(A) IPMK and SMARCB1 interaction test in yeast strain AH109 containing two reporter genes (ADE2 and HIS3). Yeast cells were co-transformed with either the GAL4-BD fusion plasmid pGBKT7 or …
Full and unedited images corresponding to panel A.
Primary data for graphs in panel C.
A volcano plot showing the relative abundance and significance (P-value) of biotinylated proteins related to the SWI/SNF complex, histones, and two negative controls (right). A dotted line within …
(A) Purified IPMK and in vitro translated FLAG-SMARCB1 were co-incubated, immunoprecipitated with an anti-FLAG antibody, and then subjected to immunoblotting. (B) Sf9 insect cells were co-infected …
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(A) IPMK and IgG were immunoprecipitated from NIH3T3 cells and then subjected to immunoblotting. (B) IPMK and IgG were immunoprecipitated from NIH3T3 cells in the presence of DSP, an established …
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(A) Schematic diagram showing the human SMARCB1 fragments used for the binding studies (B and C). The IPMK-binding sites (Rpt1 and Rpt2) are highlighted in red. (B) HEK293T cells were co-transfected …
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(A) IPMK and SMARCB1 domain interaction test in yeast strain AH109 containing the HIS3 reporter gene. Yeast cells were co-transformed with either the GAL4-BD fusion plasmid pGBKT7 or pGBKT7-IPMK and …
Full and unedited images corresponding to panel A.
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(A) RT-qPCR analysis of Ipmk expression after siRNA treatment. Error bars denote the standard error of the mean obtained from four biological replicates. The expression levels were normalized to the …
Primary data for graph in panel A.
Primary data for graph in panel C.
(A) E14Tg2a cells were fractionated into cytoplasm, nucleoplasm, and chromatin fractions. Immunoblotting was performed with IPMK, SMARCB1, and fractionation markers. (B) E14Tg2a cells were …
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(A) Heatmaps representing CUT&RUN results for BRG1 replicates (EgfpKD and IpmkKD) at BRG1 CUT&RUN peaks (EgfpKD cells) as indicated at the top. All heatmaps were aligned at 34,771 BRG1 CUT&RUN peaks …
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(A) Line plots showing the average enrichment of BRG1, nucleosome (MNase-seq, GSM5253962, and GSM5253963), and ATAC-seq signals (ATAC) at the TSSs of total genes (left) and TSSs of three promoter …
(A) Bar graphs showing the average differential BRG1 intensity (Log2 shIpmk1,2/shNT) upon shRNA-mediated Ipmk silencing at merged TSS clusters (five TSS clusters in Figure 5) with two promoter …
(A and B) Examples of BRG1 (EgfpKD and IpmkKD), IPMK CUT&RUN, and ATAC-seq (EgfpKD, IpmkKD, Control, and Brg1KD) assays at the TSSs of Rasl12 (A) and Phactr3 (B). The TSSs are marked with red boxes …
Primary data for graph in panel A.
Primary data for graph in panel B.
Primary data for graph in panel E.
Primary data for graphs in panel F.
(A) Examples of BRG1 (EgfpKD and IpmkKD), IPMK CUT&RUN, and ATAC-seq (EgfpKD, IpmkKD, Control, and Brg1KD) assays at the TSS of Me2 (left) and Sfrp2 (right). TSSs are marked with red boxes (bottom) …
Primary data for graphs in panel A.
(A and B) RT-qPCR analysis of the expression of pluripotency (A) and endodermal (B) marker genes after shRNA-mediated Ipmk silencing at the indicated time points during embryoid body (EB) formation. …
Primary data for graphs in panel A.
Primary data for graphs in panel B.
Primary data for graphs in panel C.
(A and B) RT-qPCR analysis of ectodermal (A) and mesodermal (B) marker expression after shRNA-mediated Ipmk silencing at the indicated time points during EB formation. Data are representative of at …
Primary data for graphs in panel A.
Primary data for graphs in panel B.
Primary data for graphs in panel C.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Homo-sapiens) | IPMK | Open Biosystems | NCBI Gene ID 253430 | |
Gene (Homo-sapiens) | SMARCB1 | Bioneer | NCBI Gene ID 6598 | |
Strain, strain background (Escherichia coli) | DH5α | Enzynomics | Cat# CP010 | |
Cell line (Saccharomyces cerevisiae) | AH109 | Clontech | See Materials and methods. Cell line maintained in Panbionet. | |
Cell line (M. musculus) | E14Tg2a | ATCC | Cat# CRL-1821 | |
Cell line (M. musculus) | MEF | This paper | See Materials and methods. Cell line maintained in S. Kim lab. | |
Cell line (M. musculus) | NIH3T3 | ATCC | Cat# CRL-1658 | |
Cell line (Homo-sapiens) | HEK293T | ATCC | Cat# CRL-3216 | |
Cell line (Homo-sapiens) | Flp-In T-REx–293 | Invitrogen | Cat# R78007 | |
Cell line (Spodoptera frgiperda) | Sf9 | Invitrogen | Cat# 11496015 | |
Transfected construct (M. musculus) | siRNA to Egfp | Bioneer | transfected construct (mouse) | |
Transfected construct (M. musculus) | siRNA to Ipmk | Bioneer | transfected construct (mouse) | |
Transfected construct (M. musculus) | siRNA to Smarcb1 | Dharmacon/Thermo Fisher Scientific | Cat# 4390771 | transfected construct (mouse) |
Transfected construct (M. musculus) | siRNA to Brg1 | Bioneer | transfected construct (mouse) | |
Antibody | anti-FLAG (Mouse monoclonal) | Sigma Aldrich | Cat# F1804 | WB (1:1000) |
Antibody | anti-SMARCB1 (Rabbit polyclonal) | Bethyl | Cat# A301-087 | WB (1:1000) IP (3 ug) |
Antibody | anti-BRG1 (Rabbit monoclonal) | Abcam | Cat# Ab110641 | WB (1:1000) |
Antibody | anti-BAF155 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 11,956 | WB (1:1000) |
Antibody | anti-BAF170 (Rabbit monoclonal) | Cell Signaling Technology | Cat# 12,760 | WB (1:1000) |
Antibody | anti-BAF250A (Rabbit monoclonal) | Cell Signaling Technology | Cat# 12,354 | WB (1:1000) |
Antibody | anti-PBAF/PBRM (Rabbit polyclonal) | Bethyl | Cat# A301-591A | WB (1:1000) |
Antibody | anti- α-Tubulin (Mouse monoclonal) | Sigma Aldrich | Cat# T5169 | WB (1:3000) |
Antibody | anti-IPMK (Rabbit polyclonal) | custom rabbit polyclonal | DOI: 10.1126/sciadv.1602296 | WB (1:1000) IP (3 ug) |
Antibody | anti-GST (Rabbit polyclonal) | Cell Signaling Technology | Cat# 2,622 | WB (1:1000) |
Antibody | anti-Histone H3 (mouse monoclonal) | Sigma Aldrich | 05–499 | WB (1:3000) |
Antibody | anti-GAPDH (mouse monoclonal) | Santa Cruz Biotech | Cat# sc-32233 | WB (1:3000) |
Antibody | anti-LaminB1 (mouse monoclonal) | Santa Cruz Biotech | Cat# sc-365214 | WB (1:1000) |
Antibody | anti-DPF2 (rabbit polyclonal) | Abcam | Cat# ab128149 | WB (1:1000) |
Antibody | anti-SMARCE1 (rabbit monoclonal) | Abcam | Cat# ab137081 | WB (1:1000) |
Antibody | anti-SS18L1 (rabbit polyclonal) | Abcam | Cat# ab227535 | WB (1:1000) |
Antibody | anti-ACTL6a (mouse monoclonal) | Santa Cruz Biotech | Cat# sc-137062 | WB (1:1000) |
Antibody | anti-SMARCD1 (mouse monoclonal) | Santa Cruz Biotech | Cat# sc-135843 | WB (1:1000) |
Antibody | anti-BCL7A (rabbit polyclonal) | Atlas Antibodies | Cat# HPA019762 | WB (1:1000) |
Antibody | anti-ACTB (mouse monoclonal) | Origene | Cat# TA811000 | WB (1:1000) |
Antibody | rabbit IgG isotype control (rabbit isotype control) | Invitrogen | 02–6102 | IP (3 ug) |
Recombinant DNA reagent | pGBKT7-GAL4-DNA-BD-fused IPMK (plasmid) | This paper | See Materials and methods. IPMK cloned into BD-containing pGBKT7 vector. | |
Recombinant DNA reagent | human brain cDNA activation domain (AD) library | Clontech | Cat# 630,486 | |
Recombinant DNA reagent | pLKO.1-hygro (plasmid) | Addgene | Cat# 24,150 | |
Recombinant DNA reagent | pcDNA5-Mito -V5-APEX2 (plasmid) | This paper | See Materials and methods. Kindly provided by H. Rhee lab. | |
Recombinant DNA reagent | pcDNA5/FRT/TO vector (plasmid) | Invitrogen | Cat# V652020 | |
Recombinant DNA reagent | pOG44 Flp recombinase expression vector (plasmid) | Invitrogen | Cat# V600520 | |
Recombinant DNA reagent | pCMV-GST (plasmid) | This paper | DOI: 10.1126/sciadv.1602296 | See Materials and Methods. |
Recombinant DNA reagent | pcDNA3.1-FLAG (plasmid) | This paper | DOI: 10.1126/sciadv.1602296 | See Materials and methods |
Recombinant DNA reagent | pGEX4T (plasmid) | Sigma Aldrich | Cat# GE28-9545-52 | |
Recombinant DNA reagent | pFASTBAC1 (plasmid) | Thermo Fisher Scientific | Cat# 10359016 | |
Recombinant DNA reagent | FLAG-DPF2-pCAG-IP (plasmid) | This paper | See Materials and methods. Kindly provided by J. Kim lab. | |
Sequence-based reagent | Ipmk shRNA #1 | Sigma Aldrich | TRC Clone ID TRCN0000360808 | . |
Sequence-based reagent | Ipmk shRNA #2 | Sigma Aldrich | TRC Clone ID TRCN0000360733 | |
Sequence-based reagent | shNT_F | This paper | Oligo sequence used for cloning pLKO.1 shNT (negative control) | CCGGTCCTAAG GTTAAGTCGCCCTCG CTCGAGCGAG GGCGACTTAA CCTTAGGTTTTTG |
Sequence-based reagent | shNT_R | This paper | Oligo sequence used for cloning pLKO.1 shNT (negative control) | AATTCAAAAACCT AAGGTTAAGTCGC CCTCGCTCGAG CGAGGGCG ACTTAACCTTAGGA |
Peptide, recombinant protein | Streptavidin | Thermo Fisher | Cat# 434,302 | |
Commercial assay or kit | Bradford protein assay | Bio-rad | Cat# 5000006 | |
Commercial assay or kit | BCA assay | Thermo Fisher Scientific | Cat# 23,225 | |
Commercial assay or kit | TNT Quick Coupled Transcription/Translation System | Promega | Cat# L1170 | |
Commercial assay or kit | NEXTflex ChIP-seq Library kit | Bioo Scientific | Cat# 5143–02 | |
Commercial assay or kit | Magnetic mRNA isolation kit | NEB | Cat# S1550S | |
Commercial assay or kit | NEXTflex Rapid directional RNA-seq kit | Bioo Scientific | Cat# 5138–08 | |
Chemical compound, drug | Doxycycline | Sigma Aldrich | Cat# D9891 | |
Chemical compound, drug | Hygromycin B | Gibco | Cat# 10687010 | |
Chemical compound, drug | puromycin | Gibco | Cat# A11138-03 | |
Chemical compound, drug | G418 | Gibco | Cat# 11811023 | |
Chemical compound, drug | DharmaFECT 1 | Thermo Fisher Scientific | Cat# T-2001–03 | |
Chemical compound, drug | FUGENE HD | Promega | Cat# E2311 | |
Chemical compound, drug | Lipofectamine LTX with Plus Reagent | Invitrogen | Cat# 15338100 | |
Chemical compound, drug | Turbofect | Thermo Fisher Scientific | Cat# R0533 | |
Software, algorithm | Sequest Sorcerer platform | Sage-N Research | Homo sapiens protein sequence database (20,675 entries, UniProt) | |
Software, algorithm | Scaffold | Proteome Software Inc. | Version 4.11.0 | |
Software, algorithm | Protein Prophet algorithm | Protein Prophet algorithm | DOI: 10.1021/ac0341261 |
Genes | Figure 6 | Relative Expression | Log2 (vs EgfpKD) | P-value (vs EgfpKD) | ||||
---|---|---|---|---|---|---|---|---|
EgfpKD | lpmkKD | Smarcb1KD | lpmkKD | Smarcb1KD | lpmkKD | Smarcb1KD | ||
Rasl12 | A | 1 | 0.272476547 | 0.538887496 | -1.8758 | -0.891947 | 0.00145 | 0.08695 |
Phactr3 | B and E | 1 | 0.582057348 | 0.480741151 | -0.780767 | -1.05667 | 0.05265 | 0.0278 |
Lrrc61 | F | 1 | 0.490901076 | 0.604801519 | -1.0265 | -0.725463 | 0.00215 | 0.01485 |
Arhgap44 | F | 1 | 0.602265871 | 0.491195499 | -0.731528 | -1.02563 | 0.03035 | 0.04435 |
Yeast two-hybrid screening assay using IPMK as bait.
Enriched protein complex-based sets from APEX2-mediated proximity labeling.
Appendix_ Sequence-based reagents.