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1 Supplement figure legends Figure S1. Alignments of PRDM16 amino acid sequences of mouse, human, dog, chicken, and Xenopus. Two putative CtBP-binding motifs are indicated in the boxes. Numbers represent amino acids from the N-terminus. Figure S2. mrna levels of adipogenic marker genes, adiponectin, PPAR# and ap2 (A) and brown fat-selective genes, cox8b and cidea (B) in 3T3-F442A cells expressing retroviral wild type PRDM16, CtBP-binding deficient mutant PRDM16, or vector control. mrna levels were measured by real-time PCR. * p<0.01 relative to vector control. p<0.01 relative to mutant PRDM16. Figure S3. Validation of microarray data on white fat-selective genes by real-time PCR. (A) mrna levels of indicated genes from WAT and BAT (n=6). (B) mrna levels of indicated genes from the differentiated 3T3-F442A cells expressing retroviral PRDM16 or vector control. 37

2 Figure S4. PRDM16 represses protein levels of resistin released in the culture medium of differentiated 3T3-F442A cells. Differentiated 3T3-F442A cells expressing retroviral wild type PRDM16 (WT), mutant PRDM16 (Mut) or vector control were cultured for 2 days, and resistin concentrations in the culture medium were measured by ELISA. * p<0.01. Figure S5. PRDM16/CtBP interaction is required for the repression of white fat-selective genes. PPAR! deficient MEFs expressing retroviral PPAR!2, together with wild type PRDM16 (WT), CtBP-binding deficient mutant PRDM16 (Mut), or vector control were differentiated to mature adipocytes. (A) Protein expression of the wild type or mutant forms of PRDM16 detected by Western blotting. (B) Oil-red O staining at day 6 of differentiation. (C) mrna levels of adipogenic marker genes analyzed by real-time PCR. (D) mrna levels of brown fat-selective genes (UCP-1 and PGC-1"). The cells were treated with or without camp (forskolin, 10µM) for 4 hrs. * p<0.01, p<0.05 relative to mutant. (E) mrna levels of brown fat-selective genes, cox8b and cidea. (F) mrna levels of white fat-selective genes (as indicated). * p<0.01 relative to vector control. Figure S6. mrna levels of white fat-selective genes (as indicated) in MEFs derived from CtBP double deficient (CtBP-1 -/- CtBP-2 -/-, KO) and heterozygous (CtBP-1 +/- CtBP-2 +/-, Het) embryos. Cells expressing retroviral PRDM16 or vector together with PPAR!2 were differentiated to mature fat cells, and mrna levels were measured by real-time PCR. * p<

3 Figure S7. Depletion of endogenous PRDM16 increases the mrna levels of resistin and angiotensinogen genes in brown fat cells. (A) PRDM16 mrna levels were measured in immortalized brown fat cells expressing a scrambled control-shrna (sh-scr) or shrna targeted to PRDM16 (sh-prdm16) at day 0 and day 4 of differentiation. (B) mrna levels of white fat-selective genes, resistin and angiotensinogen by real-time PCR. * p<

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11 Table S1 White-fat selective genes regulated by PRDM16 in 3T3-F442A cell ColumProbe ID Gene name Gene ID * _at angiotensinogen (serpin peptidase inhibitor, clade A, member 8) AK * _at annexin A1 NM_ * _at endothelin receptor type A BC * _at fatty acid desaturase 3 BE * _at integrin alpha 6 BM * _a_at phosphoserine aminotransferase 1 BC * _at RAS p21 protein activator 3 NM_ * _at resistin NM_ * _at serine (or cysteine) proteinase inhibitor, clade A, member 3K BC * _at tissue inhibitor of metalloproteinase 2 BF * _at WDNW-1 like protein (RIKEN cdna G20 gene) AV _a_at 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 BB _a_at A kinase (PRKA) anchor protein (gravin) 12 NM_ _at brain glycogen phosphorylase AW _at carbonic anhydrase 5b, mitochondrial NM_ _at caveolin 2 NM_ _at CDC42 effector protein (Rho GTPase binding) 3 BB _at coiled-coil domain containing 80 BG _at ectonucleotide pyrophosphatase/phosphodiesterase 2 BC _a_at endonuclease domain containing 1 BF _a_at fibroblast growth factor receptor-like 1 AF _a_at fibulin 2 BF _at fragile X mental retardation syndrome 1 homolog BG _x_at glutathione S-transferase, mu 1 NM_ _at histocompatibility 2, T region locus 23 NM_ _at inhibitor of DNA binding 3 NM_ _a_at leukotriene C4 synthase NM_ _at mitogen-activated protein kinase 6 BC _at pantothenate kinase 3 BC _at procollagen, type I, alpha 2 BF _a_at procollagen, type III, alpha 1 AW _at procollagen, type V, alpha 1 AW _at procollagen, type V, alpha 3 NM_ _a_at procollagen, type VI, alpha 2 BI _at progesterone receptor membrane component 2 BF _at RIKEN cdna N14 gene BC _at RIKEN cdna A630051L19 gene AF _at serum deprivation response BE _at solute carrier family 1 (neutral amino acid transporter), member 5 NM_ _at sortilin 1 AV _s_at transcription factor 7-like 2, T-cell specific, HMG-box BB _s_at X-box binding protein 1 C _at zinc finger protein 503 BB * validated by quantitative PCR 53

12 Table S2: Primer sequences Gene amplicon size CtBP Forward GTGCCCTGATGTACCATACCA Reverse TGATGTCGATATTGTCAAACCCG CtBP Forward TGGGTGCCATGATGTACCAC Reverse GTTGTCGTAGCCACTACCGA Resistin 167 Forward AAGAACCTTTCATTTCCCCTCCT Reverse GTCCAGCAATTTAAGCCAATGTT RAS p21 protein activator 3 (Ras p21) 111 Forward ACTCTGCCCCTTTTACGGAGA Reverse CCTATGATGGAATCCCTTCGGA Collagen, type XVIII, alpha 1 (Col18a1) 156 Forward GGAGACCCCCTACCTGAGAAG Reverse TGGCCGGACATGAAACAGC Integrin alpha 6 (Itga6) 175 Forward TGCAGAGGGCGAACAGAAC Reverse GCACACGTCACCACTTTGC Angiotensinogen (Agt) 114 Forward GCACCCTGGTCTCTTTCTACC Reverse TGTGTCCATCTAGTCGGGAGG Serine (or cysteine) proteinase inhibitor, 155 Forward AAGGAAGTCTTCACCGAACAAG clade A, member 3K (Serpina3k) Reverse GCCTTACGAATGCCACCAATAAC WDNM1 like protein 182 Forward AGTGAGACCTCTGCAGCTTTTAGG Reverse CAACTGTTTTCTTGGTCACAGAGC glutamic pyruvate transaminase 2 (Gpt2) 121 Forward AACCATTCACTGAGGTAATCCGA Reverse GGGCTGTTTAGTAGGTTTGGGTA annexin A1 (Anxa1) 112 Forward AGATCAAGGCCGCGTACTTAC Reverse GCTGGAGTTTTTAGCATAGCCA phosphoserine aminotransferase 1 (Psat1) 107 Forward AAGCCACCAAGCAAGTGGTTA Reverse GATGCCGAGTCCTCTGTAGTC endothelin receptor type A (Ednra) 100 Forward TTTCCGGGGACGGAGATCAA Reverse TCTGCTGTGGGCAATAGCC tissue inhibitor of metalloproteinase Forward ACACGCTTAGCATCACCCAG (Timp2) Reverse GGGAGGAGATGTAGCAAGGG fatty acid desaturase 3 (Fads3) 192 Forward TGCTGGTGTCTGCAACATGAT Reverse GACAGGTGCGACAGTCACATC PGC-1a promoter -185/ Forward CTCTAAGCGTTACTTCACTGAGGC Reverse CACCTGTCTTACTACAGTCCCCAG PGC-1a promoter -7325/ Forward GATAAAGAGACGAAGGCCCAAC Reverse GTCTGGAGGGTTCAGGTGATTTAC Resistin promoter -179/ Forward AGACAACGTCCTGAGAAGACAATC Reverse CCATCCTGCCTTGGATAATAAGTA Resistin promoter -3218/ Forward TTTCATTACCTCTCTTGTTGTCCC Reverse GAAGAATGAAGAGATTTCACCTGC Angiotensinogen promoter -127/ Forward CTTGGTCAAGCCTGGATTCTC Reverse CCAACCTAGACAAGCACAGCTATC Angiotensinogen promoter -5858/ Forward CTTCCCTACAGACTGGGAGATAGG Reverse GTCCAGCTTTGTATGCAGATCC 54