Transcriptional Activity of PGC-1α and NT-PGC-1α Is Differentially Regulated by Twist-1 in Brown Fat Metabolism

Brown fat expresses two PGC-1α isoforms (PGC-1α and NT-PGC-1α) and both play a central role in the regulation of cellular energy metabolism and adaptive thermogenesis by interacting with a wide range of transcription factors including PPARγ, PPARα, ERRα, and NRF1. PGC-1α consists of 797 amino acids, whereas alternative splicing of the PGC-1 α gene produces a shorter protein called NT-PGC-1α (aa 1–270). We report in this paper that transcriptional activity of PGC-1α and NT-PGC-1α is differently affected by the transcriptional regulator, Twist-1. Twist-1 suppresses PGC-1α but not NT-PGC-1α. The inhibition of PGC-1α activity by Twist-1 is mediated by direct interaction through the C-terminal region of PGC-1α (aa 353–797). Thus, the absence of the corresponding C-terminal domain in NT-PGC-1α allows NT-PGC-1α to be free from Twist-1-mediated inhibition. Overexpression of Twist-1 in brown adipocytes suppresses transcription of a subset of PGC-1α-target genes involved in mitochondrial fatty acid oxidation and uncoupling (CPT1β, UCP1, and ERRα). In contrast, NT-PGC-1α-mediated induction of these genes is unaffected by Twist-1. These findings show that differences in inhibitory protein-protein interactions of PGC-1α and NT-PGC-1α with Twist-1 lead to differential regulation of their function by Twist-1.


Introduction
The transcriptional coactivator PGC-1α was first identified as a coactivator of PPARγ in brown adipose tissue and is now known to interact with a broad range of nuclear receptors and transcription factors to regulate mitochondrial biogenesis in most tissues but also control adaptive thermogenesis, fatty acid/glucose metabolism, ROS metabolism, and muscle fiber type switching in a tissue-specific manner [1][2][3][4][5][6][7]. The function of PGC-1α among tissues is regulated by signaling inputs that increase transcription of PGC-1α and modulate the transcribed protein through tissue-specific posttranslational modifications [8][9][10][11][12][13][14]. This allows PGC-1α to function as a key regulator to link nutritional and environmental stimuli to the tissue-specific transcriptional programs.
Alternative splicing of PGC-1α produces an additional transcript that encodes a shorter isoform called NT-PGC-1α (aa 1-270) [15]. NT-PGC-1α is coexpressed with PGC-1α in metabolically active tissues and its expression is coregulated by the nutritional and environmental cues which activate the gene [15][16][17]. PGC-1α is a short-lived nuclear protein containing 797 amino acids. A variety of post-translational modifications enhance the stability and activity of PGC-1α by decreasing its targeting to the proteosome. In contrast, NT-PGC-1α is relatively stable since it is less effectively targeted to the proteosome due to lack of the C-terminal domain involved in proteosomal targeting [15]. Constitutive activation of target genes by NT-PGC-1α is effectively limited by a mechanism that sequesters NT-PGC-1α to the cytoplasm in a CRM1-dependent manner [16]. NT-PGC-1α activity is primarily modulated by increased translocation to the nucleus. PKA-dependent phosphorylation of NT-PGC-1α increases its nuclear retention and subsequent recruitment to the transcriptional complexes [16].
Another layer of regulation of PGC-1α function is mediated by direct interaction with other regulatory proteins. Previous studies have shown that p160 MBP , RIP140, and Twist-1 bind to PGC-1α and repress its transcriptional activity. p160 myb binding protein was originally identified as a protein that interacts with the regulatory domain 2 PPAR Research of PGC-1α (aa 200-400) in C2C12 myoblasts [18]. The docking of p160 MBP on PGC-1α inhibits transcription of PGC-1α target genes [9,18]. RIP140 is a transcriptional corepressor for a number of nuclear receptors in adipose tissue and skeletal muscle where it represses many PGC-1α target genes [19]. Mechanistically, RIP140 directly interacts with PGC-1α (aa 184-797) and suppresses its activity [20]. Recently, the transcription factor Twist-1 was also identified as a negative regulator of PGC-1α in brown adipose tissue. Twist-1 is a helix-loop-helix (HLH)-containing transcription factor involved in early development, apoptosis, cancer, and osteoblast differentiation [21][22][23]. A recent study reported that Twist-1 is recruited to the PGC-1α target genes by docking to the C-terminal domain of PGC-1α (aa 350-797) to negatively modulate oxidative metabolism and UCP1dependent uncoupling in brown adipose tissue [24]. Interestingly, all of these negative regulators bind to the central to Cterminal region of PGC-1α, suggesting that these regulators would have little or no inhibitory effect on NT-PGC-1α function in the nucleus.
The present study was designed to investigate the effect of known PGC-1α repressors on NT-PGC-1α function and found that Twist-1 plays a differential role in the regulation of PGC-1α and NT-PGC-1α activity in brown adipocytes. Twist-1 significantly suppressed PGC-1α-mediated activation of CPT1β, UCP1, and ERRα by docking to the C-terminal region of PGC-1α. In contrast, NT-PGC-1αdependent induction of these genes was not affected by Twist-1 due to lack of interaction with Twist-1.

Cell Cultures and Brown Adipocyte
Differentiation. COS-1 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). Immortalized PGC-1α-deficient mouse brown preadipocyte cell lines expressing empty vector, PGC-1α, or NT-PGC-1α [16] were maintained in DMEM supplemented with 10% FBS, 1% glutamine, and 1% penicillin/streptomycin. Preadipocytes were grown to confluence in culture medium supplemented with 20 nM insulin and 1 nM T3 (differentiation medium). Differentiation of brown adipocytes was induced (day 1) by incubating the cells in differentiation medium supplemented with 0.5 mM isobutylmethylxanthine (IBMX), 0.5 μM Dexamethasone, and 0.125 mM indomethacin for 48 hours. Thereafter, the cells were maintained in differentiation medium until day 7, followed by treatment with dibutyryl cAMP for 4 h.

Retroviral Infection. GP-293 cells were cotransfected
with pVSV-G and pBABE-zeo or pBABE-zeo-Twist-1 using Profection transfection system (Promega). Following transfection, the cells were incubated at 32 • C to increase viral titer. Virus-containing medium was collected, filtered through the 0.45 μm filter, and used to infect target cells. Immortalized PGC-1α-deficient mouse brown preadipocyte cells that ectopically express empty vector, PGC-1α, or NT-PGC-1α [16] were infected with the viral supernatant supplemented with 8 μg/mL polybrene. The medium was aspirated after 2 h and replaced with fresh viral supernatant, and the procedure was repeated. After 8 h of infection, the cells were replaced with fresh DMEM medium supplemented with 10% FBS, 1% glutamine, and 1% penicillin/streptomycin. Selection was initiated with zeocin (Invitrogen) 48 h after infection.

Real-Time PCR Analysis.
Total RNA was isolated from brown adipocytes using Tri-Reagent (Molecular Research Center) and RNeasy kits (Qiagen). For quantitative RT-PCR analysis, 2 μg of RNA samples were reverse transcribed using oligo dT primers and M-MLV reverse transcriptase (Promega), and 4 ng of cDNA were used in quantitative PCR reactions in the presence of a fluorescent dye (Cybergreen, Takara) on Applied Biosystems 7900 (Applied Biosystems). Relative abundance of mRNA was normalized to that of cyclophilin mRNA. The primers for Twist-1, UCP1, CPT1β, Cox7a1, ERRα, VLCAD, PPARα, and cyclophilin were previously described [15,16,24].

Twist-1 Negatively
Regulates PGC-1α but Not NT-PGC-1α. Brown adipose tissue expresses two PGC-1α isoforms (PGC-1α and NT-PGC-1α), both of which regulate transcription of mitochondrial and thermogenic genes by promoting the activity of several nuclear receptors including PPARs [15][16][17]. RIP140 and Twist-1, which have been shown to negatively regulate PGC-1α activity in adipose tissue, were  (Figure 1(a)). To assess the ability of these transcriptional regulators to modulate NT-PGC-1α activity, we carried out transient cotransfection and luciferase reporter assays as described in Materials and Methods. For a transcriptional repression assay with RIP140, Gal4-DBD-fused ERRα-LBD was used since RIP140 in part decreases transcriptional activity of full length ERRα [25]. The transcriptional activity of Gal4-ERRα-LBD was not affected by RIP140 (data not shown). Coexpression of RIP140 with PGC-1α significantly inhibited the ability of PGC-1α to increase Gal4-ERRα-LBD-mediated transcription of the reporter gene (Figure 1(b)). Similarly, RIP140 suppressed NT-PGC-1α-mediated induction of the reporter gene (Figure 1(b)), indicating that amino acids 186-270 in NT-PGC-1α are sufficient for RIP140 binding and repression. A transcriptional repression assay with Twist-1 showed that Twist-1 largely suppressed the ability of PGC-1α to increase PPARγ-mediated transcription, whereas NT-PGC-1α-dependent increase of reporter gene expression was not affected by Twist-1 (Figure 1(c)).

Twist-1 Does Not Interact with NT-PGC-1α.
Twist-1 suppresses PGC-1α activity by docking to the C-terminal domain of PGC-1α (Figure 1) [24]. To test that no repression of NT-PGC-1α activity by Twist-1 is due to lack of interaction between two proteins, Flag-Twist-1 was co-expressed with PGC-1α-HA or NT-PGC-1α-HA in COS-1 cells and immunoprecipitated with IgG and anti-Flag antibody. PGC-1α was efficiently coprecipitated with Twist-1 but not with IgG control (Figure 2(a)). In contrast, NT-PGC-1α was not coimmunoprecipitated with Twist-1 (Figure 2(b)), suggesting that Twist-1 is not recruited to NT-PGC-1α target genes.

Discussion
We previously reported that PGC-1α and NT-PGC-1α regulate a number of mitochondrial and thermogenic genes in brown adipose tissue [15][16][17]. Sympathetic stimulation of BAT by cold increases transcription of the PGC-1α gene by activating and recruiting cAMP-dependent transcription factors, ATF2 and CREB, to the PGC-1α promoter [1,27]. Subsequent normal and alternative splicings of the transcribed RNA produce comparable mRNA levels of PGC-1α and NT-PGC-1α, respectively [15]. However, two transcripts produce structurally different proteins that possess fundamental differences in their protein size, stability, and localization. These different natures of two PGC-1α isoforms require different regulatory mechanisms to increase their transcriptional activity in response to the same signaling inputs. For example, cold/cAMP-activated p38 MAPK phosphorylation leads to stabilization and activation of PGC-1α protein by preventing its proteosomal targeting [8]. In contrast, cAMP-activated PKA phosphorylation increases nuclear . Relative abundance of mRNA levels was normalized to that of cyclophilin mRNA. Data represent mean ± SEM of at least four independent experiments. * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P < 0.0001; ns: not significant.

PPAR Research
accumulation of NT-PGC-1α and subsequent recruitment to the transcriptional complexes [16].
Here we show an additional regulatory mechanism that differently modulates transcriptional activity of PGC-1α and NT-PGC-1α in the nucleus of brown adipocytes. The mode of action is mediated by direct interaction of PGC-1α with a negative regulator Twist-1, which is abundantly expressed in brown adipocytes. Twist-1 is recruited to PGC-1α target genes by docking to the C-terminal region of PGC-1α and inhibits their expression by subsequently recruiting the histone deacetylase HDAC5 to the PGC-1α target gene promoters (e.g., UCP1 and CPT1) [24]. In contrast, Twist-1 has no inhibitory effect on NT-PGC-1α-mediated induction of NT-PGC-1α target genes since NT-PGC-1α does not recruit Twist-1 to its target gene promoters. Despite potential inhibition of all PGC-1α target genes by Twist-1, Twist-1 suppresses only a subset of PGC-1α target genes, including UCP1, CPT1β, and ERRα. Twist-1 is a basic helix-loop-helix (bHLH)-containing transcription factor that binds to the canonical E-box and the related sequences in the regulatory regions of target genes [28], thus raising a possibility that the presence of potential E-boxes on the PGC-1α target gene promoters further specifies a subset of PGC-1α/Twist-1 target genes. However, it seems unlikely that subsequent docking of Twist-1 to the potential E-boxes on the PGC-1α target gene promoters is required for its inhibitory effect since Twist-1-mediated suppression does not depend on its DNA-binding activity [24]. Instead, Twist-1 exerts its transcriptional repression on PGC-1α target genes by altering chromatin conformational states by recruitment of histone deacetylases (HDAC) [24]. Thus, it is likely that subsequent recruitment of additional regulators (e.g., histone deaceylases) to the PGC-1α target gene promoters by Twist-1 is required for its suppression of PGC-1α-mediated gene expression.

Conclusion
Our findings demonstrate a differential regulation of PGC-1α and NT-PGC-1α activity by Twist-1 in brown adipocytes. Twist-1 suppresses PGC-1α-mediated transcriptional activation of a subset of PGC-1α target genes, including UCP1, CPT1β, and ERRα. In contrast, NT-PGC-1α-mediated induction of these genes is not affected by Twist-1.