Purification and identification of CtBP-1 and CtBP-2 in the native PRDM16 transcriptional complex. (A) Silver staining of the PRDM16-associated proteins. Nuclear extracts prepared from adipocytes expressing either vector (left lane) or PRDM16 (right lane) were purified using Flag M2 agarose. After extensive washing, the PRDM16 complex was eluted with 3xFlag peptide and separated by 4%–12% gradient SDS-PAGE. (B) PRDM16 complex was immunopurified from the immortalized brown fat cells and separated by SDS-PAGE. Endogenous CtBP-1 and CtBP-2 were detected by Western blotting.

PRDM16/CtBP interaction mediates the repression of the white fat-selective gene program. 3T3-F442A cells expressing retroviral wild-type PRDM16, CtBP-binding-deficient mutant PRDM16 (Mut2), or an empty vector, were differentiated to mature adipocytes. (A) Protein expression of wild-type or mutant PRDM16 detected by Western blotting. (B) Oil-red O staining of 3T3-F442A cells at day 6 of differentiation-expressing wild-type PRDM16 (WT), mutant PRDM16 (Mut), or vector control (vector). (C) Microarray analysis of the differentiated cells expressing vector (left), wild-type PRDM16 (WT, middle), or mutant PRDM16 (Mut, right). See the text for details on each group. (D) mRNA levels of brown fat-selective genes (UCP-1 and PGC-1α) analyzed by real-time PCR. The cells were treated with or without cAMP (forskolin, 10 μM) for 4 h. (*) P < 0.01 relative to control with cAMP treatment; (^†) P < 0.05 relative to control without cAMP treatment. (E) mRNA levels of white fat-selective genes (as indicated). (F) Cellular respiration of the differentiated 3T3-F442A cells expressing wild-type PRDM16, mutant PRDM16, or vector control. Total mitochondrial oxygen consumption and uncoupled respiration were measured from cells with or without cAMP treatment (0.5 mM dibutyryl cAMP) for 12 h (n = 4). (*) P < 0.01; (^†) P < 0.05.

PRDM16 recruits CtBP onto the promoters of white fat-selective genes. (A) Transcriptional activity of resistin promoter in response to wild-type PRDM16 (WT), CtBP-binding-deficient mutant PRDM16 (Mut), or vector control in 3T3-L1 cells. Luciferase constructs and each expression plasmids were transfected into the differentiated 3T3-L1 cells by electroporation. After 48 h, cells were harvested and luciferase assay was performed. Each value was normalized by β-galactosidase activity (n = 3). (*) P < 0.05; (**) P < 0.01. (B) ChIP assay showing the recruitment of PRDM16 and CtBP onto the promoters of white fat genes (resistin and angiotensinogen). Differentiated brown fat cells expressing either PRDM16 or vector were immunoprecipitated with antibody against Flag (PRDM16), CtBP, or IgG (negative control). Protein-associated DNA was amplified by primer sets designed for the indicated regions of the promoters. PCR products were separated by 2% agarose gel. Graphs on the right show quantitative results in the proximal promoter regions using real-time PCR. (C) ChIP assay of PRDM16 and CtBP on the PGC-1α promoter.

PRDM16 directly interacts with CtBP-1 and CtBP-2 through its PLDLS motif. (A) Full-length CtBP-1 (top) or PGC-1α (bottom) were ³⁵S-labeled by in vitro translation and incubated with various GST fusion fragments of PRDM16. GST beads were washed and separated by 4%–12% gradient SDS-PAGE. (B) Schematic illustration of wild-type PRDM16 (WT) and mutant forms of PRDM16. PFDLT was mutated to PFAST in Mut1, PLDLS was mutated to PLASS in Mut2, and both motifs were mutated in Mut1/2 by site-directed mutagenesis. The blue box represents the PR domain, and the red boxes show the zinc fingers of PRDM16. (C) Flag-tagged PRDM16 was transiently expressed along with CtBP-1 (left), CtBP-2 (middle), or PGC-1α (right) in COS-7 cells. PRDM16 was immunoprecipitated using Flag M2 agarose, separated by SDS-PAGE, and CtBP-1, CtBP-2, or PGC-1α was detected by Western blotting. The inputs of each assay are shown in the bottom panels.

Genetic requirement for the CtBPs in the PRDM16-mediated suppression of white fat-selective gene expression. MEFs derived from CtBP-1 and CtBP-2 double-deficient (CtBP-1^−/− CtBP-2^−/−, KO) or double heterozygous (CtBP-1^+/− CtBP-2^+/−, Het) embryos stably expressing PRDM16 or vector control together with PPARγ2 were differentiated into mature adipocytes. (A) Protein expression of PRDM16 and CtBP detected by Western blotting. (B) mRNA levels of brown fat-selective genes (UCP-1, PGC-1α, and cidea) analyzed by real-time PCR. (C) mRNA levels of white fat-selective genes (as indicated). (*) P < 0.05.

PGC-1α/β and CtBP directly compete for binding to the PRDM16 complex. (A) Fixed amounts of wild-type PRDM16 (WT) or CtBP-binding-deficient mutant PRDM16 (Mut) were expressed along with fixed amounts of CtBP (CtBP-1) and variable amounts of PGC-1α in COS-7 cells. Cell lysates were incubated with Flag M2 agarose (PRDM16) and separated by SDS-PAGE. The interaction of PRDM16 with CtBP or PGC-1α was detected by Western blotting. The inputs are shown in the bottom panels. (B) Fixed amounts of wild-type PRDM16 (WT) or mutant PRDM16 (Mut) were expressed along with fixed amounts of PGC-1α and variable amounts of CtBP. Interaction of PRDM16 with CtBP was detected as described above. (C) Wild-type PRDM16 (WT) or mutant PRDM16 (Mut) were coexpressed with PGC-1β or CtBP, and the interaction of PRDM16 with PGC-1β or CtBP was examined as described above. (D) A schematic model of brown fat determination by PRDM16. PRDM16 represses white fat-selective genes such as resistin by recruiting CtBP onto their promoters. Recruitments of PGC-1α and PGC-1β to PRDM16 trigger the dismissal of CtBP from the PRDM16 complex, leading to the robust activation of brown fat-selective genes.