Molecular Cell, Volume 46 Supplemental Information Integrative Genomics Identifies the Corepressor SMRT as a Gatekeeper of Adipogenesis through the Transcription Factors C/EBPand KAISO Sunil K. Raghav, Sebastian M. Waszak, Irina Krier, Carine Gubelmann, Alina Isakova, Tarjei S. Mikkelsen, and Bart Deplancke
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Molecular Cell, Volume 46
Supplemental Information
Integrative Genomics Identifies the Corepressor
SMRT as a Gatekeeper of Adipogenesis
through the Transcription Factors C/EBPand KAISO
Sunil K. Raghav, Sebastian M. Waszak, Irina Krier, Carine Gubelmann, Alina Isakova, Tarjei S. Mikkelsen, and Bart
Deplancke
Figure S1. SMRT KD Enhances Adipogenesis in 3T3-L1 Cells
Figure S1, Related to Figure 6 and 7. SMRT KD Enhances Adipogenesis in 3T3-L1
Cells
(A) Fold-change of SMRT mRNA expression in SMRT KD as compared to shRNA
control cells as measured by qPCR. Error bars represent the standard deviation
observed in three replicate experiments.
(B) Western blot to determine SMRT protein levels in the nuclear extracts of SMRT KD
compared to shRNA control cells. The figure constitutes a representative western
blot of three replicate experiments.
(C) Densitometric analysis of SMRT protein bands as observed in the western blot.
(D) Oil red-O staining of SMRT KD and shRNA control cells at D6. We observed
significantly enhanced differentiation in control versus SMRT KD cells, consistent
with previous results (Yu et al., 2005) and confirming the efficacy of the SMRT KD.
Figure S2. ChIP-qPCR Validation of Randomly Selected SMRT Peaks Derived from
ChIP-Seq Data
Figure S2, Related to Figure 1. ChIP-qPCR Validation of Randomly Selected SMRT
Peaks Derived from ChIP-Seq Data
(A) ChIP-qPCR for 15 randomly selected SMRT peak regions using SMRT KD and
shRNA control 3T3-L1 pre-adipocytes. The results shown are representative of two
ChIP-qPCR replicate experiments. Isotype match rabbit antibody and two genomic
regions not bound by SMRT were used to determine the non-specific ChIP
enrichment (see Supplemental Experimental Procedures for more experimental
detail and the list of primers used for qPCR).
(B) ChIP-qPCR for 25 randomly selected SMRT peak regions to validate the decrease in
SMRT binding after differentiation induction at D1. Isotype match rabbit antibody is
used to estimate the non-specific enrichment by beads used for ChIP and the
average enrichment (or lack thereof) of two genomic regions not bound by SMRT in
ChIP-Seq is used to subtract the non-specific ChIP enrichment by the SMRT
antibody (see Supplemental Experimental Procedures for more experimental detail
and the list of primers used for qPCR).
Figure S3. Position of De Novo Identified Motifs with Respect to SMRT Peak
Maxima and Further Validation of KAISO DNA Binding at SMRT Promoter-
Proximal Sites in 3T3-L1 Cells
Figure S3, Related to Figures 2 and 3. Position of De Novo Identified Motifs with
Respect to SMRT Peak Maxima and Further Validation of KAISO DNA Binding at
SMRT Promoter-Proximal Sites in 3T3-L1 Cells
(A) Positional distribution of de novo motifs with respect to SMRT peak maxima (see
Figure 2A for more details on motif statistics).
(B) SMRT tag density at regions bound by C/EBPβ at D0 and occupied by pro-
adipogenic TFs at 4h.
(C) Graph showing linear regression lines fitted to MITOMI data points corresponding to
each tested KAISO target probe sequence (Table S4D; see Supplemental
Experimental Procedures for more details). The data shown are representative of
three MITOMI replicate experiments (i.e. independent microfluidic chips).
(D) Fold decrease in Kaiso mRNA expression as determined by qPCR in KAISO KD as
compared to shRNA control cells. Error bars represent the standard deviation from
three replicate experiments.
(E) Western blot for NCoR1 and KAISO protein in NCoR1 antibody immunoprecipitated
cell lysate samples from pre-adipocytes (D0) and differentiated (D6) 3T3-L1 cells.
The presented western blot constitutes representative data from three replicate
experiments.
(F) SDS-PAGE followed by western blot for KAISO protein in SMRT antibody immuno-
precipitated cell lysate samples from shRNA control (empty vector treated), KAISO
KD, NCoR1 KD, and SMRT KD pre-adipocyte 3T3-L1 cells. Densitometric analyses
for detected KAISO bands are shown below the western blot panel. The presented
western blot constitutes representative data from two replicate experiments.
(G) SMRT ChIP in SMRT KD, KAISO KD, and shRNA control cells at promoter-
proximal SMRT binding sites containing a KAISO motif.
(H) KAISO ChIP-qPCR (using a KAISO-specific antibody from Abcam, cat no.: ab12723)
for 15 selected SMRT-bound promoter-proximal regions with a KAISO motif in
comparison to 15 SMRT-bound promoter-proximal or -distal regions lacking such a
motif. Error bars represent the standard deviation observed in two replicate
experiments.
Figure S4. Control Experiments to Examine the Significance of Open Chromatin
and Histone Mark Enrichment within SMRT-Bound Regions
Figure S4, Related to Figure 4. Control Experiments to Examine the Significance
of Open Chromatin and Histone Mark Enrichment within SMRT-Bound Regions
(A-E) Enrichment of randomized SMRT peaks within DNase I hypersensitive sites (A),
and H3K27ac (B), H3K27me3 (C), H3K4me1 (D), and H3K4me3 (E) marked
regions (see Supplemental Experimental Procedures for more details).
Figure S5. SMRT-Bound Regions Show Increased Chromatin Accessibility 24h
after 3T3-L1 Differentiation Induction and hence after SMRT Release
Figure S5, Related to Figure 4. SMRT-Bound Regions Show Increased Chromatin
Accessibility 24h after 3T3-L1 Differentiation Induction and hence after SMRT
Release
(A) Contour plot showing the dynamic changes in SMRT binding (as RPKM) and DNaseI
hypersensitive changes (i.e., chromatin accessibility) at all SMRT-bound regions in
pre-adipocytes (D0) and during differentiation (at 2h, D1, and D6). DHS data was
used from (Siersbaek et al., 2011). The degree of chromatin accessibility is
measured in terms of peak height observed at DNase I hypersensitive sites.
Horizontal and vertical line levels were arbitrarily chosen.
(B) Contour plot showing the dynamics of chromatin accessibility at SMRT D0 promoter-
proximal peak regions enriched for the KAISO motif.
Figure S6. Transcriptional Dynamics of All SMRT-Bound Genes
Figure S6, Related to Figure 5. Transcriptional Dynamics of All SMRT-Bound
Genes
(A-B) RNA pol II occupancy at promoters (-30 to +300 bp relative to TSS) of all
proximally or distally bound SMRT target genes over the course of terminal
adipogenesis.
(D-E) Gene body RNA pol II occupancy of all proximally or distally bound SMRT target
genes over the course of terminal adipogenesis.
(C-F) Randomly selected control genes show low promoter and gene body RNA pol II
densities.
Figure S7. KAISO KD Cells Exhibit Accelerated Cell Cycle Progression during the
Early Mitotic Clonal Expansion Phase of Terminal Adipogenesis
Figure S7, Related to Figure 6. KAISO KD Cells Exhibit Accelerated Cell Cycle
Progression during the Early Mitotic Clonal Expansion Phase of Terminal
Adipogenesis
(A-B) Propidium iodide (PI) staining-based FACS analysis of KAISO KD (A) and shRNA
control (B) cells during the first 24h after induction of differentiation. The presented
figure constitutes representative data from three replicate experiments.(C) Histograms
indicating the percentage of KAISO KD and shRNA control cells at each cell cycle
phase (G0/G1, S, and G2/M) during the first 24h after differentiation induction
(**P<0.01, *P<0.05; two-sided t-test). Error bars show the standard error of the mean
from three replicate experiments.
Figure S8. Simplified, Schematic Model of the Molecular Mechanisms Underlying
the Involvement of SMRT in Terminal Adipogenesis
Figure S8. Simplified, Schematic Model of the Molecular Mechanisms Underlying
the Involvement of SMRT in Terminal Adipogenesis
M indicates methylated DNA.
Supplemental Experimental Procedures
3T3-L1 Cell Culture and Differentiation
Mouse embryonic fibroblast-adipose like cells (cell line 3T3-L1) obtained from ATCC
were maintained in Dulbecco‟s modified Eagle‟s medium (DMEM, Invitrogen) containing
10% fetal calf serum (FCS; Amimed), 1X antibiotic solution (Invitrogen) and the cultures
were incubated at 37°C and 5% CO2. Cells were sub-cultured in 1:5 into new petri-
plates when they were 75-80% confluent. 3T3-L1 pre-adipocytes at 2 days post-
confluence were differentiated into adipocytes using differentiation inducing cocktail of
1µM Dexamethasone (Dex), 0.5mM isobutyl-methyl-xanthine (IBMX) and 167nM insulin
in DMEM with 10% FCS. After two days of induction with differentiation medium, cells
were washed with cell culture grade 1X phosphate buffered saline (PBS) and complete
medium containing 167nM insulin was added. Two days thereafter, fresh DMEM
medium containing FCS was added to the cells and at day six, cells were stained with
oil red-O to estimate the extent of differentiation into mature fat cells.
Chromatin Immunoprecipitation of SMRT, NCoR1, and RNA Polymerase II
Cells were collected from pre-adipocytes (D0) and at five distinct time points after
induction of differentiation (2h, D1, D4, and D6). The cells were washed two times with
1X PBS and cross-linked using 1% formaldehyde for 10 min at room temperature
followed by quenching the reaction using 125mM glycine for 5 min. After quenching, the
petri-plates were placed on ice, cells were scraped using a cell scraper and collected in
falcon tubes. The cells were then washed three times using cold 1X PBS and cell
pellets were stored at -80°C until further use. The cells were lysed in nuclei extraction
EDTA, 1mM DTT) supplemented with protease and phosphatase inhibitors (Roche).
Lysates were cleared by centrifuging at maximum speed in a tabletop refrigerated
centrifuge for 10 min. Antibodies (10 μg/IP) against SMRT (Abcam, ab-24551), NCoR1
(Abcam, ab-24552), and isotype matched rabbit antibody (Millipore) were added to the
cell lysate and incubated for 3h at 4°C while rotating. Protein-A sepharose beads (50µl)
that were washed and pre-blocked with BSA were then added to the samples and
further incubated for 2h to pull down the protein-antibody complex. The beads were
then washed 5 times with Triton X-100 IP buffer and once with 1X TE buffer. After the
last wash, beads containing immune-precipitated protein complexes were boiled in 60μl
of 2X SDS sample loading buffer. Proteins present in the eluted supernatant were
resolved on a 7.5% SDS-PAGE gel, and then transferred to a nitrocellulose membrane.
Immunoblotting was performed with anti-KAISO antibody (Abcam, ab-12723). To
confirm the SMRT and NCoR1 pull-down by the respective antibodies, the same blots
were also developed for SMRT and NCoR1. To further validate the presence of KAISO
in a SMRT-containing complex, SMRT IP in KAISO KD, NCoR1 KD, SMRT KD and
Empty vector (shRNA control) 3T3-L1 preadipocyte cells followed by western blotting for
KAISO was performed as described above. Densitometric analysis of the bands
detected in western blot was performed using AlphaDigidoc-1201 software. The stable
knockdown 3T3-L1 cells for these genes were made as detailed in the lentivirus-
mediated gene knockdown section.
Cell Cycle Analysis using FACS
The standard method of Propidium Iodide (PI) based FACS analysis was used to
observe the changes in cell cycle progression. 3T3-L1 cells (SMRT KD, KAISO KD, and
shRNA control cells) were cultured in six-well plates as described above. At specific
time points (-72h, 0h, 16h, 18.5h, 20h, 22h and 24h), the cells were trypsinized, washed
two times with 1X PBS and cooled on ice for 30 min. The cells were then fixed by drop-
wise addition of 70% ethanol, while gently vortexing the cells to avoid clumping. Before
PI staining, fixed cells were washed three times with 1X PBS and 200µl of staining
buffer (50µg/ml PI, 0.01% triton X-100, 10µg/ml RNase-A in PBS) was subsequently
added to each sample. The samples were then incubated for 45 min at 37 ºC for
staining and RNA degradation as RNA might interfere with the PI staining of DNA.
10,000 live-gated cells from each sample were then analyzed to determine their cell
cycle phase using an Accuri-6 flow cytometer (settings: FSC-SSC gate for live cell
singlet selection and FL2A histogram for PI-stained cell signal intensity). FlowJo
analysis software was then used to calculate the proportion of G0/G1, S, and G2/M cells
in each sample. Significant differences in the number of cells at a specific cell cycle
phase between the different samples were determined using a student‟s two-sided t-
test.
Effect of the PPARγ Ligand Rosiglitazone on the Differentiation Capacity of SMRT
KD 3T3-L1 Cells
An equal number of SMRT KD and shRNA control cells (50,000/well) were sub-cultured
into six-well plates and grown to confluence. Two days post-confluence, the cells were
differentiated using MDI medium (normal differentiation induction medium), 100nM &
250nM PPARγ ligand-containing medium, 167nM insulin and 1µM dexamethasone-
containing medium. After two days, 167nM insulin-containing DMEM medium with 10%
FCS was added to each well which was changed to DMEM medium containing 10%
FCS on the fourth day of differentiation. At D6, the wells were washed two times with 1X
PBS and the cells were fixed using 10% formalin in PBS. The accumulated fat droplets
inside cells were then stained using oil red-O to estimate the extent of differentiation.
List of Primers Used for ChIP-qPCR and Gene Expression Analyses
Gene Primer Sequence
control1_ChIP_F CACACAGCTGACCTCCAGAA
control1_ChIP_R AGTGGCAAGGTCTCTGCTTC
control2_ChIP_F GGGTGCTAGCCTTCCTGACT
control2_ChIP_R TCCAAGGTTCTCCCGACATA
Ampk1_ChIP_F CGGTGCTGGTGGCTAGAG
Ampk1_ChIP_R TCCTCCTAAAATGGCTACAAGG
Cdk12_ChIP_F GTGCCGTTTCGGTTTAATCT
Cdk12_ChIP_R CTAGCCTCCGCCTCACAC
Atf2_ChIP_F CATCCCTACAACCTCCAAGC
Atf2_ChIP_R GGCAGGACCATGAATTAGTGA
Atf4_ChIP_F CGCAGACCCCTGATCCTA
Atf4_ChIP_R GGCGAGTCACCTAAACCTCA
Atf6_ChIP_F CAGATCCACTCACCCCAGTC
Atf6_ChIP_R CCATGGAGTCGCCTTTTAGT
Atf7_ChIP_F ACTCGTGGGGCTGAGTTG
Atf7_ChIP_R ACTCAAGCCACGCTCACA
Camk1d_ChIP_F AAAGCACCACGTGTACAAACA
Camk1d_ChIP_R CTCTTTGCTGGGCTACCTTG
Chmp4b_ChIP_F CCTTTGACCTCTGCGAGACT
Chmp4b_ChIP_R AAGCCGGGAGTCTGAGTTTT
Crebzf_ChIP_F GGGGGTGGAAACTAGGTTTTAT
Crebzf_ChIP_R CTTTGCGGTGATGTCATAGG
Hdac4_ChIP_F CAGGGCAGTTAGGCACTCTC
Hdac4_ChIP_R TTGCCCTCAAAGCCTCAG
Hnrnpa2b1_ChIP_F ACTAACGCGTCTCCGCTTAC
Hnrnpa2b1_ChIP_R AGGAGAGTGTAGGCCCTTCC
Med1_ChIP_F GATCGCGAGATTAATCGTGTT
Med1_ChIP_R GAGACTTTGGTGCGGTTCC
Meis1_ChIP_F CCGACCAGAATGCTAGAACC
Meis1_ChIP_R TTGTGTAAGACGCGACCTGT
Ndufa11_ChIP_F CCAGTGTCATCGCAAGACC
Ndufa11_ChIP_R CTGACCTTTGCTTCCAGACC
Por_ChIP_F TTCCGAGGAGAGGATGAGG
Por_ChIP_R AAATCTCTGCTGTTGGTACGG
Rxra_ChIP_F AGTGAAACTTCCCGGAGGA
Rrxa_ChIP_R GAGAGGTGCCAGAGAACAGG
Suv39h1_ChIP_F CCTGCGCAGTAGCAAAGC
Suv39h1_ChIP_R GGCTAGCAATATGACTGACAAGG
Tle3_ChIP_F GCCGCCACATTATTTTGTTAC
Tle3_ChIP_R AGCACGAGGTCTGAACTGC
Ncoa4_ChIP_F TGTCTGGGTCGGTCTAAGGT
Ncoa4_ChIP_R GCCACTCTCGTCCTTACCG
Map3k14_ChIP_F GGGCTTTGAGGCAAGACTAA
Map3k14_ChIP_R CAAGGACAAGTGGCTCACC
Arf3_ChIP_F GGGCTATGGCAGCTAGCAC
Arf3_ChIP_R CGGAGGTTCAGGACGTGT
Nkrf_ChIP_F AACCGGTCTCCAACTTCAAA
Nkrf_ChIP_R GCAGAGTGCGTCAATGAAGA
Ccnt1_ChIP_F CGACACCCCGTAGACGAA
Ccnt1_ChIP_R AGATAGTCCCGCCCACCT
Tipin_ChIP_F GCCGCTGTCTAGGTGAGGT
Tipin_ChIP_R GGGCTCCACTTCCAAAATCT
Pfkfb2_ChIP_F GAGCTTGGTGGCATTGTTG
Pfkfb2_ChIP_R AAGGGAAGGCTTTTAATTCACC
Hnrnpk_ChIP_F CAGAGGATAATGGCGTCTGC
Hnrnpk_ChIP_R CCCCTCACCACAGAGTGC
Ets1_ChIP_F TGTTGCTATGAAGGGGAGTGT
Ets1_ChIP_R CCCAGCTCAAAGACAACAAGA
Sirt1_ChIP_F AAGAGTGAGCCACACTTACGG
SIRT1_ChIP_R ACCTCTAGGTGGCGTCCAA
Aspn_gene_F TCGATTTGTTTCCAACATGTCC
Aspn_gene_R CCGATGTCAGACCTAGATCAG
Tle1_gene_F CTCAGGAACATCAACAACAGG
Tle1_gene_R CGATGATGGCATTCAACTCTG
Tnfaip6_gene_F CAACCCACATGCAAAGGAG
Tnfaip6_gene_R TACTCATTTGGGAAGCCCG
Trpv6_gene_F CTTGTGCCAAATAACCAGGG
Trpv6_gene_R CATCAGGTGTTGGAACATCAC
Wnt5a_gene_F ACGCTTCGCTTGAATTCCT
Wnt5a_gene_R CCCGGGCTTAATATTCCAA
Cebpα_gene_F AAACAACGCAACGTGGAGA
Cebpα_gene_R GCGGTCATTGTCACTGGTC
Six1_gene_F AAGGAAAGGGAGAACACCG
Six1_gene_R TTCTGGTCTGGACTTTGGG
Akt1_gene_F AGCTCTTCTTCCACCTGTC
Akt1_gene_R GAGGTTCTCCAGCTTCAGG
Kaiso_gene_F TGCTTGGGGTAGGACTCTGA
Kaiso_gene_R TGAATGTCTGTAGCAGAAATCAGTT
Smrt_gene_F CATGAAGGTCTACAAGGACC
Smrt_gene_R TGCATAAACTTCTCACGGA
Tgfβ3_gene_F AATTACTGCTTCCGCAACC
Tgfβ3_gene_R TTTCCAGCCTAGATCCTGC
Plin4_gene_F GAGGCCTTCCAGATGACAG
Plin4gene__R CACCATGGTGTTCAAGCTC
Srf_gene_F TGAAGAAGGCCTATGAGCTG
Srf_gene_R TATACACATGGCCTGTCTCAC
Twist1_gene_F AGCTACGCCTTCTCCGTCT
Twist1_gene_R TCCTTCTCTGGAAACAATGACA
Snai2_gene_F TGCAAGATCTGTGGCAAGG
Snai2_gene_R CAGTGAGGGCAAGAGAAAGG
Itga1_gene_F AAGGCAAATGGGTTCTTATTGG
Itga1_gene_R CAACTGGACACTTATAGACATCTC
Postn_gene_F GAGGTGGAGAAACAGGAGAG
Postn_gene_R CTTCTAGGCCCTTGAACCC
Mrps6_gene_F CGAGGAGGGTATTTCCTGG
Mrps6_gene_R CTAACCACGTCAATGTCTCG
Pten_gene_F TAACTGCAGAGTTGCACAG
Pten_gene_R CAAGATCTTCACAGAAGGGT
Coup-tf1_gene_F CCAAGCATGATGCTTGTGG
Coup-tf1_gene_R CTTCTCACATACTCCTCCAGG
Fzd4_gene_F CAACTTAGTGGGACACGAG
Fzd4_gene_R AAAGGAAGAACTGCAGCTG
Fzd8_gene_F TTGAAAGCACTGGCCTTTTAC
Fzd8_gene_R AGGTGACCTGTGGCCTTAAA
Egfr_gene_F TGGAGCTATGGTGTCACTG
Egfr_gene_R TGAGATGTCACTTGCTGGG
Ebf1_gene_F TACAGCAATGGGATACGGA
Ebf1_gene_R GGCCTTCATACACTATGGC
Id2_gene_F GAGACCTGGACAGAACCAG
Id2_gene_R ATTCAGATGCCTGCAAGGA
Foxc2_gene_F CGGCTAGGACTGGACAACTC
Foxc2_gene_R CTGACAGCTCGCATTGCTC
Ccdc99_ChIP_F CACTAACTCCACCTCAGCACAG
Ccdc99_ChIP_R CCGGCGCTTACTTAGCAG
Ddx20_ChIP_F GTGGACTCGGAGGTTGTCA
Ddx20_ChIP_R CCCCGCCTCAAGTCTAAATA
Mdh2_ChIP_F CAAGCTTCTTGCGCTTCTCT
Mdh2_ChIP_R GACTCCAACGACCTCCACTC
Psmd5_ChIP_F GAGATCTTACGGAGCGAAGC
Psmd5_ChIP_R GACCGCGTTGAGAAAGGAT
Mgmt_ChIP_F AATGGCAGTAAATTCTTCAATAAGC
Mgmt_ChIP_R GGCTCATTTTCTGTGCTGTTG
Med6_ChIP_F CAGTAAAGGCGATGACTACCG
Med6_ChIP_R TTTTGACCTCCCCGCTAAC
Med23_ChIP_F TCCAAACAGGTCGCAGTTC
Med23_ChIP_R GACAGCGCTGCTTGATCC
Nfatc3_ChIP_F CTCTGGCGCTTCTTGCTC
Nfatc3_ChIP_R ACCGACCTATCGCGTGAGT
Nr4a1_ChIP_F GGAGGGGAGGAGATCCTGT
Nr4a1_ChIP_R GGAGGGGGTGTTGTAAATCC
Runx2_ChIP_F GCGAAGGAATGTGTAAACAGG
Runx2_ChIP_R AGAGGCATTTTGCGTTGTG
Prkg1_ChIP_F AGCCTAGTGAAATGTGAACAGATG
Prkg1_ChIP_R AAGGAACTCTTGGCTTATTCCAT
Prkca_ChIP_F GTCCCGTGTTGTGATGAATG
Prkca_ChIP_R TTCCAACATGAACAGCAAGC
Rarβ_ChIP_F CCTCTGGGCAGCTGATACTT
Rarβ_ChIP_R GTGCAGGAAATGCCTTTTG
Med27_ChIP_F CATTTCTTTGTCATTCACTATTAAGCA
Med27_ChIP_R TGATCTCCATCTAGGGAAGTCAT
Pld1_ChIP_R GCATAGCCTCAGCTTCCTGT
Pld1_ChIP_R AATCTGTACAGTTGCCTTTCTAATCA
Supplemental References
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