The interactome of histone deacetylase HDA19 in dark-grown Arabidopsis seedlings

Here, we describe a data set derived from an immunoprecipitation (IP)-based analysis of the histone deacetylase HDA19 interactome in etiolated Arabidopsis seedlings. HISTONE DEACETYLASE 19 (HDA19) belongs to the class 1 of the Reduced Potassium Dependence3/Histone Deacetylase-1 (RPD3/HDA1) superfamily and is possibly the most intensively studied HDA. Here, we describe a data set derived from an immunoprecipitation (IP)-based analysis of the histone deacetylase HDA19 interactome in etiolated Arabidopsis seedlings. We believe that this data set presented here provides a valuable resource for follow-up research on novel interacting partners of this central protein.


Introduction
As a central epigenetic modification, histone acetylation affects the expression of genes with a wide range of functions across all life forms (Shen et al., 2015).Histone acetylation is mediated by histone acetylases and, generally, promotes DNA-templated transcriptional activity.Histone deacetylases (HDAs) reverse this process, leading to an inactive chromatin state and decreased transcriptional activity.Acetylation and deacetylation are central regulatory switches that govern responses to various environmental cues, orchestrating chromatin dynamics and gene activity to modulate the phenotypic readout.
In this Data Report, we provide an immunoprecipitation (IP)-based analysis of the HDA19 interactome in etiolated Arabidopsis seedlings.HDA19 is directly involved in hypocotyl elongation during photomorphogenesis and, based on our findings, also in hypocotyl elongation during skotomorphogenesis (Veĺez-Bermudez and Schmidt, 2021).The exact role of HDA19 in the latter process is still under investigation.For this reason, we conducted an immunoprecipitation experiment that provides new insights regarding the role of HDA19 in skotomorphogenesis and allows for comparisons of HDA19 targets in hypocotyl elongation during skoto-and photomorphogenesis.We believe that the catalogue of HDA19-interacting proteins presented here provides a valuable resource for follow-up research on novel interacting partners of this central protein.The material used for this experiment was derived from hypocotyls of 6-day-old etiolated Arabidopsis seedlings.The dataset contains a total of 6 files, 3 independent biological replicates of each Col-0 (control plants) and 35S::HDA19-GFP plants.

Plant materials and growth conditions
Arabidopsis thaliana Col-0 and the transgenic line 35S::HDA19-GFP were used in this study.35S::HDA19-GFP lines have been described previously (Zhou et al., 2005).Seeds were soaked in 35% bleach for 5 min, washed five times for 5 min with sterile water, and resuspended in 1 mL of sterile water for further use.Seeds were subsequently placed on a growth medium (Estelle and Somerville, 1987; ES medium) containing 5 mM KNO 3 , 2 mM MgSO 4 , 2 mM Ca (NO 3 ) 2 , 2.5 mM KH 2 PO 4 , 70 mM H 3 BO 3 , 14 mM MnCl 2 , 1 mM ZnSO 4 , 0.5 mM CuSO 4 , 0.01 mM CoCl 2 , 0.2 mM Na 2 MoO 4 , and 40 mM Fe-EDTA, solidified with 0.4% Gelrite Pure.MES (1 g/L) and 1.5% (w/v) sucrose were added, and the pH was adjusted to 5.5 with KOH.Seeds were stratified on plates for 2 days at 4°C in the dark and grown at 22°C in vertical position in the dark with 70% relative humidity.

Immunoprecipitation
Experiments were carried out with the mMACS Epitope GFP tag protein isolation kit (MACSmolecular) following the manufacturer's instructions with minor modifications.Hypocotyls were collected by dissection in dark conditions within ca. 5 minutes per plate (~25 seedlings).Samples from each plate were frozen immediately in liquid nitrogen.A total of 25 plates were used to obtain 0.5 gram of tissue.Hypocotyls were ground with liquid nitrogen and resuspended in 500 mL of extraction buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2X complete protease inhibitor cocktail EDTA-free (ROCHE), 1 mM PMSF, and 50 mM MG132).The samples were incubated on ice for 30 minutes with occasional mixing and centrifuged for 20 minutes at 10,000 x g at 4°C.The supernatants were individually collected in fresh tubes, and 400 mL of each input was added to 50 ml of anti-GFP microbeads and incubated for 1 hour and 30 minutes in a mixer set to 60 rpm at 4°C, while 100 mL of each input was kept to be used for Western blots using anti-GFP as a control.The samples were eluted in 50 mL denaturing elution buffer supplied with the kit.

S-Trap sample digestion and protein identification
The protocol was conducted as described previously (Chen C. W. et al., 2023).Briefly, 50 mL eluted IP sample was resuspended in 30 µL of lysis buffer (5% SDS (w/v) in 50 mM triethylammonium bicarbonate (TEAB), pH 8.5), transferred to a 1.7 mL tube, sonicated 10 times for 10 sec each, centrifuged at 16,000 g at 4°C for 20 min, and the supernatant was collected.The IP sample protein amount was determined by using a bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA).The IP protein digestion was performed in the S-Trap micro column following the manufacturer's protocol with some modifications.Shortly, 10 mg of protein in lysis buffer was reduced and alkylated using 1.6 mL of 200 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 1.6 mL of 800 mM 2-chloroacetamide (CAA) at 45°C for 15 min.After alkylation, 3.3 µL of 55.5% (v/v) phosphoric acid (PA) was added, and the pH (~1) was controlled by means of pH paper.After acidification, the sample was mixed with 198 mL of binding buffer (100 mM TEAB in 90% (v/v), MeOH).After gentle vortexing, the sample was loaded onto an S-trap micro column and centrifuged at 4,000 g for 2 min to trap the proteins.The sample was then washed three times in the column with 150 µL of binding buffer and centrifuged at 4,000 g for 2 min each time.An additional centrifugation step (4,000 g for 2 min) was added to fully remove residual binding buffer.The S-trap column was transferred to a fresh 1.7 mL sample tube for the digestion, and 20 µL of protease solution (Lys-C + trypsin, 50 mM TEAB) was added into individual S-traps containing the samples.The cap of each S-trap was loosely closed to limit evaporative loss, and the samples were incubated for 2.5 h at 47°C.The column was removed from the incubator, and 40 µL of of three buffers were added consecutively to the column: 50 mM TEAB, elution buffer 2 (0.2% formic acid in H 2 O) and elution buffer 3 (50% acetonitrile (ACN) in ultrapure water).The column was centrifuged at 4,000 g for 2 min and the elution solution was collected in a new tube, dried by speed vacuum, resuspended in 100 µL of 0.1% formic acid, desalted, and loaded into a C18 Ziptip pipette tip.The elution was dried down under vacuum, the pellet was re-dissolved in 10 µL of 0.1% (v/v) formic acid (FA) with 3% (v/v) acetonitrile (ACN), and the liquid chromatography was performed by injecting 4 µL of sample in the LC-nESI-Q Exactive mass spectrometer model (Thermo Fisher Scientific) coupled with an on-line nanoUHPLC (Dionex UltiMate 3000 Binary RSLCnano).The Acclaim PepMap 100 C18 trap column (75 µm x 2.0 cm, 3 µm, 100 Å, Thermo Scientific) and the Acclaim PepMap RSLC C18 nano LC column (75 µm x 25 cm, 2 µm, 100 Å) were used to deliver solvent and separate tryptic peptides with a linear gradient from 5% to 35% of acetonitrile in 0.1% (v/v) formic acid for 60 min at a flow rate of 300 nl/min.The acquisition cycle for MS data was performed in the datadependent mode with a full survey MS scan followed by 10 MS/MS scans of the top 10 precursor ions from the scan.The mass spectrometer was operated in full scan mode (m/z 350-1,600) in the Orbitrap analyser at a resolution of 70,000.Data-dependent MS/MS acquisitions were performed with a 2 m/z isolation window, 27% NCE (normalized collision energy), and 17,500 resolving power.

Data analysis and identification of putative interactors
Raw data were analysed with the Proteome Discoverer ™ Software 2.2 (Thermo Fisher) using the Sequest search algorism.
The Arabidopsis protein database (Araport11) was used to conduct the searches; only high confidence proteins were selected for the analysis.All peptide spectrum matches were filtered with a q-value threshold of 0.05 (5% FDR), and the proteins were filtered with high confidence threshold (0.05 q-value, 5% FDR).Nuclear proteins identified in more of two biological replicates were considered as putative interacting partners of HDA19.Localization of the proteins was gathered from published experimental evidence or-in cases where such information was unavailable-prediction inferred from the Subcellular Location of Proteins in Arabidopsis Database (SUBA).

Protein-protein interaction network
The PPI network was constructed using STRING (https:// string-db.org).Only nucleus-located partners of HDA19 (as listed in Table 1) were considered.

Dataset description
The identification of HDA19 protein partners via IP-LC-MS/MS relies on the capacity to distinguish true interactors from non-specific binders.To produce the current IP dataset, we used a powerful system to reduce the background in the IP samples.Samples from Arabidopsis thaliana Col-0 wild-type and 35S::HDA19-GFP plants were immunoprecipitated using the MultiMACS GFP isolation kit system with µMACS MicroBeads conjugated to an anti-GFP monoclonal antibody for faster and effective magnetic labelling of GFP-tagged fusion proteins.The complete procedure is depicted in Figure 1A.The IP samples were digested, subjected to LC-MS/MS analysis, and HDA19-binding proteins were identified using the Proteome Discoverer software.The dataset provided a total of 371 putative interactors that were identified with high confidence (Supplementary Data File 1).Please note that the proteins identified in the IP using Col-0 (present in the deposited data set) were removed as background.Considering only candidate proteins that are preferentially or exclusively located to the nucleus according to experimental results or predictions, a subset of 52 putatively interacting proteins was identified in at least two biological replicates (Table 1).A GO enrichment analysis of this subset revealed that besides predicted processes such as 'histone modification', 'chromatin organization', and 'negative regulation of transcription', proteins in the categories 'multicellular organism development', 'cell cycle', 'protein modification' and 'reproduction' were overrepresented (Figure 1B).A protein-protein interaction (PPI) network constructed from this subset of proteins shows a suite of well-known partners of HDA19, including five members of the SIN-LIKE (SNL) family.SNL proteins were shown to be involved in the repression of AP2 family transcription factors that repress FLOWERING LOCUS T (FT) expression through histone deacetylation (Figure 1C) (Huang et al., 2019;Jing et al., 2021).We also identified HDC1, a component of histone deacetylase complexes that interacts with HDA6 and HDA19 (Perrella et al., 2016).A bimolecular fluorescence complementation approach revealed that HDC1 binds to the linker histone H1 (Perrella et al., 2016), which was identified as a putative interactor of HDA19 in the current dataset.The WD-40 repeat containing protein MULTICOPY SUPRESSOR OF IRA1 (MSI1), a conserved subunit of Polycomb Repressive Complex 2 (Xu et al., 2022), and PROLIFERATING CELL NUCLEAR ANTIGEN 2 (PCNA2), involved in DNA replication and damage repair (Xue et al., 2015) were identified as central nodes of the PPI network (Figure 1C).
The current dataset identified a large suite of putative novel interacting partners of a key regulator of plant development and stress responses, HDA19.The identification of SNL members, HDC1, and histone H1 can be considered as validation of the current IP assay.A surprisingly large subset of (predicted) nonnuclear proteins was identified with high confidence, suggesting that some of these proteins may transiently associate with chromatin.Besides expected binding partners such as HCD1, H1, and SNLs, we found that HDA19 interacts with proteins involved in chromatin remodelling, nuclear protein export/import, protein ubiquitination associated with DNA damage repair, and chloroplast-nucleus signalling, suggesting a wide range of largely unexplored functions of HDA19 in etiolated Arabidopsis seedlings.
FIGURE 1 Identification of HDA19-interacting proteins.(A) Experimental flow of the immunoprecipitation analysis.(B) Overrepresented GO categories of putative nuclear-localised HDA19 interactors.Plot color indicates the log10 P value of enrichment, the size indicates the frequency of the GO term in the underlying GO annotation database (plots of more general terms are larger).(C) PPI network of putative nuclear-localised HDA19 interactors.

TABLE 1
Putative nuclear-localized binding partners of HDA19.
Localization of the proteins was gathered from published experimental evidence or prediction inferred from the Subcellular Location of Proteins in Arabidopsis Database (SUBA).