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EIN2 Mediates Direct Regulation of Histone Acetylation in the Ethylene Response

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EIN2 Mediates Direct Regulation of Histone Acetylation in the Ethylene Response EIN2 mediates direct regulation of histone acetylation in the ethylene response Fan Zhanga,b,1, Likai Wanga,b,1, Bin Qic, Bo Zhaoa,b, Eun Esther Koa,b, Nathaniel D. Riggana,b, Kevin China,b, and Hong Qiaoa,b,2 aInstitute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; bDepartment of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; and cDepartment of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, CO 80309 Edited by Steven E. Jacobsen, University of California, Los Angeles, CA, and approved August 10, 2017 (received for review May 15, 2017) Ethylene gas is essential for developmental processes and stress of EBF1 and EBF2 (19, 20). In the nucleus, the EIN2-C transduces responses in plants. Although the membrane-bound protein EIN2 is signals to the transcription factors EIN3 and EIL1, which are key for critical for ethylene signaling, the mechanism by which the ethylene activation of expression of all ethylene-response genes (21, 22). We signal is transduced remains largely unknown. Here we show the recently discovered that acetylation at H3K23 and H3K14Ac is in- levels of H3K14Ac and H3K23Ac are correlated with the levels of volvedinethylene-regulatedgeneactivationinamannerthatde- EIN2 protein and demonstrate EIN2 C terminus (EIN2-C) is sufficient to pends on both EIN2 and EIN3 (23, 24). rescue the levels of H3K14/23Ac of ein2-5 at the target loci, using Here we show that the levels of H3K14/23Ac are positively CRISPR/dCas9-EIN2-C. Chromatin immunoprecipitation followed by correlated with the EIN2 protein levels and demonstrate that deep sequencing (ChIP-seq) and ChIP-reChIP-seq analyses revealed that EIN2-C is sufficient to rescue the levels of H3K14Ac and EIN2-C associates with histone partially through an interaction with H3K23Ac in ein2-5 at loci targeted using CRISPR/dCas9-EIN2-C. EIN2 nuclear-associated protein1 (ENAP1), which preferentially binds Chromatin immunoprecipitation followed by deep sequencing to the genome regions that are associated with actively expressed (ChIP-seq) and ChIP-reChIP-seq analyses reveal that EIN2-C genes both with and without ethylene treatments. Specifically, in the associates with histones partially through an interaction with presence of ethylene, ENAP1-binding regions are more accessible upon EIN2 nuclear-associated protein1 (ENAP1), which preferentially the interaction with EIN2, and more EIN3 proteins bind to the loci where ENAP1 is enriched for a quick response. Together, these results binds to the genome regions that are associated with actively reveal EIN2-C is the key factor regulating H3K14Ac and H3K23Ac in expressed genes in both the presence and absence of ethylene. response to ethylene and uncover a unique mechanism by which Specifically, in the presence of ethylene, ENAP1-binding regions ENAP1 interacts with chromatin, potentially preserving the open chro- are more accessible upon the interaction with EIN2, and more matin regions in the absence of ethylene; in the presence of ethylene, EIN3 proteins bind to the loci where ENAP1 is enriched for a EIN2 interacts with ENAP1, elevating the levels of H3K14Ac and quick response. Together, these results reveal EIN2-C is the key H3K23Ac, promoting more EIN3 binding to the targets shared with factor regulating H3K14Ac and H3K23Ac in response to ethylene ENAP1andresultinginarapidtranscriptional regulation. and uncover a unique mechanism by which ENAP1 interacts with chromatin, potentially preserving the open chromatin regions in histone acetylation | ethylene | Arabidopsis | CRISPR/dCas9 | ChIP-reChIP-seq Significance he plant hormone ethylene is essential for a myriad of Upon receipt of different cues, transcription factors bind to Tphysiological and developmental processes. It is important in specific DNA sequences to recruit the general transcriptional responses to stress, such as drought, cold, flooding, and pathogen machinery for gene expression. Chromatin modification plays a infection (1, 2), and modulates stem cell division (3). The com- central role in the regulation of gene expression by providing mon aquatic ancestor of plants possessed the ethylene signaling transcription factors and the transcription machinery with dy- pathway and the mechanism has been elucidated by analysis of namic access to an otherwise tightly packaged genome. We use Arabidopsis (4). Ethylene is perceived by a family of receptors Arabidopsis to study how chromatin perceives ethylene sig- bound to the membrane of the endoplasmic reticulum (ER) that naling, an important plant hormone in plant growth, devel- are similar in sequence and structure to bacterial two-component opment, and stress responses. We demonstrate that the histidine kinases (5–9). Each ethylene receptor has an N-terminal essential factor EIN2, which mediates ethylene signaling from the transmembrane domain, and the receptors form dimers that bind endoplasmic reticulum to the nucleus, directly regulates histone ethylene via a copper cofactor RAN1 (10, 11). Signaling from one acetylation through an interaction with a histone-binding protein. of the receptors, ETR1, induces its physical association with the This study reveals the novel mechanism of how chromatin per- ER-localized protein RTE1 (12). The ethylene receptors function ceives the hormone signals to integrate into gene regulation. redundantly to negatively regulate ethylene responses (5) via a downstream Raf-like protein kinase called CTR1 (13, 14). Author contributions: F.Z., B.Q., and H.Q. designed research; F.Z., L.W., B.Q., B.Z., E.E.K., N.D.R., and K.C. performed research; L.W. and H.Q. analyzed data; and F.Z. and H.Q. In the absence of ethylene, both the ethylene receptors and wrote the paper. CTR1 are active, and CTR1 is associated with the ER membrane The authors declare no conflict of interest. through a direct interaction with ETR1 (13). The CTR1 downstream This article is a PNAS Direct Submission. factor, EIN2, is localized to the ER membrane, where it interacts with ETR1 (15). The protein stability of EIN2 is regulated by two Freely available online through the PNAS open access option. F-box proteins, ETP1 and ETP2, which mediated its degradation by Data deposition: The data reported in this paper have been deposited in the National – Center for Biotechnology Information Sequence Read Archive (NCBI SRA) Gene Expres- the ubiquitin proteasome pathway (16). In the absence of ethylene, sion Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession nos. the CTR1-mediated phosphorylation at the C-terminal end of EIN2 GSE97288, GSE81200, and GSE83214). (EIN2-C) leads to a repression of EIN2 activity (17). In the presence 1F.Z. and L.W. contributed equally to this work. of ethylene, the EIN2-C is dephosphorylated, cleaved from the rest of 2To whom correspondence should be addressed. Email: [email protected]. the protein, and translocated into the nucleus (18) or into the P-body This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (19, 20). In the P-body, the EIN2-C mediates translational repression 1073/pnas.1707937114/-/DCSupplemental. 10274–10279 | PNAS | September 19, 2017 | vol. 114 | no. 38 www.pnas.org/cgi/doi/10.1073/pnas.1707937114 Downloaded by guest on October 2, 2021 the absence of ethylene; in the presence of ethylene, EIN2 in- teracts with ENAP1, elevating the levels of H3K14/23Ac, pro- moting more EIN3 proteins binding to the targets shared with ENAP1 and resulting in a rapid transcriptional regulation. Results EIN2 Is the Key Regulator of Histone Acetylation of H3K14 and H3K23 in Response to Ethylene. Previous work demonstrated that EIN2 is involved in the regulation of H3K14Ac and H3K23Ac levels in ethylene response (23). To explore the molecular mechanisms that underlie this regulation, we examined the levels of H3K14Ac and H3K23Ac in EIN2S645A transgenic plants, in which EIN2-C is con- stitutively localized to the nucleus, and these plants display a con- stitutive ethylene responsive phenotype (18). To our surprise, significant amounts of H3K14AcandH3K23Acwereelevatedin EIN2S645A in comparison to Col-0 plants treated with ethylene (Fig. 1A).Basedonthisresultandourprevious data (23), we speculated that EIN2-C itself is the key factor for histone acetylation in the ethylene response. We then examined the global levels of H3K14Ac and H3K23Ac in EIN2 gain-of-function plants (EIN2ox)andCol-0 and ein2-5 mutant plants treated with or without ethylene by Western blot. As previously reported, both H3K14Ac and H3K23Ac were elevated in Col-0 plants by ethylene treatment (Fig. 1B). The levels of H3K14AcandH3K23AcwerehigherinEIN2ox plants than in Col-0 plants both with and without ethylene treatments (Fig. 1B), and they were lower in ein2-5 mutant than in Col-0 plants in the absence ofethylene.Noethylene-inducedelevationsweredetectedinthe ein2-5 mutant (Fig. 1B). To confirm the Western blot result at the molecular level, we revisited previously published ChIP-seq data of H3K14AcandH3K23AcfromCol-0andein2-5 mutant plants (23). In the ein2-5 mutant, levels of both H3K14Ac and H3K23Ac were lower than in Col-0 plants, whereas no significant difference was detected for H3K9Ac (Fig. 1 C–E and Fig. S1). Taken together, these results suggest the EIN2-C has a key role in the regulation of Fig. 2. gRNA-EIN3-T/dCas9-EIN2-C partially restores the ein2-5 phenotype histone acetylation of H3K14 and H3K23 in the ethylene response. (A) Diagram showing the construction of binary vector containing both dCas9 and guide RNAs. Guide RNA expression is driven by the OsU3 promoter EIN2-C Is Able to Restore the Levels of Histone Acetylation at (39), and dCas9-EIN2-C is driven by the 35S promoter. (B) The seedling phe- ein2-5 notype of CRISPR/dCas9-EIN2-C transgenic plants is shown. Three-day-old Ethylene-Regulated Gene Loci in the Mutant. To further ex- μ amine the function of EIN2-C in histone regulation, we decided etiolated seedlings were grown on the medium with or without 10 M ACC before being photographed.
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