Our understanding of the detailed molecular mechanisms underpinning adaptation is still poor. One example for which mechanistic understanding of regulation has converged with studies of life history ...variation is
Arabidopsis thaliana FLOWERING LOCUS C
(
FLC
).
FLC
determines the need for plants to overwinter and their ability to respond to prolonged cold in a process termed vernalization. This review highlights how molecular analysis of vernalization pathways has revealed important insight into antisense-mediated chromatin silencing mechanisms that regulate
FLC
. In turn, such insight has enabled molecular dissection of the diversity in vernalization across natural populations of
A. thaliana
. Changes in both cotranscriptional regulation and epigenetic silencing of
FLC
are caused by noncoding polymorphisms at
FLC
. The
FLC
locus is therefore providing important concepts for how noncoding transcription and chromatin regulation influence gene expression and how these mechanisms can vary to underpin adaptation in natural populations.
Summary
Chromatin plays a central role in orchestrating gene regulation at the transcriptional level. However, our understanding of how chromatin states are altered in response to environmental and ...developmental cues, and then maintained epigenetically over many cell divisions, remains poor. The floral repressor gene FLOWERING LOCUS C (FLC) in Arabidopsis thaliana is a useful system to address these questions. FLC is transcriptionally repressed during exposure to cold temperatures, allowing studies of how environmental conditions alter expression states at the chromatin level. FLC repression is also epigenetically maintained during subsequent development in warm conditions, so that exposure to cold may be remembered. This memory depends on molecular complexes that are highly conserved among eukaryotes, making FLC not only interesting as a paradigm for understanding biological decision‐making in plants, but also an important system for elucidating chromatin‐based gene regulation more generally. In this review, we summarize our understanding of how cold temperature induces a switch in the FLC chromatin state, and how this state is epigenetically remembered. We also discuss how the epigenetic state of FLC is reprogrammed in the seed to ensure a requirement for cold exposure in the next generation.
Significance Statement
FLOWERING LOCUS C (FLC) regulation provides a paradigm for understanding how chromatin can be modulated to determine gene expression in a developmental context. This review describes our current mechanistic understanding of how FLC expression is genetically specified and epigenetically regulated throughout the plant life cycle, and how this determines plant life‐history strategy.
Plants use seasonal temperature cues to time the transition to reproduction. In Arabidopsis thaliana, winter cold epigenetically silences the floral repressor locus FLOWERING LOCUS C (FLC) through ...POLYCOMB REPRESSIVE COMPLEX 2 (PRC2)
. This vernalization process aligns flowering with spring. A prerequisite for silencing is transcriptional downregulation of FLC, but how this occurs in the fluctuating temperature regimes of autumn is unknown
. Transcriptional repression correlates with decreased local levels of histone H3 trimethylation at K36 (H3K36me3) and H3 trimethylation at K4 (H3K4me3)
, which are deposited during FRIGIDA (FRI)-dependent activation of FLC
. Here we show that cold rapidly promotes the formation of FRI nuclear condensates that do not colocalize with an active FLC locus. This correlates with reduced FRI occupancy at the FLC promoter and FLC repression. Warm temperature spikes reverse this process, buffering FLC shutdown to prevent premature flowering. The accumulation of condensates in the cold is affected by specific co-transcriptional regulators and cold induction of a specific isoform of the antisense RNA COOLAIR
. Our work describes the dynamic partitioning of a transcriptional activator conferring plasticity in response to natural temperature fluctuations, thus enabling plants to effectively monitor seasonal progression.
Polycomb-mediated epigenetic silencing is central to correct growth and development in higher eukaryotes. The evolutionarily conserved Polycomb repressive complex 2 (PRC2) transcriptionally silences ...target genes through a mechanism requiring the histone modification H3K27me3. However, we still do not fully understand what defines Polycomb targets, how their expression state is switched from epigenetically ON to OFF and how silencing is subsequently maintained through many cell divisions. An excellent system in which to dissect the sequence of events underlying an epigenetic switch is the
locus. Exposure to cold temperatures progressively induces a PRC2-dependent switch in an increasing proportion of cells, through a mechanism that is driven by the local chromatin environment. Temporally distinct phases of this silencing mechanism have been identified. First, the locus is transcriptionally silenced in a process involving cold-induced antisense transcripts; second, nucleation at the first exon/intron boundary of a Polycomb complex containing cold-induced accessory proteins induces a metastable epigenetically silenced state; third, a Polycomb complex with a distinct composition spreads across the locus in a process requiring DNA replication to deliver long-term epigenetic silencing. Detailed understanding from this system is likely to provide mechanistic insights important for epigenetic silencing in eukaryotes generally.
The timing of flowering is vital for plant reproductive success and is therefore tightly regulated by endogenous and exogenous cues. In summer annual Arabidopsis (
) accessions, like Columbia-0, ...rapid flowering is promoted by repression of the floral repressor
(
). This is through the activity of the autonomous pathway, a group of proteins with diverse functions including RNA 3'-end processing factors, spliceosome components, a transcription elongation factor, and chromatin modifiers. These factors function at the
locus linking alternative processing of an antisense long noncoding RNA, called
, with delivery of a repressive chromatin environment that affects the transcriptional output. The transcriptional output feeds back to influence the chromatin environment, reinforcing and stabilizing that state. This review summarizes our current knowledge of the autonomous pathway and compares it with similar cotranscriptional mechanisms in other organisms.
Analysis of how seasonal cues influence the timing of the floral transition has revealed many important principles for how epigenetic regulation can integrate a variety of environmental cues with ...developmental signals. The study of the pathways that necessitate overwintering in plants and their ability to respond to prolonged cold (the vernalization requirement and response pathways) has elaborated different chromatin regulatory pathways and the involvement of noncoding RNAs. The major target of these vernalization pathways in Arabidopsis (Arabidopsis thaliana) isFlowering Locus C(FLC). A relatively simple picture ofFLCregulation is emerging of a few core complexes and mechanisms that antagonize each other's actions. This balance provides a fine degree of control that has nevertheless permitted evolution of a wide range of natural variation in vernalization in Arabidopsis. Similar simple routes of adaptation may underlie life history variation between species.
Transcription in eukaryotic genomes generates an extensive array of non-protein-coding RNA, the functional significance of which is mostly unknown. We are investigating the link between non-coding ...RNA and chromatin regulation through analysis of FLC — a regulator of flowering time in Arabidopsis and a target of several chromatin pathways. Here we use an unbiased strategy to characterize non-coding transcripts of FLC and show that sense/antisense transcript levels correlate in a range of mutants and treatments, but change independently in cold-treated plants. Prolonged cold epigenetically silences FLC in a Polycomb-mediated process called vernalization. Our data indicate that upregulation of long non-coding antisense transcripts covering the entire FLC locus may be part of the cold-sensing mechanism. Induction of these antisense transcripts occurs earlier than, and is independent of, other vernalization markers and coincides with a reduction in sense transcription. We show that addition of the FLC antisense promoter sequences to a reporter gene is sufficient to confer cold-induced silencing of the reporter. Our data indicate that cold-induced FLC antisense transcripts have an early role in the epigenetic silencing of FLC, acting to silence FLC transcription transiently. Recruitment of the Polycomb machinery then confers the epigenetic memory. Antisense transcription events originating from 3′ ends of genes might be a general mechanism to regulate the corresponding sense transcription in a condition/stage-dependent manner.
Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the ...cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription.
Posttranslational modifications of histone tails are an important factor regulating chromatin structure and gene expression. Epigenetic memory systems have been predicted to involve mutually ...exclusive histone modifications that, through positive feedback mechanisms, generate bistable states 1, 2. How the states are interconverted is not understood, and whether the histone modifications are sufficient for epigenetic memory is still greatly debated 3. We have exploited the process of vernalization, the slow quantitative epigenetic silencing of Arabidopsis FLC induced by cold, to detail with fine temporal and spatial resolution the dynamics of histone modifications during an epigenetic switch. The profiles of H3K36me3, H3K4me3, and H3K4me2 at FLC throughout the vernalization process were compared to H3K27me3, which accumulates at a local nucleation region during the cold and spreads across the locus on return to the warm 2. We find for many phases of the vernalization process that H3K36me3 and H3K27me3 show opposing profiles in the FLC nucleation region and gene body, that H3K36me3 and H3K27me3 rarely coexist on the same histone tail, and that this antagonism is functionally important. A lack of H3K36me3 results in a fully silenced state at FLC even in the absence of cold. We therefore propose that H3K36me3 is the opposing modification to H3K27me3 in the Polycomb-mediated silencing of FLC. However, the lack of an absolute mirror profile predicted from modeling suggests that their antagonistic roles contribute a necessary, but not sufficient, component of the mechanism enabling switching between and inheritance of epigenetic states.
•Modeling suggests that epigenetic systems involve bistable states•These bistable states involve mutually exclusive histone modifications•H3K36me3 and H3K27me3 appear to be opposing modifications at FLC•H3K36me3/H3K27me3 balance at FLC “registers” cold exposure
Yang et al. investigate histone modification dynamics during the cold-induced epigenetic switching that occurs at the FLC locus during vernalization. H3K36me3 shows an opposing profile to H3K27me3 at the nucleation region, and Yang et al. propose that the dynamic balance between these two modifications is a register of cold exposure.
Long noncoding RNAs (IncRNAs) have been proposed to play important roles in gene regulation. However, their importance in epigenetic silencing and how specificity is determined remain controversial. ...We have investigated the cold-induced epigenetic switching mechanism involved in the silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC), which occurs during vernalization. Antisense transcripts, collectively named COOLAIR, are induced by prolonged cold before the major accumulation of histone 3 lysine 27 trimethylation (H3K27me3), characteristic of Polycomb silencing. We have found that COOLAIR is physically associated with the FLC locus and accelerates transcriptional shutdown of FLC during cold exposure. Removal of COOLAIR disrupted the synchronized replacement of H3K36 methylation with H3K27me3 at the intragenic FLC nucleation site during the cold. Consistently, genetic analysis showed COOLAIR and Polycomb complexes work independently in the cold-dependent silencing of FLC. Our data reveal a role for IncRNA in the coordinated switching of chromatin states that occurs during epigenetic regulation.
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