Inositol polyphosphate multikinase physically binds to the SWI/SNF complex and modulates BRG1 occupancy in mouse embryonic stem cells
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Republic of Korea
- Department of Chemistry, Seoul National University, Republic of Korea
- UNIST Central Research Facilities (UCRF), Ulsan National Institute of Science and Technology (UNIST), Republic of Korea
- KAIST Stem Cell Center, KAIST, Republic of Korea
- KAIST Institute for the BioCentury, KAIST, Republic of Korea
Figures
Figure 1 with 1 supplement
Identification of SMARCB1 as an IPMK-interacting protein via unbiased screening assays.
(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 …
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Figure 1—source data 1
Full and unedited images corresponding to panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig1-data1-v2.pdf
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Figure 1—source data 2
Primary data for graphs in panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Various subunits of the SWI/SNF complex and histones are IPMK-proximal/interacting proteins.
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 …
Figure 2 with 1 supplement
IPMK binds to SMARCB1 and other components of the SWI/SNF complex.
(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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Figure 2—source data 1
Full and unedited blots corresponding to panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-data1-v2.zip
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Figure 2—source data 2
Full and unedited blots corresponding to panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-data2-v2.zip
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Figure 2—source data 3
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-data3-v2.zip
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Figure 2—source data 4
Full and unedited blots corresponding to panel D.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-data4-v2.zip
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Figure 2—source data 5
Full and unedited blots corresponding to panel E.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-data5-v2.zip
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Figure 2—source data 6
Full and unedited blots corresponding to panel F.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-data6-v2.zip
Figure 2—figure supplement 1
IPMK physically binds to SMARCB1 and other subunits of the SWI/SNF complex.
(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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Figure 2—figure supplement 1—source data 1
Full and unedited blots corresponding to panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data1-v2.zip
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Figure 2—figure supplement 1—source data 2
Full and unedited blots corresponding to panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data2-v2.zip
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Figure 2—figure supplement 1—source data 3
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data3-v2.zip
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Figure 2—figure supplement 1—source data 4
Full and unedited blots corresponding to panel D.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data4-v2.zip
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Figure 2—figure supplement 1—source data 5
Full and unedited blots corresponding to panel E.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data5-v2.zip
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Figure 2—figure supplement 1—source data 6
Full and unedited blots corresponding to panel F.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data6-v2.zip
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Figure 2—figure supplement 1—source data 7
Full and unedited blots corresponding to panel G.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data7-v2.zip
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Figure 2—figure supplement 1—source data 8
Full and unedited blots and gels corresponding to panel H.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data8-v2.zip
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Figure 2—figure supplement 1—source data 9
Full and unedited blots and gels corresponding to panel I.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig2-figsupp1-data9-v2.zip
Figure 3 with 1 supplement
Identification of the distinct domains required for IPMK-SMARCB1 interactions.
(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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Figure 3—source data 1
Full and unedited blots corresponding to panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-data1-v2.zip
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Figure 3—source data 2
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-data2-v2.zip
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Figure 3—source data 3
Full and unedited blots corresponding to panel D.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-data3-v2.zip
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Figure 3—source data 4
Full and unedited blots corresponding to panel F.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-data4-v2.zip
Figure 3—figure supplement 1
Domain maps of the interaction between IPMK and SMARCB1.
(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 …
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Figure 3—figure supplement 1—source data 1
Full and unedited images corresponding to panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 1—source data 2
Full and unedited blots corresponding to panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-figsupp1-data2-v2.zip
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Figure 3—figure supplement 1—source data 3
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-figsupp1-data3-v2.zip
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Figure 3—figure supplement 1—source data 4
Full and unedited blots corresponding to panel D.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-figsupp1-data4-v2.zip
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Figure 3—figure supplement 1—source data 5
Full and unedited blots corresponding to panel E.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig3-figsupp1-data5-v2.zip
Figure 4 with 2 supplements
IPMK-BRG1 co-localize at promoters-TSSs/enhancers, and IPMK regulates the genomic localization of BRG1.
(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 …
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Figure 4—source data 1
Primary data for graph in panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-data1-v2.xlsx
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Figure 4—source data 2
Primary data for graph in panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Chromatin fraction assay and BRG1 at enhancers.
(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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Figure 4—figure supplement 1—source data 1
Full and unedited blots corresponding to panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-figsupp1-data1-v2.zip
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Figure 4—figure supplement 1—source data 2
Full and unedited blots corresponding to panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-figsupp1-data2-v2.zip
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Figure 4—figure supplement 1—source data 3
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-figsupp1-data3-v2.zip
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Figure 4—figure supplement 1—source data 4
Full and unedited blots corresponding to panel D.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-figsupp1-data4-v2.zip
Figure 4—figure supplement 2
mRNA expression of SWI/SNF complex subunits, replicates of BRG1 CUT&RUN and ATAC-seq, and IPMK rescue experiments.
(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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Figure 4—figure supplement 2—source data 1
Full and unedited blots corresponding to panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-figsupp2-data1-v2.zip
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Figure 4—figure supplement 2—source data 2
Full and unedited blots corresponding to panel F.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig4-figsupp2-data2-v2.zip
Figure 5 with 1 supplement
IPMK affects chromatin accessibility at promoter-TSS by regulating BRG1 localization.
(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 …
Figure 5—figure supplement 1
IPMK plays an important role in the maintenance of chromatin accessibility at promoter-TSS by regulating BRG1 localization.
(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 …
Figure 6 with 1 supplement
Alterations in BRG1/ATAC upon IpmkKD affect gene expression.
(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 …
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Figure 6—source data 1
Primary data for graph in panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig6-data1-v2.xlsx
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Figure 6—source data 2
Primary data for graph in panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig6-data2-v2.xlsx
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Figure 6—source data 3
Primary data for graph in panel E.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig6-data3-v2.xlsx
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Figure 6—source data 4
Primary data for graphs in panel F.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig6-data4-v2.xlsx
Figure 6—figure supplement 1
Alterations in BRG1/ATAC upon IpmkKD affect gene expression.
(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) …
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Figure 6—figure supplement 1—source data 1
Primary data for graphs in panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 1 supplement
Effect of IPMK on the expression of endodermal marker genes and our proposed model depicting the function of IPMK.
(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. …
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Figure 7—source data 1
Primary data for graphs in panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig7-data1-v2.xlsx
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Figure 7—source data 2
Primary data for graphs in panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig7-data2-v2.xlsx
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Figure 7—source data 3
Primary data for graphs in panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig7-data3-v2.xlsx
Figure 7—figure supplement 1
Effect of IPMK and BRG1 on ectodermal and mesodermal marker genes and proposed model depicting the physical interaction of IPMK and nucleosome-bound SWI/SNF complex.
(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 …
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Figure 7—figure supplement 1—source data 1
Primary data for graphs in panel A.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig7-figsupp1-data1-v2.xlsx
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Figure 7—figure supplement 1—source data 2
Primary data for graphs in panel B.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig7-figsupp1-data2-v2.xlsx
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Figure 7—figure supplement 1—source data 3
Primary data for graphs in panel C.
- https://cdn.elifesciences.org/articles/73523/elife-73523-fig7-figsupp1-data3-v2.xlsx
Author response image 1
Tables
Appendix 1—key resources table
| 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 |
Author response table 1
Table shows the actual values and calculated significance corresponding to Author response image 1.
| 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 |
Additional files
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Supplementary file 1
Yeast two-hybrid screening assay using IPMK as bait.
- https://cdn.elifesciences.org/articles/73523/elife-73523-supp1-v2.xlsx
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Supplementary file 2
Enriched protein complex-based sets from APEX2-mediated proximity labeling.
- https://cdn.elifesciences.org/articles/73523/elife-73523-supp2-v2.xlsx
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Supplementary file 3
Appendix_ Sequence-based reagents.
- https://cdn.elifesciences.org/articles/73523/elife-73523-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/73523/elife-73523-transrepform1-v2.docx
