Inositol polyphosphate multikinase physically binds to the SWI/SNF complex and modulates BRG1 occupancy in mouse embryonic stem cells

  1. Jiyoon Beon
  2. Sungwook Han
  3. Hyeokjun Yang
  4. Seung Eun Park
  5. Kwangbeom Hyun
  6. Song-Yi Lee
  7. Hyun-Woo Rhee
  8. Jeong Kon Seo
  9. Jaehoon Kim
  10. Seyun Kim  Is a corresponding author
  11. Daeyoup Lee  Is a corresponding author
  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Republic of Korea
  2. Department of Chemistry, Seoul National University, Republic of Korea
  3. UNIST Central Research Facilities (UCRF), Ulsan National Institute of Science and Technology (UNIST), Republic of Korea
  4. KAIST Stem Cell Center, KAIST, Republic of Korea
  5. KAIST Institute for the BioCentury, KAIST, Republic of Korea
8 figures, 2 tables and 4 additional files

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 …

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 …

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
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
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
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
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
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 …

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
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
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
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
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
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
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
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
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 …

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
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
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
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 …

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
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
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
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
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 …

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 …

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
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
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
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 …

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
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 …

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) …

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. …

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 …

Author response image 1

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo-sapiens)IPMKOpen BiosystemsNCBI Gene ID 253430
Gene (Homo-sapiens)SMARCB1BioneerNCBI Gene ID 6598
Strain, strain background (Escherichia coli)DH5αEnzynomicsCat# CP010
Cell line (Saccharomyces cerevisiae)AH109ClontechSee Materials and methods.
Cell line maintained in Panbionet.
Cell line (M. musculus)E14Tg2aATCCCat#
CRL-1821
Cell line (M. musculus)MEFThis paperSee Materials and methods.
Cell line maintained in S. Kim lab.
Cell line (M. musculus)NIH3T3ATCCCat#
CRL-1658
Cell line (Homo-sapiens)HEK293TATCCCat#
CRL-3216
Cell line (Homo-sapiens)Flp-In T-REx–293InvitrogenCat# R78007
Cell line (Spodoptera frgiperda)Sf9InvitrogenCat# 11496015
Transfected construct (M. musculus)siRNA to EgfpBioneertransfected construct (mouse)
Transfected construct (M. musculus)siRNA to IpmkBioneertransfected construct (mouse)
Transfected construct (M. musculus)siRNA to Smarcb1Dharmacon/Thermo Fisher ScientificCat# 4390771transfected construct (mouse)
Transfected construct (M. musculus)siRNA to Brg1Bioneertransfected construct (mouse)
Antibodyanti-FLAG (Mouse monoclonal)Sigma AldrichCat# F1804WB (1:1000)
Antibodyanti-SMARCB1
(Rabbit polyclonal)
BethylCat# A301-087WB (1:1000)
IP (3 ug)
Antibodyanti-BRG1
(Rabbit monoclonal)
AbcamCat# Ab110641WB (1:1000)
Antibodyanti-BAF155 (Rabbit monoclonal)Cell Signaling TechnologyCat# 11,956WB (1:1000)
Antibodyanti-BAF170 (Rabbit monoclonal)Cell Signaling TechnologyCat# 12,760WB (1:1000)
Antibodyanti-BAF250A (Rabbit monoclonal)Cell Signaling TechnologyCat# 12,354WB (1:1000)
Antibodyanti-PBAF/PBRM (Rabbit polyclonal)BethylCat# A301-591AWB (1:1000)
Antibodyanti-
α-Tubulin (Mouse monoclonal)
Sigma AldrichCat# T5169WB (1:3000)
Antibodyanti-IPMK (Rabbit polyclonal)custom rabbit polyclonalDOI: 10.1126/sciadv.1602296WB (1:1000)
IP (3 ug)
Antibodyanti-GST (Rabbit polyclonal)Cell Signaling TechnologyCat# 2,622WB (1:1000)
Antibodyanti-Histone H3 (mouse monoclonal)Sigma Aldrich05–499WB (1:3000)
Antibodyanti-GAPDH (mouse monoclonal)Santa Cruz BiotechCat# sc-32233WB (1:3000)
Antibodyanti-LaminB1 (mouse monoclonal)Santa Cruz BiotechCat# sc-365214WB (1:1000)
Antibodyanti-DPF2 (rabbit polyclonal)AbcamCat# ab128149WB (1:1000)
Antibodyanti-SMARCE1
(rabbit monoclonal)
AbcamCat# ab137081WB (1:1000)
Antibodyanti-SS18L1
(rabbit polyclonal)
AbcamCat# ab227535WB (1:1000)
Antibodyanti-ACTL6a (mouse monoclonal)Santa Cruz BiotechCat# sc-137062WB (1:1000)
Antibodyanti-SMARCD1 (mouse monoclonal)Santa Cruz BiotechCat# sc-135843WB (1:1000)
Antibodyanti-BCL7A (rabbit polyclonal)Atlas AntibodiesCat# HPA019762WB (1:1000)
Antibodyanti-ACTB (mouse monoclonal)OrigeneCat# TA811000WB (1:1000)
Antibodyrabbit IgG isotype control (rabbit isotype control)Invitrogen02–6102IP (3 ug)
Recombinant DNA reagentpGBKT7-GAL4-DNA-BD-fused IPMK
(plasmid)
This paperSee Materials and methods.
IPMK cloned into BD-containing
pGBKT7 vector.
Recombinant DNA reagenthuman brain cDNA activation domain (AD) libraryClontechCat# 630,486
Recombinant DNA reagentpLKO.1-hygro (plasmid)AddgeneCat# 24,150
Recombinant DNA reagentpcDNA5-Mito -V5-APEX2 (plasmid)This paperSee Materials and methods.
Kindly provided by H. Rhee lab.
Recombinant DNA reagentpcDNA5/FRT/TO vector (plasmid)InvitrogenCat# V652020
Recombinant DNA reagentpOG44 Flp recombinase expression vector (plasmid)InvitrogenCat# V600520
Recombinant DNA reagentpCMV-GST (plasmid)This paperDOI: 10.1126/sciadv.1602296See Materials and Methods.
Recombinant DNA reagentpcDNA3.1-FLAG (plasmid)This paperDOI: 10.1126/sciadv.1602296See Materials and methods
Recombinant DNA reagentpGEX4T (plasmid)Sigma AldrichCat#
GE28-9545-52
Recombinant DNA reagentpFASTBAC1 (plasmid)Thermo Fisher ScientificCat#
10359016
Recombinant DNA reagentFLAG-DPF2-pCAG-IP (plasmid)This paperSee Materials and methods.
Kindly provided by J. Kim lab.
Sequence-based reagentIpmk shRNA #1Sigma AldrichTRC Clone ID TRCN0000360808.
Sequence-based reagentIpmk shRNA #2Sigma AldrichTRC Clone ID TRCN0000360733
Sequence-based reagentshNT_FThis paperOligo sequence used for cloning pLKO.1 shNT (negative control)CCGGTCCTAAG
GTTAAGTCGCCCTCG
CTCGAGCGAG
GGCGACTTAA
CCTTAGGTTTTTG
Sequence-based reagentshNT_RThis paperOligo sequence used for cloning pLKO.1 shNT (negative control)AATTCAAAAACCT
AAGGTTAAGTCGC
CCTCGCTCGAG
CGAGGGCG
ACTTAACCTTAGGA
Peptide,
recombinant
protein
StreptavidinThermo FisherCat# 434,302
Commercial assay or kitBradford protein assayBio-radCat# 5000006
Commercial assay or kitBCA assayThermo Fisher ScientificCat# 23,225
Commercial assay or kitTNT Quick Coupled Transcription/Translation SystemPromegaCat# L1170
Commercial assay or kitNEXTflex ChIP-seq Library kitBioo ScientificCat# 5143–02
Commercial assay or kitMagnetic mRNA isolation kitNEBCat# S1550S
Commercial assay or kitNEXTflex Rapid directional RNA-seq kitBioo ScientificCat# 5138–08
Chemical compound, drugDoxycyclineSigma AldrichCat# D9891
Chemical compound, drugHygromycin BGibcoCat#
10687010
Chemical compound, drugpuromycinGibcoCat#
A11138-03
Chemical compound, drugG418GibcoCat#
11811023
Chemical compound, drugDharmaFECT 1Thermo Fisher ScientificCat#
T-2001–03
Chemical compound, drugFUGENE HDPromegaCat# E2311
Chemical compound, drugLipofectamine LTX with Plus ReagentInvitrogenCat# 15338100
Chemical compound, drugTurbofectThermo Fisher ScientificCat# R0533
Software, algorithmSequest Sorcerer platformSage-N ResearchHomo sapiens protein sequence database (20,675 entries, UniProt)
Software, algorithmScaffoldProteome Software Inc.Version 4.11.0
Software, algorithmProtein Prophet algorithmProtein Prophet algorithmDOI: 10.1021/ac0341261
Author response table 1
Table shows the actual values and calculated significance corresponding to Author response image 1.
GenesFigure 6Relative ExpressionLog2 (vs EgfpKD)P-value (vs EgfpKD)
EgfpKDlpmkKDSmarcb1KDlpmkKDSmarcb1KDlpmkKDSmarcb1KD
Rasl12A10.2724765470.538887496-1.8758-0.8919470.001450.08695
Phactr3B and E10.5820573480.480741151-0.780767-1.056670.052650.0278
Lrrc61F10.4909010760.604801519-1.0265-0.7254630.002150.01485
Arhgap44F10.6022658710.491195499-0.731528-1.025630.030350.04435

Additional files

Download links