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Defining Proximity Proteomics of Post-Translationally Modified Proteins by Antibody-Mediated Protein A-APEX2 Labeling

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Defining Proximity Proteomics of Post-Translationally Modified Proteins by Antibody-Mediated Protein A-APEX2 Labeling Defining proximity proteomics of post-translationally modified proteins by antibody-mediated protein A-APEX2 labeling Xinran Li1#, Jiaqi Zhou1#, Wenjuan Zhao1#, Qing Wen1#, Weijie Wang1, Huipai Peng1, Kelly J. Bouchonville3, Steven M. Offer2, 3, 4,5, Zhiquan Wang2*, Nan Li1*, Haiyun Gan1* 1 Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China 2 Devision of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN 55905 USA 3 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905 USA 4 Mayo Clinic College of Medicine, Rochester, MN 55905 USA 5 Mayo Clinic Cancer Center, Rochester, MN 55905 USA # These authors contributed equally to this work. * Corresponding author * Zhiquan Wang: E-mail address: [email protected]. * Nan Li: E-mail address: [email protected]. * Haiyun Gan: E-mail address: [email protected]; Tel: +86-0755-26653824 Supplementary Figures: a b BSA(ng) pA-APEX2 (µl) (1:5 dilution) 0.5 1 2 3 4 5 180 180 130 130 100 100 Expected Sizes: pA-APEX2 70 BSA:66.5 KDa 70 55 pA-APEX2 before DTT:~73 KDa 55 pA-APEX2 after DTT:~48 KDa 40 40 35 35 25 25 15 15 c d e * * * * * * Ponceau S Streptavidin-HRP Ponceau S Streptavidin-HRP Ponceau S Streptavidin-HRP Supplementary Figure 1. Purified pA-APEX is enzymatically active toward whole-cell lysate. a, Purification process of pA-APEX2. b, the concentration of pA-APEX2 was determined by BSA standards, 4μg/μl. c, pA-APEX2 labeling in vitro. pA-APEX2 and indicated supplements for the peroxidase reaction were incubated with whole-cell lysates as indicated, and the reaction product was analyzed by western blot. d, Titration of reaction time and supplements for the pA-APEX2 reaction. e, Western blot analysis of H3K27me3-mediated pA-APEX2 proximity labeling protein in living cells by change labeling time or secondary antibody, Whole-cell lysate was analyzed by Ponceau Stain (left) and Western blot (right). “*” denote endogenous biotinylated proteins. H3K27me3 log2(fc) > 1 Methyltransferase log2(fc) > 0 Histone deacetylase complex Transcription elongation factor Transferase complex Transcriptional repressor complex Methyltransferase complex THO complex MLL1 complex Histone methyltransferase complex Replication Replication fork Transcription regulator complex fork Transcription export complex Histone deacetylation Transcriptional repressor Transcription Transcription regulator export Supplementary Figure 2. The proximal proteome of H3K27me3-interacting proteins identified by AMAPEX. Network analysis of H3K27me3 interactomes according to the major cellular component GO terms (N proteins = 104, of all 484). Individual proteins are shown as nodes, and interactions are shown as edges. The interactions were retrieved from the STRING database with interaction score > 0.4. a Arid2 ChIP-Seq b Arid2 H3K27me3 Brd7 ChIP-Seq 2415 526 988 H3K27me3 ChIP-Seq SRGAP2D c Arid2 ChIP-Seq d Brd7 H3K27me3 Brd7 ChIP-Seq 4410 395 1140 H3K27me3 ChIP-Seq FRG2C LINC00960 e Nsd2 ChIP-Seq f Nsd2 H3K27me3 H3K27me3 ChIP-Seq 294 270 452 ARHGEF18 Supplementary Figure 3. The interactions between H3K27me3 and Arid2, Brd7, or Nsd2 are verified by ChIP-seq. a, c, e, Representative co-localization of ChIP-seq peaks between H3K27me3 and Arid2, Brd7 or Nsd2. b, d, f, Venn diagrams showing the number of H3K27me3 peaks overlapping with Arid2, Brd7, or Nsd2 peaks. The ChIP-seq datasets for Arid2, Brd7, and H3K27me3 are from MCF-7 cells. The ChIP-seq dataset for Nsd2 and H3K27me3 are from K-562 cells. All of the bigwig files and peak files were download from https://chip-atlas.org/. a b c * * * * * * Supplementary Figure 4. Antibody-mediated specific biotin labeling by pA-APEX2. MEF cells were briefly crosslinked with 0.1% formaldehyde before the antibody-directed pA- APEX2 biotinylation reaction. Whole cell lysates were extracted, and biotinylated proteins were purified using streptavidin beads. Whole-cell lysates (input), flow throughs, and immunoprecipitated (IP) proteins were analyzed by western blot. a, H3K4me3, b, H3K9me3, c, H4K12ac. “*” denote endogenous biotinylated proteins. - H2O2 IgG BAC-GFP ChromID +H2O2 PcG protein complex Nuclear pore a d Nuclear ubiquitin ligase complex log2(fc) > 1 Rif1 Identified MCM complex Orc3 Replication fork Transferase complex log2(fc) > 0 Pogz Not identified Kinesin complex Spliceosomal complex LRWD1 Methyltransferase complex Cbx3 Ahdc1 Champ1 Methyltransferase complex Wiz Rnf2 1xe11 Smchd1 Zmym4 b H3K9me3 Hnrnpa2b1 PCC = 0.97 Nono Sfpq Uhrf1 2 Ncl Hnrnpab Cbx5 Ubiquitin ligase complex Hells Ptbp1 Phc2 Replication Setdb1 Orc1 fork Ring1 of RepeatIntensities Adnp Bmi1 Atrx MCM complex Chaf1b Dnmt1 Chd4 c Intensities of Repeat 1 1xe10 Cbx8 Ehmt1 Chaf1a Chromatin Cbx4 Chromosomal region Lrif1 Nuclear body Mecp2 Nuclear periphery Hnrnpr Spindle Trip12 Nuclear chromosome MCM complex Tpx2 Spliceosomal complex Top2a Replication fork Kinesin complex Spliceosomal complex Shprh Nuclear ubiquitin ligase complex Usp3 Cytoplasmic stress granule Sgo2 Kinesin complex Zfp292 Protein serine/threonine phosphatase complex Cbx2 Condensed chromosome Mki67 Nuclear pore Numa1 Nuclear inclusion body Hdgfl2 Synaptobrevin 2-SNAP-25-syntaxin-1a complex Brwd1 DNA repair complex Baz1b Nucleolar part Tmpo Nuclear exosome (RNase complex) Adnp2 Nuclear pore Psip1 Zfp512b Supplementary Figure 5. The proximal proteome of H3K9me3 identified by AMAPEX. a, H3K9me3-interacting proteins identified by AMAPEX. Heatmap showing the enrichment as log2- fold intensity change of interacting proteins relative to the controls as indicated. Data are shown as Z scores. Blocks in blue represent the enrichment of proteins identified by ChromID and BAC-GFP in previous publications. b, The reproducibility of two biological replicates of pA-APEX2 experiments in the identification of H3K9me3-interacting proteins was calculated by Pearson correlation coefficient (PCC). c, The top 20 enriched cellular component GO terms for the H3K9me3 proximal proteins. Bar plots represent the -log10 (p value) of the enriched terms. d, Network analysis of H3K9me3 interactomes according to the major cellular component GO terms (N proteins = 83, of all 486). Individual proteins are shown as nodes, and interactions are shown as edges. The interactions were retrieved from the STRING database with interaction score > 0.4. Proteins were selected based on min 1.0 log2-FC in two pA-APEX2 experiments. Proteins detected in pA-APEX2 experiments with log2-FC > 1 are shown as orange nodes, proteins with log2-FC > 0 and detected in ChromID or BAC- GFP are shown as light-yellow nodes, and proteins detected in ChromID or BAC-GFP but not in pA- APEX2 experiments are shown as white nodes. Transcription regulator - H O IgG DNA repair complex log2(fc) > 1 BAC-GFP ChromID +H2O2 2 2 a b d Transferase complex Spliceosomal complex log2(fc) > 0 Sin3a PML body Dido1 beta-catenin-TCF complex Bptf 1xe10 SWI/SNF superfamily-type complex Rbbp4 Spliceosome Chd1 H3K4me3 Methyltransferase complex Kmt2a PCC = 0.94 Transcription elongation factor Hdac1 Tcf20 U2-type precatalytic spliceosome Usp36 SWI/SNF complex Ruvbl1 Srfbp1 Nuclear speck Wiz Znf148 2 Repeat Transcription regulator Rnf2 Wdr5 Tet1 Xrcc1 Nsd3 Sap130 Intensitiesof Kdm2b Hcfc1 Mki67 Klf4 Nop14 Kdm2a Nop2 1xe10 Pbrm1 Intensities of Repeat 1 Morc3 Ppp1r10 Morc2a Rsl1d1 Nipbl c Orc1 PML body beta-catenin-TCF Paf1 Chromatin Phf2 Nuclear body Nol8 Spliceosomal complex Supt6h Prp19 complex DNA repair Bcor Nuclear periphery Ddx21 Histone deacetylase complex Jmjd1c Euchromatin Atad5 Chromosomal region Nuclear speck Atrx Beta-catenin-TCF complex Ing5 PML body Med1 Transcription elongation factor complex Ino80 U2-type prespliceosome Ing4 Ints12 Integrator complex Ankrd11 Cytoplasmic ribonucleoprotein granule Ddx18 Chromosome, telomeric region Brd4 Germ cell nucleus Aff4 DNA repair complex Cxxc1 Mediator complex Ep300 Sex chromosome Dek Ash2l Arid2 Methyltransferase SWI/SNF complex Identified Not identified Supplementary Figure 6. The proximal proteome of H3K4me3 identified by AMAPEX. a, H3K4me3-interacting proteins identified by AMAPEX. Heatmap showing the enrichment as log2- fold intensity change of interacting proteins relative to the controls as indicated. Data are shown as Z scores. Blocks in blue represent the enrichment of proteins identified by ChromID and BAC-GFP in previous publications. b, The reproducibility of two biological replicates of pA-APEX2 experiments identifying H3K4me3-interacting proteins. The Pearson correlation coefficient (PCC) between two replicates was calculated. c, The top 20 enriched cellular component GO terms for the H3K4me3- proximal proteins. Bar plots represent the -log10(p value) of the enriched terms. d, Network analysis of H3K4me3 interactomes according to the major cellular component GO terms (N proteins = 91, of all 193). Individual proteins are shown as nodes, and interactions are shown as edges. The interactions were retrieved from the STRING database with interaction score > 0.4. Proteins were selected based on min 1.0 log2-FC in two pA-APEX2 experiments. Proteins detected in pA-APEX2 experiments with log2-FC > 1 are shown as orange nodes, proteins with log2-FC > 0 and detected in ChromID or BAC-GFP are shown as light-yellow nodes, and proteins detected in ChromID or BAC- GFP but not in pA-APEX2 experiments
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