Prolonged cold (vernalization) epigenetically silences the Arabidopsis floral repressor FLOWERING LOCUS C (FLC) by depositing trimethylation of histone H3 lysine 27 (H3K27me3), first at a small nucleation region and then across the gene body, and this repressed state is stably inherited through cell divisions after temperatures rise. Jiang and Berger (Science, 2017) showed that restoration of H3K27me3 after each round of DNA replication depends on replication-coupled deposition and K27 trimethylation of histone variant H3.1. Two linked questions arising from this finding remain unresolved: (i) what mechanistically distinguishes and drives the nucleation, spreading, and maintenance phases at FLC — in particular, whether spreading of H3K27me3 across the gene body is independent of maintenance at the initial nucleation site, as their histone H3.1 knock-down (h3.1kd) data suggested — and (ii) how H3K27me3-mediated silencing can be established or maintained in mitotically quiescent, non-replicating plant tissues, such as mature leaves, where the replication-coupled H3.1 pathway cannot operate. A decade of intense work has identified individual components of each phase (VAL1 recruitment, the cis cold memory element and its bivalent chromatin readers, VEL-protein polymerization, PICKLE-dependent spreading, H1-facilitated replication-coupled restoration), but no integrated account experimentally dissociates the three phases at FLC, the postulated non-histone memory elements remain unidentified, and no plant mechanism for replication-independent establishment of the mark in quiescent cells has been found.

Background

Vernalization — the acquisition of flowering competence after a prolonged period of cold — is a classic model of environmental epigenetic memory in plants. The memory is stored at the FLOWERING LOCUS C (FLC) gene, a strong repressor of flowering: prolonged cold progressively silences FLC, and the silencing persists through subsequent warm growth so that plants flower in spring. The molecular carrier of this memory is trimethylation of histone H3 at lysine 27 (H3K27me3), a repressive histone modification deposited by Polycomb Repressive Complex 2 (PRC2), the histone methyltransferase complex named after the Polycomb-group genes of Drosophila that maintain repressed states through cell divisions. Cold exposure first triggers H3K27me3 over a small nucleation region near the FLC transcription start site — a 'cold memory element' of roughly three nucleosomes — after which the mark spreads across the gene body and is then maintained over many cell cycles (De Lucia et al. 2008; Angel et al. 2011; Yuan et al. 2016; Yang et al. 2017). Because every round of DNA replication halves the nucleosomal histone pool, H3K27me3 must be rapidly restored on newly deposited histones for the mark to function as heritable memory. Jiang and Berger (2017) showed that in plants this restoration is coupled to DNA replication: the replication-dependent histone variant H3.1 (deposited at the replication fork by the CAF-1 chaperone complex, in contrast to the replication-independent variant H3.3) is efficiently trimethylated at K27 during S phase, and this H3.1-specific pathway is required to propagate H3K27me3 silencing from mother to daughter cells. Two consequences of that study define the present problem. First, in H3.1 knock-down (h3.1kd) plants, cold still nucleates H3K27me3 at the FLC transcription start site, but spreading across the gene body and long-term maintenance are impaired; from this the authors suggested that spreading of H3K27me3 at FLC might be independent of maintenance at the initial nucleation site, leaving the mechanistic requirements that distinguish nucleation, spreading, and maintenance unresolved. Second, because the identified pathway is replication-coupled, it cannot operate in mitotically quiescent tissue: mature Arabidopsis leaves, which lack replicating cells, do not accumulate H3.1 after vernalization and show impaired H3K27me3 enrichment at FLC — consistent with the earlier observation of Finnegan and Dennis (2007) that vernalization-induced H3K27me3 at FLC is not maintained in mitotically quiescent cells — yet plants clearly establish stable epigenetic silencing in developmental contexts where cell division is limited. Subsequent work has clarified parts of the machinery: the transcriptional repressor VAL1 recruits PRC2 to the nucleation region (Qüesta et al. 2016); the cold memory element carries bivalent active (H3K4me3) and repressive (H3K27me3) marks that are read by an assembly of bivalent chromatin readers to drive the cold-induced switch (Gao et al. 2023); the cold-induced accessory proteins VIN3 and VRN5 polymerize through head-to-tail VEL domains that promote nucleation (Fiedler et al. 2022; Schulten et al. 2024); CK2 kinase signalling activates PRC2 during prolonged cold (Zeng et al. 2025); cold repositions nucleosomes at the nucleation region to expose the transcription start site for silencing (Montez et al. 2025); and on the spreading side, the chromatin remodeler PICKLE condenses nucleosomes in spreading regions to give PRC2 access to unmodified nucleosomes (Liang et al. 2024), while histone H1 compaction facilitates replication-coupled H3K27me3 restoration (Liu et al. 2023). Mathematical modeling shows that a purely histone-based read/write mechanism anchored at a three-nucleosome nucleation region cannot by itself explain the observed stability of memory, implying additional, still-unidentified protein memory elements that persist through replication (Lövkvist et al. 2021). Related concepts exist in animals — stepwise post-replication re-methylation of Polycomb domains every cell cycle (Veronezi and Ramachandran 2024) and division-independent Polycomb switching in non-dividing cells (Pease et al. 2021; McCole et al. 2025) — but no plant mechanism for replication-independent establishment or maintenance of H3K27me3 in quiescent tissue has been identified (reviews: Hugues et al. 2020; Joly and Jacob 2023; Gao and He 2023; Baile and Calonje 2024; Montez et al. 2026).

Problem Statement

The problem is to determine, mechanistically, how cold-induced Polycomb memory at Arabidopsis FLC is built and inherited, through two linked questions arising from the findings of the source study (Jiang and Berger, Science 2017) that remain unresolved and share one objective — explaining the write, extension, and inheritance of the H3K27me3 domain at FLC and its dependence on DNA replication: (1) What molecular components and requirements distinguish the nucleation, spreading, and maintenance phases of H3K27me3 at FLC during and after vernalization? In particular, is spreading of H3K27me3 across the FLC gene body mechanistically independent of maintenance at the initial nucleation site, as suggested by the h3.1kd phenotype in which cold-induced nucleation at the transcription start site occurs while gene-body spreading and long-term maintenance are impaired; and which phases genuinely require the replication-coupled H3.1 modification pathway? (2) By what mechanism, if any, is H3K27me3-mediated (or equivalent Polycomb-based) silencing established or maintained in mitotically quiescent, non-replicating plant tissues such as mature leaves, where the replication-coupled H3.1 pathway cannot operate?

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