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SUPPLEMENTAL MATERIALS
FIGURE LEGENDS
Figure S1. Effects of HOXC10 in adipocytes and C2C12 myoblasts
(a) qPCR analysis of HOXA10, HOXC10, and HOXD10 in BAT, SubQ and VISC. (b) qPRC analysis
of HOXC10 and PRDM16 that were used for Figure 1b. (c) Absorbance reading of Oil-red O staining
in 3T3-L1 adipocytes expressing either empty vector control or FLAG-HOXC10 at day 6 of adipocyte
differentiation. (d) qPCR analysis of brown fat and adipogensis genes in adipocytes derived from SVF
of BAT transduced with either empty vector control or FLAG-HOXC10. (e) A schematic diagram
depicting the mutation on each of the three helices of the homeobox domain of HOXC10. qPCR
analysis of genes in 3T3-L1 adipocytes transduced with either empty vector control, FLAG-HOXC10,
FLAG-HOXC10 DBM1, FLAG-HOXC10 DBM2, or FLAG-HOXC10 DBM3. Cells were treated with 10
µM Forskolin for 2 hrs before harvest. (f) Oil-red O staining in adipocytes derived from SVF of
subcutaneous WAT transduced with HOXC10 shRNA or a control shRNA. (g) qPCR analysis of
muscle-specific genes in C2C12 myoblasts transduced with HOXC10 shRNA, a control shRNA, or
expressing FLAG-PRDM16 at day 6 of myotube differentiation. (h) Immunoblot and densitometry
analysis for Myf5, MEF2A, and FLAG in C2C12 myoblasts transduced with HOXC10 shRNA, a control
shRNA, or expressing FLAG-PRDM16 at day 6 of myotube differentiation. GAPDH served as a
loading control. All data are presented as means ±SEM; *p<0.05, **p<0.01; n=4
Figure S2. HOXC10 has no effect on BAT.
(a) qPCR analysis for PRDM16, PGC1α, UCP1, and HOXC10 mRNA in BAT of mice after 4 weeks of
intravascular administration of 4 x 1012 GC of AAV-FLAG HOXC10 or AAV-Emerald vectors at room
temperature (RT) and or exposure to 4ºC for 7 days. Cold exposure was done with male 8-week-old
mice, n=5 per group. (b) qPCR analysis of genes in BAT of AAV-transduced mice after exposure to
4ºC for 7 days. n=8 (c) Immunoblot and densitometry analysis of genes in BAT of AAV-transduced
mice after exposure to 4ºC for 7 days. n=6. All data are presented as means ±SEM; *p<0.05,
**p<0.01, ***p<0.001. (d) Immunoblot of UCP1, PGC1α and 14-3-3 as loading control of 3T3-L1
adipocytes treated without or with 10M forskolin for 6h. Lysates from BAT were included as a
positive control.
Figure S3. Expression of AAV-FLAG HOXC10 in BAT has no effect on glucose metabolism and
brown fat genes.
(a) Rectal temperature of male mice at RT and after 7 days of exposure at 4ºC. Readings were taken
after 4 weeks of intrainterscapular BAT administration of 5 x 10 9 GC of AAV-FLAG HOXC10 or AAVEmerald vectors. n=7-10 per group (b) qPCR analysis of brown fat-specific genes in BAT of AAVtransduced mice after exposure to 4ºC for 7 days. n=6 (c) Immunoblot and densitometry analysis for
PRDM16, UCP1, PGC1 , Cox7a1, FLAG, and GFP in BAT of AAV-transduced mice after exposure
to 4ºC for 7 days. HSP90 served as loading control. n=8 (d) Intraperitoneal glucose tolerance test in
AAV-transduced mice at RT and after 7 days of exposure at 4ºC. n=7-9 per group. All data are
presented as means ±SEM; *p<0.05, **p<0.01, ***p<0.001
Figure S4. PRDM16 partially rescues HOXC10 suppression effects.
(a-d) qPCR analysis of genes in adipocytes derived from SVF of subcutaneous WAT co-transduced
with either empty vector control or FLAG-HOXC10 with either empty vector control or FLAG-PRDM16.
Cells were treated with 10 µM Forskolin for 6 hrs before harvest. Data are presented as means ±SE;
*p<0.05, **p<0.01; n = 3
Table S1. Differently expressed genes in adipocytes expressing HOXC10
Genes whose expression was down-regulated in adipocytes expressing HOXC10
aldo-keto reductase family 1, member C12 /// aldo-keto reductase family 1, member C13
ATP-binding cassette, sub-family A (ABC1), member 1
prostaglandin E receptor 4 (subtype EP4)
coiled-coil-helix-coiled-coil-helix domain containing 6
G protein-coupled receptor 64
Ral GTPase activating protein, alpha subunit 2 (catalytic)
nudix (nucleoside diphosphate linked moiety X)-type motif 12
guanine nucleotide binding protein (G protein), alpha inhibiting 1
RAB GTPase activating protein 1-like
RIKEN cDNA 2310035C23 gene
glutamate receptor, ionotropic, AMPA3 (alpha 3)
glutamyl aminopeptidase
prolactin receptor
citrate lyase beta like
signal recognition particle receptor, B subunit /// transferrin
glutaredoxin 3
cytochrome P450, family 2, subfamily r, polypeptide 1
headcase homolog (Drosophila)
zinc finger protein 606
hydroxysteroid dehydrogenase like 2
synaptosomal-associated protein 23
carnitine acetyltransferase
24-dehydrocholesterol reductase
predicted gene 3776 /// glutathione S-transferase, alpha 1 (Ya) /// glutathione S-trans
interleukin 7
serum deprivation response
acyl-Coenzyme A dehydrogenase family, member 11
fatty acid desaturase 3
RIKEN cDNA A930007A09 gene
embryonal Fyn-associated substrate
lysophosphatidic acid receptor 4
solute carrier family 25, member 32
phenylalanine-tRNA synthetase 2 (mitochondrial)
Ras association (RalGDS/AF-6) domain family member 2
suppressor of Ty 20
coiled-coil domain containing 91
synaptonemal complex protein 3
phosphorylase kinase gamma 1
RIKEN cDNA 4632427E13 gene
solute carrier family 7 (cationic amino acid transporter, y+ system), member 11
avian musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene homolog
insulin-like growth factor binding protein 2
solute carrier family 1 (high affinity aspartate/glutamate transporter), member 6
peptidase domain containing associated with muscle regeneration 1
folliculin interacting protein 1
suprabasin
family with sequence similarity 13, member C
carboxylesterase 2G
sodium channel, voltage-gated, type VII, alpha
early growth response 3
apolipoprotein E
RIKEN cDNA 5730508B09 gene
ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit /// ATP synthase sub
RIKEN cDNA 1110059M19 gene
phytanoyl-CoA hydroxylase interacting protein-like
butyrylcholinesterase
pyruvate carboxylase
endonuclease/exonuclease/phosphatase family domain containing 1
carboxymethylenebutenolidase-like (Pseudomonas)
alcohol dehydrogenase 1 (class I)
eyes absent 4 homolog (Drosophila)
solute carrier family 7 (cationic amino acid transporter, y+ system), member 11
protease, serine, 23
mitochondrial tumor suppressor 1
amylase 1, salivary
SH3-binding kinase 1
clusterin
regulator of G-protein signaling 2
enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase
RWD domain containing 3
ATP-binding cassette, sub-family D (ALD), member 2
potassium voltage-gated channel, shaker-related subfamily, beta member 1
solute carrier family 2 (facilitated glucose transporter), member 4
CAP-GLY domain containing linker protein family, member 4
tenascin C
glutamate-ammonia ligase (glutamine synthetase)
alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide
expressed sequence BB144871
MAP7 domain containing 2
nemo like kinase
ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B)
regulator of cell cycle
V-set and transmembrane domain containing 2A
cannabinoid receptor 1 (brain)
RIKEN cDNA C730049O14 gene
cytochrome P450, family 4, subfamily b, polypeptide 1
adipocyte-related X-chromosome expressed sequence 1 /// adipocyte-related X-chromosome
pleiotrophin
sorbitol dehydrogenase
alcohol dehydrogenase, iron containing, 1
3-oxoacid CoA transferase 1
actin, alpha 2, smooth muscle, aorta
glutathione S-transferase, alpha 4
aldo-keto reductase family 1, member C14
glycine-N-acyltransferase
chordin-like 1
acyl-CoA synthetase long-chain family member 1
retinol saturase (all trans retinol 13,14 reductase)
kelch-like 24
sorbin and SH3 domain containing 1
galactosidase, beta 1-like 2
UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2
acid phosphatase 1, soluble /// low molecular weight phosphotyrosine protein phosphatas
activin A receptor, type IC
succinate receptor 1
adrenergic receptor, beta 3
adenomatosis polyposis coli down-regulated 1
natriuretic peptide receptor 3
3-hydroxyisobutyryl-Coenzyme A hydrolase
NEDD4 binding protein 2-like 1
carbonic anhydrase 3
chemokine (C-X-C motif) ligand 3
ATPase, Na+/K+ transporting, beta 1 polypeptide
delta-like 1 homolog (Drosophila)
nucleic acid binding protein 1
ABI gene family, member 3 (NESH) binding protein
carbonic anhydrase 5b, mitochondrial
family with sequence similarity 213, member A
aldehyde dehydrogenase family 1, subfamily A7
cytochrome P450, family 1, subfamily a, polypeptide 1
transmembrane and immunoglobulin domain containing 1
thyroid hormone responsive
aquaporin 7
Genes whose expression was up-regulated in adipocytes expressing HOXC10
serum amyloid A 1
resistin
cellular retinoic acid binding protein I
serum amyloid A 2
preproenkephalin
chemokine (C-C motif) ligand 8 /// c-C motif chemokine 8-like
microfibrillar-associated protein 4
fin bud initiation factor homolog (zebrafish)
complement factor D (adipsin)
collagen and calcium binding EGF domains 1
RIKEN cDNA 0610040B09 gene
regulator of G-protein signaling 5
G protein-coupled receptor 149
regulator of G-protein signaling 4
protein phosphatase 1, regulatory (inhibitor) subunit 3C
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2
serine (or cysteine) peptidase inhibitor, clade A, member 3G
thioredoxin 1
chondroitin sulfate proteoglycan 5
MDS1 and EVI1 complex locus
adenosine A2b receptor
tumor necrosis factor receptor superfamily, member 4
G protein-coupled receptor 116
NSL1, MIND kinetochore complex component, homolog (S. cerevisiae)
netrin 1
calcium/calmodulin-dependent protein kinase kinase 2, beta
RIKEN cDNA 1810019D21 gene
lymphocyte antigen 6 complex, locus C1 /// lymphocyte antigen 6 complex, locus C2
neurotensin
leucine rich repeat containing 15
RIKEN cDNA 4930553P13 gene
sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F
regulator of telomere elongation helicase 1
UDP-glucose ceramide glucosyltransferase
TPX2, microtubule-associated protein homolog (Xenopus laevis)
fructose bisphosphatase 2
progesterone receptor
RNA binding protein, fox-1 homolog (C. elegans) 3
microphthalmia-associated transcription factor
RIKEN cDNA 4833447P13 gene
retinol dehydrogenase 16 /// retinol dehydrogenase 18, pseudogene
late cornified envelope 1A2
cytochrome P450, family 2, subfamily b, polypeptide 10
RIKEN cDNA 1700055N04 gene /// aldehyde dehydrogenase 3 family, member B2
acyl-Coenzyme A dehydrogenase family, member 10
ribosomal modification protein rimK-like family member B
thioesterase superfamily member 4
insulin-like growth factor 2 mRNA binding protein 1
arylsulfatase i
TNF receptor-associated factor 6
Supplemental Experimental Procedures
Plasmids
The full-length cDNA sequence for human HOXC10 (Accession # BC001293) was cloned into pLentiHIKO lentiviral vector and pBabe-puro retroviral vector with a 3x FLAG tag sequence at the Nterminus. The homeodomain mutant (DBM1/2/3) of HOXC10 was created by site-directed
mutagenesis. The retroviral vector FLAG PRDM16 was obtained from Addgene (#15504). For
lentivirus-mediated shRNA expression, the sequence targeting the mouse HOXC10 in C2C12
myoblasts (5’ CACCTCGGATAACGAAGCTAA 3’) and mouse HOXC10 in SVF (5’
GAGATCAAGACAGAGCAAAGC 3’) were cloned into pLKO3.1 vector. The PRDM16 promoter (2076
bp) was amplified by PCR using primers (5’ GCGGGTACCCAGTTCTCCAAATCTCAAGG 3’ and 5’
GATTCCGCGAGCCGACACCAGATCTCGC 3’) and cloned into pGL3 Basic Vector using KpnI and
BglII restriction enzyme sites. AAV-FLAG HOXC10 and AAV-Emerald vectors were generated by
amplification of the transgenes and cloned into the pAAV MSC2 vector obtained from Addgene
(#46954). A short version of the adipocyte protein 2 (mini/aP2) promoter (1, 2) was cloned into the
vector to drive the expression of the transgenes.
Protein isolation and Western blotting
Whole-cell lysates were isolated from cells with 2% sodium dodecyl sulphate (SDS) in phosphatebuffered saline (PBS) containing protease inhibitor cocktail (Complete, Roche), while whole-cell
lysates from tissues were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5%
sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) containing protease inhibitor cocktail. Lysates
were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore) and probed with antiHOXC10 (ab153904; Abcam), anti-PRDM16 (ab106410; Abcam), anti-UCP1(ab23841; Abcam), antiPGC1α (ab54481; Abcam), anti-CEBPβ (ab32358; Abcam), anti-Myf5 (ab125078; Abcam), antiMEF2A (ab109420; Abcam), anti-Cox7a1 (ab123591; Abcam), anti-FABP4 (3544; Cell signalling),
anti-GAPDH (5174; Cell signalling), HSP90 (4874; Cell signalling), anti-pan 14-3-3 (SC629; Santa
Cruz), anti-GFP (SC9996; Santa Cruz), and anti-FLAG (F1804; Sigma-Aldrich) primary antibodies,
followed by secondary detection with a horseradish peroxidise (HRP)-conjugated species-specific
antibody (GE Healthcare). Immunoreactive bands were detected with Clarity western ECL (Bio-rad) or
WesternBright Sirius (Advansta). Densitometry was performed with the ImageJ software (NIH).
Gel shift assays
The 256bp DNA probe containing the HOXC10 binding site was PCR using 5’ biotinylated primers (5’
AGCTCTGAGGCTTTTCTCTGG 3’ and 5’ GCAACAAGATTCAGAGCTGTC 3’), and incubated (5 fmol)
with nucleus extract in binding buffer accordingly to Lightshift Chemiluminescent EMSA kit (Pierce).
Two hundred-fold molar excess of unlabelled probe was added to the binding reaction for competition
assays. DNA-protein complexes were separated by electrophoresis on a 5% PAGE gel, and then
transferred onto nylon membrane, crosslinked and detected with Chemiluminescent Nucleic Acid
Detection Module (Pierce). For supershift experiments, normal rabbit IgG (sc2077; Santa cruz), antiHOXC10 (ab153904; Abcam), anti-HOXC8 (ab86236; Abcam) antibodies were incubated with the
DNA-protein complexes before separation by electrophoresis.
Chromatin immunoprecipitation assays
3T3-L1 adipocytes expressing FLAG-tagged HOXC10 were fixed with 1% formaldehyde for 10 min at
room temperature, before quenching with 125 mM glycine for 5 min. Cells were rinsed with PBS and
nuclei were isolated prior to lysis with 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA and 0.5%
SDS supplemented with protease inhibitor cocktail. Chromatin was sheared by sonication and diluted.
Lysates were then precleared with protein A/G Sepharose beads (Invitrogen). Aliquots of input were
removed. Protein-DNA complexes were immunoprecipitated with 2 µg of antibody overnight at 4ºC
with normal mouse IgG (sc2025; Santa cruz) or anti-FLAG. Complexes were captured with protein
A/G Sepharose beads blocked with bovine serum albumin and salmon sperm DNA, washed and
eluted with 1% SDS/0.1M NaHCO3. Crosslinking was reversed by overnight incubation at 4ºC,
followed by proteinase K and RNAase digestion and purification using the phenol/choloroform method.
Eluted DNA were analysed by PCR using primers in Table S2.
Oil-Red-O staining
Cells were fixed with 4% paraformaldehyde for 10 min, rinsed with PBS, and incubated for 50 min
with a 60/40 solution of 0.5% Oil-Red-O (Sigma-Aldrich) in isopropanol before cells were rinsed in
water and imaged. To obtain absorbance reading, Oil-red-O was eluted with 100% isopropanol for 10
min and OD measured at 500 nM.
Histology
Adipose tissues were isolated and fixed in 10% neutral-buffered formalin, dehydrated, and embedded
in paraffin for sectioning. Sections were stained with haematoxylin and eosin or were probed with
antibodies for UCP1 (ab23841; Abcam).
Table S2: Primers used for real time PCR analysis and ChIP assays
Gene
Adiponectin
CEBPβ
CideA
Cox7a1
Cox8b
Dio2
Elvol3
Emerald
FABP4
FLAG
HOXA10
HOXC10
HOXD10
MCK
MEF2A
Myf5
Myf6
Myg
MyoD
PGC1α
PPARγ
PRDM16
TBP
UCP1
ChIP
Primers
PRDM16-P1
PRDM16-P2
PRDM16-P3
PRDM16-P4
PRDM16-P5
PRDM16-P6
PRDM16-P7
PRDM16-P8
PRDM16-P9
PRDM16P10
PRDM16P11
Forward Primer 5’-3’
CAC CGC AGA CGA CAG GAA G
TCG GGA CTT GAT GCA ATC C
ATC ACA ACT GGC CTG GTT ACG
CAG CGT CAT GGT CAG TCT GT
GAA CCA TGA AGC CAA CGA CT
CAG TGT GGT GCA CGT CTC CAA TC
TCC GCG TTC TCA TGT AGG TCT
CGA CGG CAA CTA CAA GAC CC
GAT GCC TTT GTG GGA ACC T
CTA CAA AGA CCA TGA CGG
CTA CTA CGC CCA CGG TGG
AGC TAA AGA GGA GAT AAA GGC
TGG AAT GCA AAC CTG TGG
GCA AGC ACC CCA AGT TTG A
AAC CGA CAG GTT ACT TTT AC
CAG CCC CAC CTC CAA CTG
ATC AGC TAC ATT GAG CGT CTA CA
AGC GCA GGC TCA AGA AAG TGA
ATG
CGC CAC TCC GGG ACA TAG
CCC TGC CAT TGT TAA GAC C
TCA GCT CTG TGG ACC TCT CC
CAC GGT GAA GCC ATT CAT ATG CG
ACC CTT CAC CAA TGA CTC CTA TG
CTG CCA GGA CAG TAC CCA AGC G
Forward Primer 5’-3’
Reverse Primer 5’-3’
GCA CCT GCA CCA GGG C
AAA CAT CAA CAA CCC CGC
TAC TAC CCG GTG TCC ATT TCT
AGA AAA CCG TGT GGC AGA GA
GCG AAG TTC ACA GTG GTT CC
TGA ACC AAA GTT GAC CAC CAG
GGA CCT GAT GCA ACC CTA TGA
GGC TGT TGT AGT TGT ACT CCA G
CTG TCG TCT GCG GTG ATT T
GCT CCG CGT ACG AGT TCG
CGA CGG CGC TTC ATT ACG
TCT TTC TCC AAT TCC AGC G
ATC TGT TCG GGT CTG TCC
ACC TGT GCC GCG CTT CT
TCT TAA CGT CTC AAC GAT AT
GGG ACC AGA CAG GGC TGT TA
CCT GGA ATG ATC CGA AAC ACT TG
CTG TAG GCG CTC AAT GTA CTG
GAT
GAA GTC GTC TGC TGT CTC AAA GG
CAT TTG TCT CTG CTG CTG TTC C
ACC CTT GCA TCC TTC ACA AGC
CTC CGA TGC TTG TTG AGG G
TGA CTG CAG CAA ATC GCT TGG
TCT GTC TGG ACT TCA TCA GC
Reverse Primer 5’-3’
CAG TTC TCC AAA TCT CAA GGG
CTG CCT AGT ACT ATA AAC GG
AGC TCT GAG GCT TTT CTC TGG
ACC CCT GCG AGG GCT CAG G
TGC TCT TGC CCA GCC AGG
CTC CTG AAA GGG GCT AGC
TGA GGA CCG ACG GCT CCA G
TTA GGG CGC GTT GGC CCT G
GGA AGT CGC GCA GAA ACC
CGA GCT GCG CTG AAA AGG
CCT TTC TCA GTC TCT ACC TGG
AAT GGA GAG TGC TGC TGG G
GCA ACA AGA TTC AGA GCT GTC
CTT CAC ATT CGG AGC TCA GG
AGT ACC GGG TGC GGA AGC
AGC TCC AAG CTC TGC CAC C
AAG CTT TCG TTC GCC GTC G
TGT GGC TCC TGC CCA AGC
GCC TTC TCG GTC TTC ACC
CTC CTT GAG CCC AGA AGC
CAA ACT GAC AAT GCT AGG G
GAC CGC GCG GTC GAC C
Supplemental References
1.
Graves RA, Tontonoz P, Spiegelman BM. Analysis of a tissue-specific enhancer: ARF6
regulates adipogenic gene expression. Mol Cell Biol. 1992;12(3):1202-8.
2.
Ross SR, Graves RA, Greenstein A, Platt KA, Shyu HL, Mellovitz B, et al. A fat-specific
enhancer is the primary determinant of gene expression for adipocyte P2 in vivo. Proc Natl Acad Sci
U S A. 1990;87(24):9590-4.