Epigenetic switch from repressive to permissive chromatin in response to cold stress

Abstract

Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47 Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance.

Keywords: CUL4-based E3 ligase; HOS15; cold stress response; derepression; histone acetylation.

Fig. 1.

HOS15 directly interacts with HD2C. (A) HOS15 interacts with HD2C by yeast two-hybrid assay with HOS15 and HD2C as bait and prey, respectively. (B) HOS15 binds to HD2C in vivo. Tobacco plants were infiltrated with Agrobacterium harboring 35S::HOS15-FLAG and 35S::HD2C-GFP for transient expression. Protein extracts (input) were immunoprecipitated (IP) with anti-FLAG, and resolved by SDS/PAGE. Immunoblots were developed with anti-FLAG and anti-GFP to detect HOS15 and HD2C fusions, respectively. (C) HOS15 interacts with HD2C in vivo. HOS15 and HD2C were fused to C terminal of CLuc and N terminal of NLuc for split luciferase complementation assays. Agrobacterium carrying 35S::CLuc-HOS15 and 35S::HD2C-NLuc were infiltrated into tobacco leaves for transient expression. After 2 to 3 d, the bottom side of the tobacco leaves were sprayed with 1 mM luciferin, and bioluminiscence was detected. Combination of CLuc-RAR1 and SGT1a-NLuc is included as a positive control. Images shown are representative of three biological replicates of three individual experiments. (D) HOS15 interacts with HD2C in vivo. Shown are the results of BiFC analyses performed with constructs of VYNE-HOS15 and VYCE-HD2C, which were transiently expressed in Arabidopsis protoplasts. Nuclei were stained with DAPI, and YFP fluorescence was detected under the confocal microscope. (Scale bars, 10 μm.)

Fig. 2.

HD2C is involved in freezing stress response. (A‒C) hd2c is tolerant to freezing stresses. Three-week-old plants pretreated with cold (4 °C for cold-acclimation) or not (nonacclimation) were exposed to freezing temperatures as indicated. (B) Survival ratio was determined with nonacclimated (−4 °C) or cold-acclimated (−6 °C) plants in 7 d after freezing treatment. The data are the means of three technical replicates with SD (n = 25 for each replicate: *P < 0.05; **P < 0.01; Student’s t test). (C) Electrolyte leakages of nonacclimated (A, Left) or acclimated (A, Right) plants were measured at indicated temperatures. Error bars are SD (n = 6). (D) Relative transcript levels of cold responsive genes are higher in hd2c mutant upon cold treatment. Two-week-old plants including wild-type (white bar) or hos15-2 (hatched bar) and hd2c-1 (gray bars) or HD2C_OX (black bars) were treated with cold (0 °C) for indicated periods. Total RNA was isolated and transcript levels of CORs were measured by qRT-PCR and normalized to that of ACTIN2. Bar represent means ± SD from three biological replicates with three technical repeats each.

Fig. 3.

HOS15 is a component of CUL4-based Ub E3 ligase complexes. (A) HOS15 interacts with DDB1 directly in yeast two-hybrid assays. Assays were performed with DDB1B protein as prey and HOS15 as bait. (B and C) HOS15 interacts with DDB1 proteins in vivo. Total proteins (input) extracted from tobacco plants transiently expressing HOS15-FLAG and DDB1A-HA (B) or DDB1B-HA (C) were immunoprecipitated (IP) with anti-HOS15. Immunoblots were carried out with anti-HOS15 and anti-HA to detect HOS15-FLAG and DDB1A-HA or DDB1B-HA, respectively. (D) HOS15 interacts with CUL4 in vivo. Co-IP of HOS15 and DDB1B or CUL4. Total proteins from 12-d-old 35S::Flag-DDB1B/ddb1a and 35S::Flag-CUL4 plants were pulled down with anti-HOS15. Anti-FLAG was used to detect DDB1B and CUL4. (E) Cold enhances the interaction of HOS15 and CUL4. Total protein extracts from CUL4 overexpressors exposed to cold stress (4 °C) for 24 h were pulled down with anti-HOS15.

Fig. 4.

Cold-induced degradation of HD2C is mediated by HOS15. (A) The protein abundance of HD2C is reduced upon cold stress. Nuclear proteins extracted from 12-d-old HD2C overexpressors treated with cold stress (0 °C) for the indicated periods were applied to immunoblots with anti-GFP. Hintone3 (H3) was used as loading controls. (B) Relative HD2C mRNA levels in HD2C overexpressors are not reduced upon cold. Total RNA was extracted and gene expression of selected genes was checked by qRT-PCR analysis. Results are from three biological replicates and values represent means ± SD (n= 9). (C) Cold reduces the biofluorescence from 7-d-old HD2C-GFP overexpressing plants exposed to cold (0 °C) for 6 h. (D) Cold-induced degradation of HD2C is impaired in hos15-2. Ten-day-old wild-type and hos15-2 plants were treated with cold (0 °C) for 15 h in the presence of proteasome inhibitor MG132 (50 μM). Nuclear proteins were applied to immunoblot with anti-HD2C. (E) The poly-ubiquitination of HD2C is blocked in hos15-2 mutant. Seven-day-old plants (HD2C-GFP and hos15-2 HD2C-GFP) were treated with cold (4 °C) for 12 h in the presence of MG132. Total proteins were incubated with Ub-binding p62 resin or with empty agarose resin (negative control). Anti-Ub was used to detect ubiquitinated proteins in input protein extracts and pulled-down (PD) samples. Anti-GFP allowed the detection of HD2C-GFP and its ubiquitinated forms Ub(n)-HD2C-GFP.

Fig. 5.

HD2C and HOS15 associate to the promoter locus of COR15A. (A) Structure of the COR15A promoter and amplicon regions (I to V) used for ChIP. The arrow indicates the TSS. White boxes mean CBF binding cis elements, and gray box denotes 5′UTRs. (B) Cold-induced H3 acetylation of the COR15A promoter locus is impaired in hd2c mutants. Chromatin from wild-type, hd2c-1, hos15-2, and hos15-2hd2c-1 plants treated with cold (0 °C) for 24 h were immunoprecipitated with anti-H3Ac antibody. A control reaction was processed in parallel with rabbit lgG. ChIP and input-DNA were applied to real-time qPCR using primers specifically targeting to the COR15A promoter region, IV. Error bars indicate SE (n = 3). The experiments were repeated two times with similar results. (C and D) HOS15 and HD2C associates to regions IV and V of the COR15A promoter, CBF cis element regions. Chromatin complexes from wild-type plants were immunoprecipitated with anti-HOS15 (C) or anti-HD2C (D). A control reaction was processed in parallel with rabbit lgG. ChIP and input-DNA samples were quantified by real-time qPCR using primers specific to the different regions (I to V) of the COR15A. (E and F) Cold enhances the binding of HOS15 protein to COR15A promoter but reduces that of HD2C protein. ChIP assay was carried out using anti-HOS15 (E) and anti-HD2C (F) with wild-type plants treated with cold (0 °C, for 24 h). (G and H) HOS15 is required for HD2C association to COR genes chromatin and vice versa. Four-week-old seedling (22 °C) or 4-wk-old seeding after cold (0 °C) treatment 1 d were used for isolating input chromatin. ChIP data from wild-type and hos15-2 or hd2c-1 plants. Epitope-tagged HD2C chromatin complex was immunoprecipitated with anti-HD2C antibody or HOS15 chromatin complex was immunoprecipitated with anti-HOS15. A control reaction was processed in parallel with rabbit lgG. ChIP and input-DNA samples were quantified by real-time qPCR using promoter specific to the different region of the COR15A genes. The structures of the COR15A gene as well as the position of the primer used for qRT-PCR corresponding to the distal promoter regions are marked on the diagram at the top. The ChIP results are presented as fold-enrichment of nontarget DNA. Error bars indicate SE (n = 3). The experiments were repeated at least two times with similar results.

Fig. 6.

Cold-induced binding of CBF proteins to COR15A promoter is affected by HOS15 and HD2C. (A) HOS15 interacts with CBF proteins using yeast split Ub assay. Yeast cells cotransformed with Nub (Vector) or Cub-HOS15 and CBF1-Nub-RUra3P, CBF2-Nub-RUra3P, or CBF3-Nub-RUra3P were spotted on selective media (–HTU and -HTU+5-FOA). Pictures were taken after 4-d incubation at 30 °C. (B) HOS15 makes a complex with CBF proteins upon cold treatment. Total protein extracts from wild-type exposed to cold (0 °C) for 24 h were subjected to gel-filtration, using a Superdex 200 10/300 column. Each eluate [0.5 mL 50 mM Tris⋅Cl (pH7.5) and 100 mM NaCl] was TCA-precipitated, and analyzed by immunoblots with anti-CBFs and anti-HOS15 antibodies. (C and D) Cold-induced binding of CBF proteins to cold-responsive gene COR15A promoter regions, II (C), and IV (D) is reduced and enhanced in hos15-2 and hd2c-1, respectively. Chromatin from wild-type, hos15-2, and hd2c-1 plants treated with cold (0 °C) for 24 h were immunoprecipitated with anti-CBF antibody (***P < 0.001). Similar results were obtained from three independent experiments. (E and F) Binding of HOS15 and HD2C proteins to cold-responsive gene COR15A promoter regions, II, and IV is reduced and in cbf1/2/3 mutant, respectively. Chromatin from wild-type and cbf1/2/3 mutant plants treated with cold (0 °C) for 24 h were immunoprecipitated with anti-HOS15 (E) or anti-HD2C (F) antibodies. Similar results were obtained from three independent experiments.

Fig. 7.

Model for the HOS15-mediated chromatin remodeling in response to cold stress. In the absence of cold stress, HOS15 forms a complex with HD2C to repress COR gene expression by hypoacetylation of COR chromatin. Under cold stress conditions, HOS15 recruits CUL4 to form a CRL4^HOS15 complex, resulting in degradation of HD2C via the Ub–proteasome system. Dissociation of HD2C by CRL4^HOS15 results in the hyperacetylation of H3 on COR chromatin and induces the association of CBF transcription factors to the COR promoters via HOS15, thereby increasing CORs expression and cold tolerance. The unknown factor (question mark) recruiting HOS15 to COR genes under temperate conditions might also be CBFs expressed at basal levels (see Discussion for details).