differences in expression between Col-0 and *vrn2-5* were calculated by Student's t test (*p* < 0.05 *, *p* < 0.01 **, *p* < 0.001 ***).

(E and F) Representative histochemical staining from a dissected 4-week-old Col-0/*pVRN2::VRN2-GUS* rosette. The rosette was dissected to expose the meristem, leaf primordia (leaves 10 and 11), and leaves 6–9, which are arranged in developmental order from youngest (left) to oldest (right). Scale bar in (D), 500 µm; in (E), 5 mm.

(F) Hypothetical model of VRN2-mediated repression of growth in *Arabidopsis*. In the dividing cells of the hypoxic shoot meristem and emerging leaves, stable VRN2-PRC2 deposits H3K27me3 at core PIF4 target genes (e.g., *HAT4/YUC9*). (ii) In the expanding cells of normoxic leaf tissues, where VRN2 is degraded, this H3K27me3 mark is stable and facilitates the conditional repression of these genes by the red-light-activated phyB-VIL1 module. This restricts the expression of *SAURs* in the cell expansion zone in response to light. The green-to-yellow gradient follows the transition of division-mediated to expansion-mediated growth across developmental time as leaves emerge and grow.
Nonetheless, the question remains as to how proteolytic control of VRN2 across leaf development contributes to its light-dependent regulation of growth. As highlighted in our hypothetical model (Figure 6G), stable VRN2 in the hypoxic SAM, emerging primordia, and young leaves would ensure that H3K27me3 is deposited at target loci. Since chromatin marks need to be replenished following mitosis, a continual pool of VRN2 in this region would lead to the maintenance of H3K27me3 at these loci as cells continually divide. As cells enter the expansion zone, H3K27me3 replenishment is no longer needed as cell division stops. This histone mark might then act as a scaffold/binding target for the phyB/VIL1 module or other light-responsive regulators to introduce dynamic regulation in normoxic tissues. Indeed, it was previously shown that H3K27me3 levels in the *HAT4* REs remain constant across the dark-light cycle<sup>38,62</sup>—a finding we corroborated (Figure 5G)—indicating that H3K27me3 mark is stably imprinted prior to dynamic VIL1-mediated chromatin loop formation. This spatiotemporally separated model is further reinforced by our observation that ectopic VRN2 accumulation does not lead to gain-of-function phenotypes—e.g., *prt6-1* mutant rosettes and hypocotyls are not significantly smaller than Col-0 plants (Figures 1E and 2D), and *pSAUR19::GUS* levels are not reduced in *prt6-1* relative to Col-0 (Figure 3A).

H3K27me3 levels in VRN2-PRC2 targets remained relatively stable across the dark-light cycle (Figure 5G). This might seem counterintuitive since many of these genes are actively transcribed in the dark and switched off in the light in Col-0. However, there is increasing evidence that the classic “on/off” antagonism of different histone marks is too simplified, and that, paradoxically, histone modifications linked to transcriptional activation and repression can co-localize in some genes.<sup>6,12,68</sup> For example, approximately one third of all H3K27me3-marked genes in *Arabidopsis* also carry the positive H3K4me3 mark, including several of the *vrn2-5* UP genes identified in this study (e.g., *HAT4*, *FLC*, *PIL1*, *PRE1*, *HFR1*, and several *IAAs*; Figures 2 and S2).<sup>8,11,12,69</sup> This so-called chromatin bivalency is found at hundreds of loci linked to various stress responses in plants and is proposed to keep genes poised for signal-responsive expression or repression.<sup>7,9,10,68,70</sup> This is true of *VIN3*, for example, which is induced in response to cold without removal of H3K27me3.<sup>71,72</sup> Another study examining dehydration response genes found that H3K27me3 levels remained relatively stable independent of their expression status.<sup>70</sup> Activation of these genes was instead associated with increased H3K4me3 levels, without any H3K27me3 depletion. Interestingly, a recent study showed that *HAT4* induction in the dark coincides with increased H3K4me3 deposition.<sup>69</sup> It has been posited that stable H3K27me3 levels are important for the correct regulation of certain environment-responsive genes, setting the dynamic range of their transcription by limiting their expression under some conditions without preventing their activation by others. This would certainly be beneficial for hub genes such as *HAT4* that act as convergence points for light and temperature signals, allowing for rapid transcriptional changes in response to the prevailing environmental conditions.

In summary, we have identified a developmentally encoded role for the polycomb protein VRN2 in restricting the expression of growth-promoting genes in the light. This additional layer of regulation acts to ensure effective suppression of these genes

in response to the environment and reinforces the notion that phytochrome-triggered degradation of PIFs alone is not sufficient to effectively switch off PIF signaling. Our work provides insights into the role of chromatin modifications in regulating environment-responsive gene expression in plants and identifies a mechanism that potentially connects developmentally encoded hypoxia gradients to the epigenetic control of growth.

## Limitations of the study

The VRN2-GUS and VRN2-YFP reporter lines used here reveal clear post-translational enrichment of VRN2 in the SAM, leaf primordia, and the basal region of young leaves, which is mediated by the PRT6/N-degron pathway of proteolysis. This suggests that there might be a gradient of hypoxia within the developing leaf. Direct measurement of intracellular O₂ levels—for example, using an O₂ sensing Clark electrode or high-resolution genetically encoded hypoxia reporters—will help to directly resolve this. Moreover, the use of cell-type- or tissue-specific complementation lines to further test the hypothesis that VRN2-PRC2 function in the hypoxic dividing cells of the shoot is sufficient to restore epigenetic control of PIF signaling in *vrn2* will help to further support the spatial model we propose.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Daniel James Gibbs (d.gibbs@bham.ac.uk).

### Materials availability

All reagents developed in this study can be obtained from the lead contact upon reasonable request and with a completed material transfer agreement.

### Data and code availability

* Raw mRNA sequencing, protein (VRN2-YFP) ChIP, and histone (H3K27me3) ChIP data have been deposited at the Gene Expression Omnibus (GEO: GSE266049).

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank William Gray (University of Minnesota, USA) for the *pSAUR19::GUS* line, Daan Weits (Utrecht University, the Netherlands) for the *pHRPE5X::GUS* line, Hannes van Haeren (VIB, Ghent, Belgium) for advice with rosette quantification, Jie Song (Imperial College London, UK) and Anna Schulten and Robert Maple (John Innes Centre, UK) for advice on ChIP-seq, and Rashmi Sasidharan (Utrecht University, the Netherlands) for early discussions on red/far-red light signaling. D.J.G. was supported by a European Research Council (ERC) Starting grant (GasPlant-715441) and Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/V008587/1. E.K. was supported by BB/X009653/1. S.H. was supported by the Netherlands Organization for Scientific Research (019.201EN.004) and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy (CIBSS-EXC-2189-Project ID 390939984). M.A.S. was supported by a BBSRC-funded MIBTP-DTP studentship.

## AUTHOR CONTRIBUTIONS

D.J.G. and R.O. designed the study and its experiments. R.O., A.-M.L., A.J.R., A.K., T.X., O.A., M.A.S., C.D.M., S.H., E.K., and D.J.G. conducted experiments. R.O., A.-M.L., S.H., E.K., and D.J.G. analyzed data. S.H., E.K., and
D.J.G. secured funding for the work. D.J.G. and R.O. wrote the paper with input from all authors.

### DECLARATION OF INTERESTS

The authors declare no competing interests.

### STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* Arabidopsis growth and transgenic lines

* **METHOD DETAILS**

* Phenotyping assays

* Histochemical staining and leaf pavement cell size

* gDNA extraction for genotyping

* Western blotting

* Co-immunoprecipitation

* qRT-PCR

* RNA-sequencing and analysis

* ChIP sequencing

* ChIP sequencing Analysis

* ChIP qPCR

* Confocal microscopy

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

### SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.devcel.2025.03.001.

Received: April 29, 2024

Revised: October 29, 2024

Accepted: March 3, 2025

Published: March 26, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Anti-GFP</td><td>Sigma-Aldrich</td><td>Cat#:SAB4301138; RRID: AB_2750576</td></tr><tr><td>Anti-GUS</td><td>Sigma-Aldrich</td><td>Cat#:G5420; RRID: AB_477020</td></tr><tr><td>Anti-PIF4</td><td>Agrisera</td><td>Cat#:AS16 3955</td></tr><tr><td>Anti-FIE</td><td>Agrisera</td><td>Cat#:AS12 2616; RRID: AB_3676233</td></tr><tr><td>Anti-phyB</td><td>Prof. Akira Nagatani</td><td>Yamaguchi et al.<sup>76</sup></td></tr><tr><td>Anti-H3</td><td>Abcam</td><td>Cat#:ab1791; RRID: AB_302613</td></tr><tr><td>Anti-H3K27me3</td><td>Millipore</td><td>Cat#:17-622; RRID: AB_916347</td></tr><tr><td>Anti-Rabbit IgG (HRP)</td><td>Santa Cruz</td><td>Cat#:sc-2004; RRID: AB_631746</td></tr><tr><td>Anti-Mouse IgG (HRP)</td><td>Santa Cruz</td><td>Cat#:sc-358914; RRID: AB_10915700</td></tr><tr><td>Anti-Goat IgG (HRP)</td><td>Promega</td><td>Cat#:PA1 28664; RRID: AB_430838</td></tr><tr><td>Anti-GFP</td><td>Abcam</td><td>Cat#:ab290; RRID: AB_303395</td></tr><tr><td>Protein-A magnetic Dynabeads</td><td>ThermoFisher</td><td>Cat#:10002D</td></tr><tr><td>Protein-A agarose</td><td>Abcam</td><td>Cat#:ab193254</td></tr><tr><td>GFP-Trap® agarose</td><td>Chromotek</td><td>Cat#:gta20</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Murashige and Skoog (MS) medium</td><td>Sigma-Aldrich</td><td>Cat#:M5524</td></tr><tr><td>Pierce™ ECL Western blotting substrate</td><td>ThermoFisher</td><td>Cat#:32106</td></tr><tr><td>GlycoBlue™ Coprecipitant</td><td>ThermoFisher</td><td>Cat#:AM9515</td></tr><tr><td>Hygromycin B</td><td>ThermoFisher</td><td>Cat#:10687010</td></tr><tr><td>X-gluc</td><td>PhytoTech</td><td>Cat#:X877</td></tr><tr><td>Toluidine Blue O</td><td>Sigma Aldrich</td><td>Cat#:T3260</td></tr><tr><td>MG-132</td><td>Sigma Aldrich</td><td>Cat#:M7449</td></tr><tr><td>Complete Protease Inhibitor Cocktail</td><td>Sigma Aldrich</td><td>Cat#:11836170001</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>Raw and analysed data</td><td>This study</td><td>GEO: GSE266049</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>Col-0</td><td>N/A</td><td>N/A</td></tr><tr><td>vm2-5</td><td>Nottingham Arabidopsis Stock Centre</td><td>SALK_201153</td></tr><tr><td>prt6-1</td><td>Nottingham Arabidopsis Stock Centre</td><td>SAIL_1278_H11</td></tr><tr><td>vm2-5/prt6-1</td><td>Gibbs et al.<sup>23</sup></td><td>N/A</td></tr><tr><td>pif4-2</td><td>Nottingham Arabidopsis Stock Centre</td><td>SAIL_1288_E07</td></tr><tr><td>vm2-5/pif4-2</td><td>This study</td><td>N/A</td></tr><tr><td>vil1-1</td><td>Nottingham Arabidopsis Stock Centre</td><td>SALK_136506</td></tr><tr><td>vm2-5/vil1-1</td><td>This study</td><td>N/A</td></tr><tr><td>Col-0/pSAUR19::GUS</td><td>Prof. William M. Gray</td><td>Spartz et al.<sup>44</sup></td></tr><tr><td>vm2-5/pSAUR19::GUS</td><td>This study</td><td>N/A</td></tr><tr><td>prt6-1/pSAUR19::GUS</td><td>This study</td><td>N/A</td></tr><tr><td>vil1-1/pSAUR19::GUS</td><td>This study</td><td>N/A</td></tr><tr><td>vm2-5/vil1-1/pSAUR19::GUS</td><td>This study</td><td>N/A</td></tr><tr><td>vm2-5/prt6-1/pSAUR19::GUS</td><td>This study</td><td>N/A</td></tr><tr><td>vm2-5/pVRN2::VRN2-YFP</td><td>This study</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>See Table S4</td><td></td><td>N/A</td></tr></tbody></table>

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Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Recombinant DNA</td><td></td><td></td></tr><tr><td>pVRN2::VRN2</td><td>This study</td><td>pVRN2::VRN2-YFP</td></tr><tr><td>Software and algorithms</td><td></td><td></td></tr><tr><td>DEseq2</td><td>Jean Finnegan et al.<sup>71</sup></td><td>https://bioconductor.org/packages/release/bioc/html/DESeq2.html</td></tr><tr><td>MACS2</td><td>Greb et al.<sup>74</sup></td><td>https://github.com/macs3-project/MACS</td></tr><tr><td>SICER2</td><td>Zhang et al.<sup>77</sup></td><td>https://zanglab.github.io/SICER2/</td></tr><tr><td>ChIPSeeker</td><td>Li et al.<sup>75</sup></td><td>https://www.bioconductor.org/packages/devel/bioc/vignettes/ChIPseeker/inst/doc/ChIPseeker.html</td></tr><tr><td>LeafNet</td><td>Yang et al.<sup>68</sup></td><td>https://github.com/zhouyulab/leafnet</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Arabidopsis growth and transgenic lines

Arabidopsis seed were sown directly to compost (reduced peat Sinclair potting media, vermiculite, perlite, 4:2:1) or on ½ Murishage and Skoog (MS) medium with 1% agar. Plants were grown in a Weiss Technik fitotron SGC 120 growth chamber under long (16h) day conditions at 22°C (100 mol m⁻²·s⁻¹). *vrn2-5*, *prt6-1*, *prt6-1 vrn2-5*, *pif4-2* and *vil1-1* (also known as *vrn5-8*) were described previously.<sup>23,38,73,74</sup> Lines expressing *pSAUR19::GUS*<sup>44</sup> were generated through crossing. The *pVRN2::VRN2-GUS/vrn2-5* and *pVRN2::VRN2-GUS/prt6-1* genotypes were previously reported.<sup>23</sup> The *vrn2-5/pVRN2::VRN2-YFP* complementation line was generated by amplifying the genomic sequence of *VRN2*, including ~2kb of promoter, by PCR ligating into pDONR221 and then mobilising into pGWB540 before transforming into *vrn2-5* mutant plants using the floral dip method. Homozygous transgenic plants were selected by growing seedlings on ½ MS media containing 15µg/ml Hygromycin B. Briefly, seed were sown on square plates and after stratification for 48 hours at 4°C were transferred to the light at 22°C for 4 hours before being wrapped in aluminium foil for a further 48 hours to promote skotomorphogenesis. Aluminium foil was then removed, and plants were allowed to grow normally in the light for 7 days. Seedlings developing true leaves were selected for propagation and genotyped via PCR.

## METHOD DETAILS

### Phenotyping assays

For rosette measurements, plants were grown in compost under long day (16h L: 8h D) conditions (100 mol m⁻²·s⁻¹) for 3 weeks. Leaves were removed sequentially by developmental number and transferred to 1% agar plates before imaging. For hypocotyl length measurements, sterilised seed were sown on ½ MS medium and stratified at 4°C for 48 hours. Plates were transferred to the light and grown for 7 days before photographs were taken. Photos of etiolated seedlings were taken at the indicated timepoints. To evaluate growth at 28°C, seed were stratified on ½ MS plates and then grown for 7 days at either 22°C or 28°C. In all cases, images were analysed using ImageJ.

### Histochemical staining and leaf pavement cell size

Plants expressing either *pVRN2::VRN2-GUS* or *pSAUR19::GUS* were stained at timepoints/conditions indicated. Plant material was incubated at 37°C for 1 h - 8 h in the dark suspended in GUS staining solution (1% potassium ferricyanide, 1% potassium ferrocyanide, 100 mM PBS pH 7.0, 2 mM X-gluc, 0.1% v:v Triton X-100. Seedlings were de-stained in clearing fixative (3:1 ethanol:acetic acid, 1% v:v Tween®) overnight. Cleared samples were mounted in 60% glycerol and imaged using a GX optical microscope. For leaf cell size measurements, leaf 3 from representative 2-week-old Col-0 and *vrn2-5* plants were dissected and cleared using destaining fixative. Leaves were then incubated in Toluidine blue O solution (0.1% w:v toluidine blue, 0.1% v:v Triton X-100) at 37°C for 5 minutes, rinsed, and mounted in water. 2 images for 10 leaves of each genotype were captured from the left and right expansion zones of the midrib respectively using a GX optical microscope. Cell size and number were quantified using the Python tool LeafNet.<sup>75</sup>

### gDNA extraction for genotyping

Individual leaves of 3-week-old plants were harvested and frozen in liquid N₂. Tissues were ground using a Qiagen Tissue Lyser II and gDNA was extracted using the Qiagen DNeasy® Plant Mini kit according to manufacturer instructions. Purified DNA was used as template for PCR with respective primers to genotype segregating transgenic plants.

### Western blotting

Proteins were extracted from whole Arabidopsis seedlings or dissected rosette leaves grown at 22°C under long day conditions (100 mol m⁻²·s⁻¹). Tissues were ground in tubes using a Qiagen Tissue Lyser II and mixed with protein extraction buffer I (150 mM
Tris-HCl pH7.5, 150 mM NaCl, 5 mM EDTA, 2 mM EGTA, 5% v:v glycerol, 0.1% SDS, 1% Triton X-100, 1x Complete Protease Inhibitor Cocktail (Roche)) at a ratio of 1:4. Protein quantification was performed using Bradford reagent (A6932, ITW Reagents) against a BSA standard curve. Total proteins were separated using SDS-PAGE and transferred to a PVDF membrane. PageRuler™ Plus pre-stained protein ladder (ThermoFisher) was used for reference of protein size. After transfer, membranes were blocked in 5% milk in TBS supplemented with 0.1% Tween®-20 for at least an hour. Membranes were probed overnight with antibodies in 5% milk in TBS supplemented with 0.1% Tween®-20; anti-GFP (1:1,000, Sigma-Aldrich, SAB4301138), anti-GUS (1:2000, Sigma-Aldrich, G5420), anti-PIF4 (1:1000, Agrisera, AS16 3955), anti-phyB (1:3000, a gift from Prof. Akira Nagatani's lab⁷⁵), anti-rabbit IgG conjugated to HRP (1:5000, Santa Cruz; sc-2004), anti-mouse IgG conjugated to HRP (1:5000, Santa Cruz; sc-358914), anti-goat IgG conjugated to HRP (1:10,000, Promega, PA1 28664), anti-H3 (1:2500, Abcam; ab1791), anti-H3K27me3 (1:1000, Millipore; 17-622).

**Co-immunoprecipitation**

Seedlings were grown on ½ MS agar plates under LD conditions at 22°C for 13 days. At Zt 0 on the 14th day, plates were either transferred to total darkness, or to 10 µmol m⁻²·s⁻¹ red light for 4 hours before being flash frozen. Protein extracts were generated as above from 1 g of ground seedling powder and incubated for 2 hours with 10 µl GFP-Trap® agarose (ChromoTek). Beads were washed using protein extraction buffer I and protein complexes were eluted by heating samples at 95°C in protein loading buffer.

**qRT-PCR**

Whole seedlings were flash frozen at indicated timepoints for mRNA extraction. Tissues were ground in tubes using a Qiagen Tissue Lyser II and mRNA was extracted using RNeasy Plant Mini kit (Qiagen) according to manufacture instructions. mRNA was on-column DNase treated with DNAse Set (Qiagen). mRNA concentration was quantified using a NanoDrop spectrophotometer and 1 µg of cDNA was synthesised using the qScript cDNA Supermix kit. Quantitative real-time PCR was performed using a QuantStudio™ 5 Real-Time PCR System with Brilliant II low ROX Sybr Green (Agilent). Reactions were performed with at least 2 technical replicates and 3 biological replicates were performed for each experiment. Gene expression is expressed as 2⁻ΔCT relative to ACTIN1 and UBIQUITIN-CONJUGATING ENZYME 8. Primers for genes tested are shown in Table S4.

**RNA-sequencing and analysis**

For seedling RNA sequencing, plants were grown for 10 days on vertical ½ MS plates at 22°C under long day conditions (100 mol m⁻²·s⁻¹) before being flash frozen in liquid N₂ (3 biological replicates). Seedlings were ground in liquid N₂ using a pestle and mortar and mRNA was extracted from 100 mg of leaf powder using the RNeasy Plant Mini kit (Qiagen) according to manufacture instructions. mRNA was on-column DNase treated with DNAse Set (Qiagen). Library preparation was performed by Glasgow Polyomics using the Illumina TruSeq mRNA kit (polyA selection). Paired end sequences of length 75 bp were generated using the HiSeq 4000 platform. Adapter ligated mRNA was trimmed and filtered using fastp with settings -l 50 and -q 20. Reads were aligned to the Arabidopsis genome (TAIR10) using bowtie and analysed using Kallisto³ before differential gene expression analysis was performed using the R package DEseq2.⁴ Genes in *vm2-5* or *prt6-1 vm2-5* were considered upregulated if they had an average log₂ fold change of at least 0.58 across 3 biological replicates, corresponding to an upregulation of at least 1.5 relative to Col-0 and *prt6-1*, respectively.

For rosette RNA sequencing, plants were grown for 3 weeks at 22°C under long day conditions (100 mol m⁻²·s⁻¹) in compost mix (reduced peat Sinclair potting media, vermiculite, perlite, 4:2:1) before being flash frozen in liquid N₂. 4 independent biological replicates were performed, each of which consisted of tissue from a pool of 5 plants per genotype. Plants were ground in liquid N₂ using a pestle and mortar and mRNA was extracted from 100 mg of leaf powder using the RNeasy Plant Mini kit (Qiagen) according to manufacture instructions. mRNA was on-column DNase treated with DNAse Set (Qiagen). Library preparation was performed by Novogene, UK. At least 20 million paired end sequences of length 150 bp per sample, per replicate, were generated using the Novoseq 6000 sequencing platform. Adapter removal, quality control, alignment, read counting and differential gene expression analysis were performed using HISAT2.⁷⁷ Differential gene expression analysis was performed in R studio using the EdgeR⁷⁸ and DESeq2⁷⁹ packages. Genes in *vm2-5* were considered upregulated if they had an average log₂ fold change of at least 0.58, corresponding to an upregulation of at least 1.5 relative to Col-0 plants. Gene ontology enrichment analysis was performed using the R package TopGO⁵. Venn diagrams were created using Venny 2.0 (https://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html).

**ChIP sequencing**

For VRN2-YFP protein ChIP, 6g of 13-day old seedlings grown on ½ MS plates at 22°C under long day photoperiodic conditions (100 µmol m⁻²·s⁻¹) were harvested into 3 miracloth bags and washed in SDW. Seedlings were crosslinked by vacuum infiltration for 15 minutes in PBS supplemented with 1% formaldehyde. The crosslinking reaction was quenched by adding 2M glycine to a final concentration of 125 mM and applying a vacuum for a further 5 minutes. Seedlings were then washed in SDW 4 times before being patted dry to remove excess moisture and flash frozen in liquid nitrogen. Tissues were ground in liquid nitrogen using a pestle and mortar for at least 10 minutes. Nuclei were isolated using 70 ml HONDA buffer (20 mM HEPES pH 7.4, 440 mM sucrose, 1.25% w:v Ficoll, 2.5% w:v Dextran-T40, 10mM MgCl₂, 0.5% v:v Triton X-100, 5 mM DTT, 1x Complete Protease Inhibitor Cocktail (Roche), 25 µM MG-132 (Sigma Aldrich)). After addition of HONDA buffer, samples were gently rotated at 4°C for 15 minutes. Extracts were centrifuged at 3,500 g for 30 minutes at 4°C to pellet nuclei. The supernatant was then removed and nuclei were gently resuspended in 1.5 ml of HONDA buffer and split into 3 x 1.5 ml DNA LoBind tubes (Eppendorf). Samples were centrifugated at 2,000 g for 10 minutes to pellet nuclei and the supernatant was removed. This washing process was repeated 2 times for each sample. Nuclei
<figure><img src="image_29.png" /></figure>

were resuspended in nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% v:v SDS, 1x Complete Protease Inhibitor Cocktail (Roche), 25 µM MG-132 (Sigma Aldrich)) and sonicated using a Bioruptor® Plus (Diagenode) for 3 x 5 minutes on low setting to lyse nuclei and shear chromatin to generate fragments between 200 and 800 bp, centring around 500 bp. Samples were then centrifuged at 14,500 g for 5 minutes at 4°C to pellet cell debris. Chromatin extracts were transferred to 15 ml LoBind tubes and diluted 1:10 in ChIP dilution buffer (15 mM Tris-HCl pH 8, 1.2 mM EDTA, 1.1% v:v Triton X-100), and precleared with 50 µl of Protein-A agarose (ab193254) by gentle rotation at 4°C. For each biological sample, 3 IPs were performed independently from the equivalent of 2 g of chromatin and pooled during DNA cleanup. Prior to immunoprecipitation 150 µl of chromatin extract was taken as input control. 4 µg of anti-GFP (ab290, Abcam) antibody was incubated with chromatin extracts for 3 hours and gently rotated at 4°C. VRN2-antibody complexes were immunoprecipitated overnight using 50 µl of Protein-A agarose. Beads were sequentially washed twice each by rotating for 5 minutes at 4°C with low salt (150 mM NaCl, 0.1% v:v SDS, 1% v:v Triton X-100, 4mM EDTA, 20 mM Tris-HCl pH 8.0), high salt (500 mM NaCl, 0.1% v:v SDS, 1% v:v Triton X-100, 4mM EDTA, 20 mM Tris-HCl pH 8.0) and TE (2 mM EDTA, 10 mM Tris-HCl pH 8.0) buffers. DNA-protein complexes were eluted twice with 200 µl ChIP elution buffer (1% v:v SDS, 100 mM NaHCO₃) by incubating for 15 minutes at 65°C. Samples were reverse crosslinked overnight by incubating at 65°C with 200 mM NaCl before treatment with Proteinase K for 1 hour at 65°C. ChIPed DNA was purified using phenol, chloroform, isoamyl alcohol 25:24:1 (v/v/v) (Sigma) followed by precipitation at -20°C overnight in 100% EtOH with 90 mM NaOAc and 1µl GlycoBlue™ Coprecipitant (ThermoFisher). Precipitated DNA was centrifuged at 21,000 g for 1 hour at 4°C and washed twice with 70% EtOH. The DNA pellet was resuspended in 50 ul of TE buffer and cleaned up further using the ChIP DNA Clean and Concentrator™ kit according to manufacture instructions. Library preparation and sequencing was performed by Glasgow Polyomics. A minimum of 1 ng of ChIPed DNA was processed using the NEBNext® Ultra™II DNA Library Prep kit according to manufacturer instructions. Adapter ligated DNA was amplified with 10 rounds of PCR and sequenced 2x100bp using the Illumina NextSeq2000 sequencing system.

For histone H3K27me3 ChIP sequencing 1 g of 11-day old seedlings grown in 1/2 MS plates were harvested in 50 mL falcon tubes and washed twice in SDW. Seedlings were crosslinked by vacuum infiltration in PBS containing 1% (v/v) formaldehyde 15 min. Crosslinking reaction was quenched by the addition of a final concentration of 125 mM glycine and applying further vacuum for 5 min. Crosslinked seedlings were rinsed 4 times with SDW and patted dried and flash frozen. Tissues were ground with mortor and pestle and resuspended in 30 mL extraction buffer 1 (10 mM Tris-HCl, pH 8.0, 5 mM βmercaptoethanol, 0.4 M sucrose, protease inhibitor cocktail (cOmplete, Roche), vortexed, incubated for 5 min and filtered through 2 layers of Miracloth. Filtered solution was centrifuge at 2880 g for 20 min at 4°C and the pellet resuspended in 1 mL extraction Buffer 2 (10 mM Tris-HCl, pH 8.0, 0.25 M sucrose, 10 mM MgCl2, 1% (v/v) Triton X-100, 5 mM β-mercaptoethanol, 1x protease inhibitor cocktail (cOmplete, Roche)). Solution was transferred to a new 1.5 mL Eppendorf tube and centrifuged at 12000 xg for 10 min at 4°C. Pellet was resuspended in 300 µL extraction buffer 3 (10 mM Tris-HCl, pH 8.0, 1.7 M sucrose, 0.15% (v/v) Triton X-100, 2 mM MgCl2, 5 mM β-mercaptoethanol, 1x protease inhibitor cocktail (cOmplete, Roche)). The resuspended pellet was overlayed onto 900 µL extraction buffer 3 and centrifuge at 16000 xg for 1h at 4°C. The chromatin pellet was resuspended in 320 µL nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% (w/v) SDS, 1x protease inhibitor cocktail (cOmplete, Roche)) and transferred to a DNA LoBind tube. An aliquot of 10 µL (unsheared chromatin) was saved for later quality check. Chromatin sonicated 3 times for 5 min (30 sec on/off intervals) in a Bioruptor® Plus (Diagenode) at the low setting to generate DNA fragments in the range of 200-800 bp. Sheared chromatin was centrifuged at 16000 xg for 5 min and supernatant kept. An aliquot of 10 µL (sheared chromatin) was saved for later quality check. Dynabeads Protein A magnetic beads (15 µL per sample) were washed three times in ChIP dilution buffer (1.1% (v/v) Triton X-100, 1.2 mM EDTA, 16.7 mM TrisHCl, pH 8.0, 167 mM NaCl, 1x protease inhibitor cocktail (cOmplete, Roche). Antibodies were added to the beads in 50 µL ChIP dilution buffer (4 µg anti-H3K27me3, Millipore 17-622) and incubated for 1 h at 4°C. The beads were washed 3 times with ChIP dilution buffer. 10% of the chromatin solution was kept as input sample. The remaining chromatin was diluted 10 times in ChIP dilution buffer and 900 µL were added to each antibody bound beads and incubated by rotation overnight at 4°C. Each sample was washed 2x 5min with 1 mL Low salt wash buffer (150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0), and 1x 5min with 1 mL high salt wash buffer (500 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0). 1 mL of Lithium Chloride wash (0.25 M LiCl, 1% (v/v) NP-40, 1% (w/v) sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) was added and incubated for 5 min followed by the addition of 1 mL of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Beads were finally washed with 1 mL TE buffer for 5 min. The immune complexes were eluted from the beads with 2x 250 µL 1% SDS, 0.1 M NaHCO₃ for 20 min at 65°C. Reverse crosslinking was performed by the addition of a final concentration of 0.3 M NaCl and incubated overnight at 65°C. DNA was purified with 500 µL phenol, chloroform, isoamyl alcohol 25:24:1 (v/v/v) (Sigma) and precipitated with 1.35 mL ice cold 100% EtOH, 50 µL NaAc 3 M, pH 5.2, 2 µL glycoblue, incubated 72h at -80°C. Samples were centrifuged at maximum speed for 5 min and a pellets were washed 2 times with 75% EtOH followed by air drying.

## ChIP sequencing Analysis

For VRN2-YFP protein ChIP, adapters ligated to raw paired-end reads were trimmed and filtered using FastP with minimum length set to 50 bp and minimum q score of 20. Trimmed reads were aligned to the TAIR10 assembly of the Arabidopsis genome using bwa-mem of the Barrows-Wheeler Alignment Tool⁸⁰ with default parameters. Alignments were subject to an additional filter in which reads were removed if they were unmapped or had a mapping quality score of <30. Prior to peak calling, a blacklist of highly duplicated regions was removed from all alignment files.⁸¹ MACS2⁸² was used to call enriched regions with the settings -g 101274395 -f BAMPE -nomodel -keep-dup auto -q 0.05. Consensus peaks for VRN2-YFP were considered if they were observed to have an FDR of < 0.05 and an enrichment of at least 1.5 fold in at least 2 independent biological replicates. The closest gene to each
peak was annotated using the annotatePeak function in the R package ChIPSeeker⁸³ with priority set to promoters, 5UTR, 3UTR, exon, intron, downstream and intergenic regions. For differential H3K27me3 ChIP, adapters ligated to raw single-end reads were trimmed and filtered using FastP⁸⁴ with minimum length set to 50 bp and minimum q score of 20. Trimmed reads were aligned to the TAIR10 assembly of the Arabidopsis genome using bwa-mem of the Burrows-Wheeler Alignment Tool⁸⁰ with default parameters. Alignments were subject to an additional filter in which reads were removed if they were unmapped or had a mapping quality score of <30. Prior to peak calling, a blacklist of highly duplicated regions was removed from all alignment files.⁸¹ SICER²⁸⁵ was used to perform differential peak calling with the sicer DF function, with settings -efg 0.95 -significant reads. Consensus hypomethylated peaks in *vrn2-5* were considered if they were observed to have an FDR of < 0.05 and a relative fold change difference of at least 80% of WT in at least 2 independent biological replicates. The closest gene to each peak was annotated using the annotatePeak function in the R package ChIPSeeker⁸³ with priority set to promoters, 5UTR, 3UTR, exon, intron, downstream and intergenic regions. Where peaks mapped to multiple genes, only the closest was considered. To visualise data, bigwig files were created using the DeepTools⁸⁶ function bamCoverage to bin reads into 20 bp windows which were normalised by reads per genome coverage (RPGC). Gene specific enrichment was evaluated using IGV.

## ChIP qPCR

The ChIP PCR protocol has previously been described.⁶³ Samples were handled exactly as for the VRN2-YFP ChIP sequencing, with some differences. Isolated nuclei were resuspended in RIPA buffer (1% NP-40 (v:v), 0.1% sodium deoxycholate (v:v), 0.1% SDS (v:v), 1x Complete Protease Inhibitor Cocktail (Roche), 25 µM MG-132 (Sigma Aldrich) in PBS). Nuclei were lysed by sonication on high setting for 5 minutes on a cycle of 30s on, 30s off, twice. For VRN2-YFP samples, chromatin was sheared for 15 minutes by treatment with the endonuclease benzonase; for H3K27me3 ChIP, nuclei were lysed by sonication and DNA fragmentation was achieved using 3 cycles of 30s on 30s off for 5 minutes on low setting. For VRN2-YFP, the IP step was performed overnight using 4 µg of anti-GFP (ab290, Abcam) and Protein-A agarose (ab193254) which had been blocked with salmon sperm DNA (10 mg/ml, ThermoFisher). For H3K27me3, the IP was performed overnight with 3 µg of anti-H3K27me3 (Millipore, 17-622) or anti-H3 (Abcam, ab1791) respectively with Protein A magnetic Dynabeads. For each qPCR, four technical repeats were performed from a minimum of 2 independent biological replicates. Enrichment values were calculated by normalising the % input to the enrichment of 5SrDNA which served as a negative control.

## Confocal microscopy

Protein localisation was performed using confocal microscopy in seedlings stably expressing VRN2-YFP, or transiently in *Nicotiana benthamiana* leaves. VRN2-YFP seeds were surface sterilised and stratified in the dark at 4°C for 3 days, and then grown on ½ MS 0.8 % agar-containing media for 3 days in the dark. Seedlings were exposed to 24 h of red light (1 µmol m⁻²·s⁻¹) prior to imaging. Transient expression was performed by infiltrating 4 week old *N. benthamiana* leaves with pVRN2::VRN2-YFP, 35S::phyB-CFP using *A. tumefaciens* (GV3101). Prior to imaging, plants were dark adapted for 24 h before treatment with red light (10 µmol m⁻²·s⁻¹) for 4 hours. Seedlings (Arabidopsis) or leaf discs (*N. benthamiana*) were mounted onto glass microscope slides containing a droplet of dH₂O (without fixation) and imaging was performed using a Leica SP8 confocal microscope. YFP (VRN-YFP) and CFP (phyB-CFP) fluorescence was monitored using excitation at 514 nm (YFP) or 405 nm (CFP) and emission between 550 nm and 595 nm (YFP) 460 or 505 nm (CFP).

## QUANTIFICATION AND STATISTICAL ANALYSIS

ImageJ was used to analyse hypocotyl length, leaf area and epidermal cell area. Statistically significant differences were calculated by either Students t-test (Figures 2G, 2H, 5F, and 6D) or one/two way ANOVA followed by Tukey's test (Figures 1E, 2D, 2E, 4C-4E, 5H, 5G, 6A, S1A, S1B, S1G, S2D, and S3D) using R studio. Information pertaining to the number of biological and technical repeats for each assay can be found in respective figure legends. In all bar charts, error bars represent the SEM; all box and whisker plots describe (from top to bottom) the maximum, 75th percentile, median, 25th percentile and minimum values of the data.