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A cis cold memory element and a trans epigenome reader mediate Polycomb silencing of FLC by vernalization in Arabidopsis

Nature Genetics volume 48, pages 1527–1534 (2016)Cite this article

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Abstract

Some plants acquire competence to flower in spring after experiencing a seasonal temperature drop—winter cold, in a process termed vernalization. In Arabidopsis thaliana, prolonged exposure to cold induces epigenetic silencing of the potent floral repressor locus FLOWERING LOCUS C (FLC) by Polycomb group (PcG) proteins, and this silencing is stably maintained in subsequent growth and development upon return to warm temperatures. Here we show that a cis-regulatory DNA element in the nucleation region for PcG silencing at FLC and two homologous trans-acting epigenome readers, VAL1 and VAL2, control vernalization-mediated FLC silencing. The sequence-specific readers recognize both the cis element (termed the cold memory element) and a repressive mark, trimethylation of histone H3 at lysine 27 (H3K27me3), and directly associate with LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), leading to establishment of the H3K27me3 peak in the nucleation region at FLC during vernalization. Thus, our work describes a mechanism for PcG-mediated silencing by a DNA sequence-specific epigenome reader.

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Figure 1: Identification of a 47-bp FLC silencing element.
Figure 2: Phenotypic and expression analyses of FLC transgenes bearing a mutation in the FLC silencing element.
Figure 3: VAL1 and VAL2 recognize the Sph/RY motifs in the FLC silencing element and repress FLC expression.
Figure 4: Analysis of VAL1 protein function.
Figure 5: VAL1 and VAL2 associate with LHP1 and meditate LHP1 binding to FLC chromatin.

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Acknowledgements

We thank M. Suzuki (University of Florida) for kindly providing val1 and val2 seeds and the Flanders Interuniversity Institute for Biotechnology (Belgium) for providing pBGW vector. This work was supported in part by funding from the Chinese Academy of Sciences and from the Ministry of Science and Technology of China (grant 2016YFA0503200 to J.D.).

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  1. Wenya Yuan, Wannian Yang & Yizhong Wang

    Present address: Present addresses: Novolife Biomedical Research Co. Ltd., Beijing, China (W. Yuan) and School of Life Sciences, Huazhong Normal University, Wuhan, China (W. Yang and Y.W.).,

Authors and Affiliations

  1. Shanghai Center for Plant Stress Biology, CAS Center for Excellence of Molecular Plant Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Wenya Yuan, Xiao Luo, Zicong Li, Yizhong Wang, Rui Liu, Jiamu Du & Yuehui He

  2. Department of Biological Sciences, National University of Singapore, Singapore

    Wenya Yuan, Wannian Yang & Yuehui He

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Contributions

Y.H. conceived the project. W. Yuan, X.L., Z.L., W. Yang, Y.W., and R.L. performed the experiments. W. Yuan, X.L., Z.L., W. Yang, Y.W., R.L., J.D., and Y.H. analyzed the data. Y.H. wrote the manuscript.

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Correspondence to Yuehui He.

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Integrated supplementary information

Supplementary Figure 1 A cis-regulatory region in the first intron of FLC mediates transcriptional repression.

(a) Relative FLC transcript levels in the indicated transgenic seedlings (in the FRI flc-3 background). A total of eight independent T1 lines for each transgene were examined. (b) The 10-bp TTCTGCATGG sequence mediates FLC repression independently of the autonomous pathway gene FCA. FCA represses FLC expression to promote flowering3. WT-FLC and FLC ∆10 (transgenes) were introduced into fca flc-3; the 10-bp deletion caused further FLC upregulation and consequent late flowering. “Non-flower” denotes plants that had not flowered after 3 months of growth in long days. (c) Relative mRNA levels of the GUS fusion with part of FLM and the 5′ UTR of SPY in the indicated T1 transgenic plants. 35–38 independent T1 seedlings from each line were pooled for RNA extraction. Error bars, s.d. of three biological repeats. (d) FLC mRNA levels in T1 plants expressing the indicated transgenes. 30, 28, and 30 independent T1 seedlings were pooled for RNA extraction from WT-FLC, FLCm1, and FLCm2, respectively. In a, c, and d, FLC levels were normalized to those of the endogenous control, TUB2. In a and d, error bars, s.d. of triplicate quantifications. (e) Box plots of the flowering times of the indicated lines (T1 generation; in the flc-2 background). Center lines represent medians, box limits represent the 25th and 75th percentiles, and whiskers represent the ranges of data; red dots are data points.

Supplementary Figure 2 Schematic of the FLC locus.

On top is the sequence of the 47-bp FLC silencing element (Sph/RY motifs are in red; numbers correspond to nucleotide positions, with A of the ATG codon as +1). Arrows indicate a transcription start site (TSS); boxes correspond to exons. VRE denotes a 289-bp region around the COLDAIR TSS, the deletion of which disrupts the maintenance of vernalization-mediated FLC repression upon return to warm temperature31. COOLAIR RNAs mainly consist of three isoforms denoted by purple31.

Supplementary Figure 3 VAL1 and VAL2, but not VAL3, RAV1, or VRN1, recognize and bind to the Sph/RY-bearing FLC silencing element.

WT-72 bp, m1.m1, and m2.m2 denote the 72-bp FLC fragment (+451 to +522, where A of the ATG codon is +1), a mutant fragment with both Sph/RY motifs mutated with the m1 mutation, and a fragment with both motifs mutated to the m2 mutation, respectively.

Supplementary Figure 4 Analyses of val1, val2, val3, and val1 val2 mutants.

(a) Box plots of the flowering times of the indicated lines in long days. Box plots show the median line, interquartile range (box limits), whiskers (extending 1.5 times the interquartile range), and data points (red cycles). Fifteen plants were scored for each genotype. (b) Phenotypes of 4-d-old val1 val2 seedlings. The selfed progeny of a weak val1 val2 double mutant exhibit a 1:9 ratio of strong to weak mutants (a total of 267 mutants were scored at an early seedling stage). Scale bars, 1 mm. (c) pVAL1-VAL1:Flag rescued the val1 val2 double-mutant phenotype. Shown are 3-week-old plants grown in long days. Scale bars, 1 cm.

Supplementary Figure 5 Analyses of COOLAIR and COLDAIR RNA levels during the course of vernalization in wild-type and val1 val2 seedlings.

(a,b) Relative COOLAIR (a) and COLDAIR (b) RNA levels. RNA levels were normalized to those of PP2A; error bars, s.d. of two biological repeats.

Supplementary Figure 6 Analysis of FLCm1 copy number by qPCR.

An FLC intron I region from wild type (Col) or the indicated transgenic lines (T3 homozygotes and in the flc-2 background) was quantified, and signal was first normalized to that of the single-copy reference gene FLOWERING LOCUS T, followed by normalization to that of the endogenous FLC in wild type. Error bars, s.d. of triplicate quantification. Both FLCm1-1 and FLCm1-2 bear a single T-DNA insertion.

Supplementary Figure 7 VAL1 and VAL2 can form homodimers.

Yeast two-hybrid assays with full-length VAL1 and VAL2 were conducted.

Supplementary Figure 8 Analysis of His-SUMO-VAL1N.

(a) Schematic of the VAL1 protein. The gray bar denotes VAL1N. (b) Affinity-purified His-SUMO-VAL1N from E. coli. Purifications were analyzed by SDS–PAGE. (c) His-SUMO-VAL1N, but not His-SUMO, binds to H3K27me2 and H3K27me3, as demonstrated by histone 3 peptide pulldown assays.

Supplementary Figure 9 Working model for the regulatory CME–VAL1/VAL2–H3K27me3 interactions to mediate Polycomb silencing of FLC by vernalization.

Before vernalization, FLC expression in early seedlings is subject to dynamic control by the active histone 3 lysine 36 methyltransferase EFS and a repressive core PRC2 complex recruited by CME–VAL1/VAL2–LHP1. During long exposure to cold, more VAL proteins (and LHP1) bind to the CME-containing nucleation region, leading to the recruitment of PHD (VIN3)–PRC2 to establish the H3K27me3 peak at this region (in one or both FLC alleles) and generate a positive feedback loop, resulting in silencing of an FLC allele. Upon return to warm temperature, the silenced FLC allele bearing repressive H3K27me3 is read by VAL1/VAL2–LHP1 through binding to both the cis element CME and H3K27me3, enabling the instruction of an allele-specific repressive chromatin state for its own inheritance during mitotic cell divisions.

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Yuan, W., Luo, X., Li, Z. et al. A cis cold memory element and a trans epigenome reader mediate Polycomb silencing of FLC by vernalization in Arabidopsis. Nat Genet 48, 1527–1534 (2016). https://doi.org/10.1038/ng.3712

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