## 3 Results
### Prdm16 Directly Binds and Activates _Ucp1_ Promoter  
As a transcription factor, Prdm16 often regulates gene expression by binding to the promoter region of the target gene. To determine the mechanism by which Prdm16 regulates _Ucp1_, we performed a double luciferase reporter experiment to quantitate promoter activation. Compared to the empty vector control, co-transfection of _p_CMV-Prdm16 with the \(-2.0\) kb _Ucp1_ promoter-luciferase resulted in an obvious activation of the _Ucp1_ promoter (Figure 4A) (_p_ < 0.01).  
Figure 2: C3G was able to up-regulate the expression of _Prdm16_ gene both in vitro and vivo. (**A**) Effect of C3G at different concentrations on _Prdm16_ gene expression in C3H10T12 cells. (**B**,**C**) Gene expression profile in BAT, real-time PCR analysis of thermogenic-related genes, including _Prdm16_, PPAR\(\alpha\) in db/db mice model (**B**) and mice with DIO model (**C**) (_n_ = 6–8). Values represent means SD. Error bars represent SD; mean values with asterisk or different letters are significantly different (* means \(p\) < 0.05; ** means \(p\) < 0.01).  
Figure 4: Prdm16 capable of activating _Ucp1_ promoter (**A**) Effect of Prdm16 on _Ucp1_ promoter (double luciferase report gene method) (**B**) PCR electrophoresis diagram and sequencing comparison diagram of pLacZi-Ucp1 bacterial solution. M: [PERSON] 2008 PLA DD:Lane 1–4: PCR products from the picked four single-colony bacterial solutions. **C**) PCR electrophoresis diagram and sequencing comparison diagram of pJG-Prdm16 bacterial solution. M: [PERSON] 2008 PLA DD: Lane 1–4: PCR products from the pickeded four single-colony bacterial solutions. **D**) Effect of Prdm16 on _Ucp1_ promoter (yeast one-hybrid assay) (**E**) Clone electrophoretic images of Ucp1 promoter with different lengths. **F**) PGL3-Basic enzyme digestion electrophoresis diagram **G**) PCR electrophoresis map of bacterial solution after ligation of _Ucp1_ promoter with different lengths to pGL3-Basic. **H**) Effects of Prdm16 on promoters of different fragments _Ucp1_. Compared with control, ** means \(p\) < 0.01; *** means \(p\) < 0.001.  
Figure 3: Overexpression of _Prdm16_ upregulates _Ucp1_ gene expression in brown adipose cells (**A**) Clone _Prdm16_ electrophoretic map, M: [PERSON] 2008 PLA DD; Lane 1–8: _Prdm16_ amplification product. **B**) PCR electrophoresis diagram of pCMV-Prdm16 bacterial solution; M: [PERSON] 2008 PLA DD; Lane 1–11: PCR products from the picked bacterial solutions of 11 single colonies. **C**) Comparative diagram of pCMV-Prdm16 sequencing results. **D**) Effect of overexpression of _Prdm16_ on _Ucp1_ gene expression C3H10T12 cells. **(E**) Effects of shPRDM16 on Ucp1 gene expression during C3H10T12 cell differentiation. Compared with control, ** means \(p\) < 0.01.  
This interaction of _Prdm16_ with the _Ucp1_ promoter was subsequently reverified. A _p_LacZi-Ucp1 reporter vector and _p_]G-Prdm16 fusion expression vector were constructed and transfected into yeast for blue-steak screening (Figure 4B-D). Similar to the positive control group of Plazzi+ and PJG+, treatment groups transfected with _p_LacZi-_Ucp1_ and _p_]G-Prdm16 were blue and dark in color. The results verified by the single yeast hybrid assay showed that _Prdm16_ could directly bind to the _Ucp1_ promoter.  
To start defining how Prdm16 activates the _Ucp1_ promoter, 5'deletions of the _Ucp1_ promoters driving a luciferase reporter were generated and co-transfected along with _p_CMV-Prdm16 into 293 cells (Figure 4E-G). All _Ucp1_ promoter constructs deleted down to \(-500\) bp, thus showing remarkable activation upon _Prdm16_ co-transfection. However, _Ucp1_ promoter activation by Prdm16 was lost when the promoter was deleted to \(-150\) bp, indicating that Prdm16 functions through the sequence from \(-500\) to \(-150\) bp (Figure 4H).