EIN2-dependent regulation of acetylation of histone H3K14 and non-canonical histone H3K23 in ethylene signalling

Abstract

Ethylene gas is essential for many developmental processes and stress responses in plants. EIN2 plays a key role in ethylene signalling but its function remains enigmatic. Here, we show that ethylene specifically elevates acetylation of histone H3K14 and the non-canonical acetylation of H3K23 in etiolated seedlings. The up-regulation of these two histone marks positively correlates with ethylene-regulated transcription activation, and the elevation requires EIN2. Both EIN2 and EIN3 interact with a SANT domain protein named EIN2 nuclear associated protein 1 (ENAP1), overexpression of which results in elevation of histone acetylation and enhanced ethylene-inducible gene expression in an EIN2-dependent manner. On the basis of these findings we propose a model where, in the presence of ethylene, the EIN2 C terminus contributes to downstream signalling via the elevation of acetylation at H3K14 and H3K23. ENAP1 may potentially mediate ethylene-induced histone acetylation via its interactions with EIN2 C terminus.

Figure 1. Acetylation at H3K14 and H3K23 is up-regulated by ethylene.

(a) Global histone acetylation at indicated lysines of histone H3 in wild-type Col-0 seedlings grown in the dark for 3 days and then treated with or without 4 h ethylene gas. Total histones were subjected to immunoblotting with antibodies that recognize indicated histone H3 acetylations. Immunoblotting with anti-histone H3 was used as a loading control. (b–c) ChIP quantitative real time PCR detection of H3Ac, H4Ac, H3K14Ac and H3K23Ac in indicated EIN3-R genes and EIN3-NR genes in 3-day-old etiolated Col-0 seedlings with or without ethylene treatment. Precipitation with IgG pre-immune serum served as a control. Data represent relative fold changes. Each experiment has three biology replicates with similar result. (d) The levels of H3K9Ac (2 replications), H3K14Ac (1 replication) and H3K23Ac (2 replications) in Col-0 in response to ethylene examined by Standard ChIP-Seq assays (2 or 1 replication). Heat map showing ethylene-regulated peaks of H3K9Ac, H3K14Ac and H3K23Ac. (e–h) Standard ChIP-seq assays showing that the elevated enrichment of H3K14Ac and H3K23Ac in EIN3-R (4 representative genes indicated in the figure) in Col-0 treated with ethylene and no significant enrichment of H3K9Ac is observed. Binding levels are indicated by reads per kilobase per million reads in sample (RPKM). Col-0 seedlings grown in the dark for 3 days with or without 4 h ethylene gas treatment for ChIP-seq. Dash boxes highlight the difference enrichment of H3K9Ac, H3K14Ac or H3K23Ac. EIN3-R, represents ethylene-regulated EIN3 targets; EIN3-NR represents non-ethylene-regulated EIN3 targets. Dash boxes highlight the difference enrichment of H3K9Ac, H3K14Ac or H3K23Ac. Different letters were used to indicate statistically significance difference (P≦0.05 Student's t-test=3) within the same genes.

Figure 2. EIN2 is important for the elevation of H3K14Ac and H3K23Ac in the presence of ethylene.

(a–b) ChIP quantitative real time PCR detection of H3K14Ac and H3K23Ac enrichment in Col-0, ein2-5, ein3-1eil1-1 3-day-old etiolated seedlings treated with air or 4 h ethylene gas. Precipitation with IgG pre-immune serum served as a control. Data represent the relative fold change. EIN3-R, represents ethylene-regulated EIN3 targets; EIN3-NR represents non-ethylene-regulated EIN3 targets. Each experiment has three biology replicates with similar result. (c) The levels of H3K14Ac (2 replications) and H3K23Ac (2 replications) in ein2-5 in response to ethylene examined by Standard ChIP-seq assays. Heat map showing ethylene-regulated peaks of H3K14Ac and H3K23Ac. (d,e) Standard ChIP-seq assays showing that ethylene- induced enrichment of H3K14Ac and H3K23Ac in EIN3-R genes is abolished in ein2-5 mutant. Binding levels are indicated by reads per kilobase per million reads in sample (RPKM). Col-0 seedlings grown in the dark for 3 days with or without 4 h ethylene gas treatment for Chip-seq. Dash boxes highlight the difference enrichment of H3K9Ac, H3K14Ac or H3K23Ac. (f) The expression of ethylene-regulated genes is positively associated with the enrichment of H3K14Ac and H3K23Ac. Total RNAs were extracted from 3-day-old etiolated seedlings from indicated genotypes and gene expression was analyzed by qualitative RT-PCR (3 biological replicates). EIN3-R, represents ethylene-regulated EIN3 targets; EIN3-NR represents non-ethylene-regulated EIN3 targets. Different letters were used to indicate statistically significance difference (P≦0.05 Student's t-test=3) within the same genes.

Figure 3. EIN2 C-terminal end interacts with ENAP1 and ENAP1 is involved in ethylene response.

(a) Yeast two-hybrid assay revealed an interaction between the EIN2 C-terminal end and ENAP1. Growth on selective plates lacking leucine, histidine, tryptophan with 20 mM 3-AT (−Leu, −Trp, −His, +3-AT) (left panel) and an X-Gal filter assay was performed to verify the interaction between EIN2 CEND and ENAP1 (right panel). (b) In vitro GST pull-down showing the interaction between EIN2 CEND and ENAP1 and ENAP2. (c) Both ENAP1 and ENAP2 are localized to the nucleus. The images were taken from the roots of 3-day-old etiolated seedlings of pENAP1:ENAP1-YFP and pENAP2:ENAP2-YFP under a confocal microscope. (d) ENAP1 interacts with EIN2 CEND in vivo in the presence of ethylene gas. Total protein extracts from 35 S:ENAP1-YFP-HA transgenic plants treated with or without ethylene were immunoprecipitated with an anti-HA antibody. The EIN2 CEND was detected by anti-EIN2 CEND antibody. The immunoprecipitation with IgG from 35S:ENAP1-YFP-HA transgenic plants treated with ethylene was used as a control. (e) Phenotype of ENAP1 gain-of-function (ENAp1ox) and ENAP1-deficient amiR-ENAP1/enap2 mutants. The plants indicated in the figure were grown 3 days on MS with or without 10 μM ACC before being photographed. (f) ENAP1ox plants show transcriptional activation of ethylene-response genes. Total RNA was prepared from 3-day-old etiolated seedlings of ENAP1ox or wild-type (Col-0) plants treated with air or 4 h ethylene. Differentially expressed genes were identified by fragments per kilobase per million reads (FPKM) filter<0.1, requiring a twofold change comparing the indicated conditions with P<=0.05 after Benjamini–Hochberg correction.

Figure 4. ENAP1 interacts with histone H3 and regulates histone acetylation of H3K14 and H3K23.

(a) In vitro GST pull-down assay showing that ENAP1 interacts with histone H3 and H2B. (b) GST-labelled ENAP1 interacts with histone H3 in nucleosomes isolated from cow cell extracts. Purified GST-ENAP1 from E. coli was incubated with calf thymus nucleosomes, the pull-down products were subjected to immunoblotting with anti-histone H3 antibody. (c) The SANT domain is required for the interaction between ENAP1 and histone H3. GST-labelled full-length ENAP1 or truncated ENAP1ΔSANT expressed from E. coli was used for GST pull-down. The pull-down products were subjected to immunoblotting with anti-histone H3 antibody. (d) RNA-seq data collected from Fig. 3g in ENAP1ox or wild-type (Col-0) plants treated with air or 4 h ethylene was analyzed for the up- and down-regulated gene numbers. (e) Total histone extractions from 3-day-old etiolated seedlings of Col-0 and ENAP1ox seedlings were subjected to the immunoblotting with antibodies indicated. Immunoblotting with anti-histone H3 antibody served as a loading control. (f) ChIP-qPCR to quantify H3K14Ac and H3K23Ac enrichment in Col-0, ENAP1ox and amiR-ENAP1/enap2 plants treated with air or 4 h ethylene gas in indicated EIN3-R genes and EIN3-NR genes. Precipitation with IgG pre-immune serum served as a control. Data represent the relative fold change. Each experiment has three biology replicates with similar result. (g). Quantitative PCR detection of EIN3 target gene expression in Col-0, ENAP1ox and amiR-ENAP1/enap2 plants treated with air or 4 h ethylene gas (three biological replicates). EIN3-R, represents ethylene-regulated EIN3 targets; EIN3-NR represents non-ethylene-regulated EIN3 targets. Different letters were used to indicate statistically significance difference (P≦0.05 student's t-test=3) within the same genes.

Figure 5. Partial rescue of the ENAP1ox phenotype by ein2-5.

(a) The phenotype of ENAP1ox in the ein2-5 mutant background. The 3-day-old etiolated seedlings were grown on MS with (right panel) or without (left panel) 10 μM ACC before being photographed. (b) ChIP-qPCR to quantify H3K14Ac and H3K23Ac enrichment in Col-0, ENAP1ox, ein2-5 and ENAP1ox/ein2-5 plants treated with air or 4 h ethylene gas in indicated EIN3-R genes and EIN3-NR genes. Precipitation with IgG pre-immune serum served as a control. Data represent the relative fold change. Each experiment has three biology replicates with similar result. (c) Quantitative PCR detection of EIN3 target gene expression in Col-0, ENAP1ox, and ENAP1ox/ein2-5 seedlings grown in air or 4 h ethylene gas (three biological replicates). EIN3-R, represents ethylene-regulated EIN3 targets; EIN3-NR represents non-ethylene-regulated EIN3 targets. Different letters were used to indicate statistically significance difference (P≦0.05, Student's t-test=3) within the same genes.

Figure 6. Partial rescue of the ENAP1ox phenotype by ein3-1eil1-1.

(a) The phenotype of ENAP1ox/ein3-1eil1-1 mutant. The 3-day-old etiolated seedlings of the plants indicated in the figure were grown on MS with (right panel) or without (left panel) 10 μM ACC before photographed. (b) In vitro GST pull-down showing the interaction between EIN3 and ENAP1. GST-tagged ENAP1 and MBP-tagged EIN3 purified from E. coli were used. (c) ENAP1 interacts with EIN3 in vivo in the presence of ethylene. Total protein extracts from 35S:ENAP1-YFP-HA/pEIN3:EIN3-FLAG transgenic plants treated with or without ethylene treatment were immunoprecipitated with an anti-HA antibody. EIN2 was detected with an anti-EIN2 CEND antibody, and EIN3 was detected using an anti-FLAG antibody. (d) ChIP-qPCR to quantify H3K14Ac and H3K23Ac enrichment in Col-0, ENAP1ox, ein3-1eil1-1 and ENAP1ox/ein3-1eil1-1 plants treated with air or 4 h ethylene gas in indicated EIN3-R genes and EIN3-NR genes. Precipitation with IgG pre-immune serum served as a control. Data represent the relative fold change. Each experiment has three biological replicates which showed similar results. (e) Quantitative PCR detection of EIN3 target gene expression in Col-0, ENAP1ox, ein3-1eil1-1 and ENAP1ox/ein3-1eil1-1 seedlings grown in air or 4 h ethylene gas (three biological replicates). EIN3-R, represents ethylene-regulated EIN3 targets; EIN3-NR represents non-ethylene-regulated EIN3 targets. Different letters were used to indicate statistically significance difference (P≦0.05 Student's t-test=3) within the same genes.