Uncover Ucp1's BAT - obesity - related mechanism-tow-int

Tow-Int Tech helps to uncover the regulatory mechanism of uncoupling protein 1 (Ucp1) in brown adipose tissue (BAT), providing potential targets for the treatment of obesity and metabolic diseases.

Recently, researchers such as Duo Su and Tingting Jiang studied the regulatory mechanism of the Ucp1 gene in brown adipose tissue (BAT). Through various experimental methods, they identified the distal enhancer of Ucp1, analyzed its effects on thermogenesis and mitochondrial function, and published a research paper titled "Identification of a distal enhancer of Ucp1 essential for thermogenesis and mitochondrial function in brown fat" in the journal Communications Biology under Nature. This study provides new targets for the treatment of obesity and related metabolic diseases.

1. Research Background

Brown adipose tissue (BAT) regulates body temperature through non - shivering thermogenesis, and uncoupling protein 1 (Ucp1) is a key protein in this process. The three - dimensional structure of the genome is crucial for gene expression, and enhancers can regulate gene expression. However, the enhancer - mediated regulatory mechanism of Ucp1 remains unclear.

2. Experimental Methods

2.1 4C - seq analysis: Conduct 4C - seq experiments on interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) of mice to analyze the chromatin interactions of Ucp1, and determine reliable interaction sites and differential interaction sites.

2.2 Enhancer identification: Analyze the potential enhancers of Ucp1 by combining public datasets. Verify their activities through luciferase reporter assays, and analyze the histone modifications and chromatin interactions in the enhancer regions.

2.3 Functional studies: Use the CRISPR - dCas9 - KRAB system to inhibit the enhancers, and detect indicators such as Ucp1 expression, mitochondrial function, and lipid metabolism. Study the regulatory effects of related proteins and factors on the enhancers through methods such as RNA interference and ChIP - qPCR. In vivo, inhibit the Ucp1 enhancer in iBAT by lentivirus injection, and observe the effects on mouse body temperature, energy metabolism, and mitochondrial function.

Through methods such as RNA interference (siRNA - mediated knockdown of RAD21) and 3C - qPCR, study the regulatory effect of the chromatin loop mediated by cohesin on the interaction between Ucp1 - En4 and the Ucp1 promoter. Hi - C data analysis shows that the two are located in the same TAD. After knocking down RAD21, the interaction strength between Ucp1 - En4 and the Ucp1 promoter decreases, and Ucp1 expression also significantly decreases, indicating that cohesin is crucial for this interaction and the regulation of Ucp1 expression.

Use methods such as ChIP - qPCR to study the regulatory effects of EBF2 and CBP on the activity of the Ucp1 - En4 enhancer. DNA pull - down and ChIP - seq analyses identified the transcription factors that bind to Ucp1 - En4. ChIP - qPCR verified the binding of EBF2 and CBP and their effects on the H3K27ac level. Luciferase reporter assays showed that the two factors synergistically increase the transcriptional activity of Ucp1 - En4, and this regulatory mechanism is conserved in mice and humans.

Presents the experimental results of inhibiting Ucp1 - En4 in iBAT in vivo. Immunofluorescence microscopy confirmed successful lentivirus infection. qRT - PCR and Western blot were used to detect the expression changes of Ucp1 and lipogenesis - related genes. Metabolic studies and infrared imaging were used to analyze indicators such as mouse oxygen consumption, thermogenesis, and body temperature. It was found that inhibiting Ucp1 - En4 affects iBAT thermogenesis but does not affect whole - body energy consumption. Transmission electron microscopy and qPCR were used to detect mitochondrial morphology and function, indicating that mitochondrial function is impaired under cold - adapted conditions.

3. Experimental Results

3.1 Characteristics of the Ucp1 chromatin interaction landscape: There are differences in Ucp1 chromatin interactions between iBAT and eWAT. There are more interaction sites in iBAT, and some sites are up - regulated in iBAT. Four iBAT - specific Ucp1 - active enhancers were identified, three of which are activated by cold stimulation.

3.2 Functional verification of enhancers: Ucp1 - En4 and Ucp1 - En6 can regulate Ucp1 expression in brown adipocytes. Inhibiting these two enhancers reduces Ucp1 expression, affects mitochondrial function, leads to an increase in lipid droplet size and an increase in the expression of lipogenesis - related genes.

3.3 Regulatory mechanism: The interaction between Ucp1 - En4 and the Ucp1 promoter is regulated by a chromatin loop mediated by cohesin, and the RAD21 subunit is involved. EBF2 and CBP synergistically regulate the activity of Ucp1 - En4, and this mechanism is conserved in mice and humans.

3.4 In vivo experimental results: Inhibiting Ucp1 - En in iBAT in vivo reduces Ucp1 expression, affects the thermogenic capacity and mitochondrial function of iBAT, and decreases the body temperature of mice, but does not affect whole - body energy consumption.

4. Research Significance

This study reveals the regulatory mechanism of Ucp1 in BAT, identifies key enhancers, provides potential targets for the treatment of obesity and metabolic diseases, and deepens our understanding of the molecular mechanism of thermogenesis in brown fat.

5. Experimental Instruments Used in the Experiment

5.1 High - throughput sequencing platform: Sequences the 4C - seq library to analyze the chromatin interactions of Ucp1 in iBAT and eWAT, determine its interaction sites and differences, etc., laying the foundation for subsequent research.

5.2 Microplate luminometer: Measures the luciferase activity and plays a role in the experiment to evaluate the activity of potential Ucp1 enhancers. The effectiveness of the enhancer is judged by detecting the luciferase activity.

5.3 Real - time fluorescence quantitative PCR instrument: Conducts qRT - PCR experiments to detect gene expression levels, such as detecting the expression of Ucp1, Rad21, and lipogenesis - related genes (Acly, Acaca, Fasn), etc., to analyze the effects of related factors on gene expression.

5.4 Image processing and analysis software: Observes iBAT samples by transmission electron microscopy to detect mitochondrial morphology and structure. It was found that after inhibiting Ucp1 - En4, under specific conditions, the mitochondria were swollen and the cristae were irregular, indicating impaired mitochondrial function.

5.5 Transmission electron microscope: Performs animal fluorescence imaging. After mice are infected with lentivirus, it observes the infection and expression of the virus in iBAT to confirm the effectiveness of the experimental operation.

5.6 Animal energy metabolism monitoring system (Tow-Int Tech model: EM - 8M - WA)  Measures the whole - body energy metabolism of mice, including indicators such as oxygen consumption (VO₂) and thermogenesis, to study the impact of Ucp1 - En4 on the overall energy metabolism of mice.

5.7 Infrared thermal imaging camera: Obtains thermal images of mice and analyzes them through software to detect changes in mouse body temperature. It was found that after inhibiting Ucp1 - En4, the back skin temperature and rectal temperature of mice decreased, indicating its impact on body temperature regulation.

6. An animal energy metabolism system (Tow-Int Tech) is used in the study. 

It is used to measure the whole - body energy metabolism of mice, including metabolic parameters such as oxygen consumption (VO₂), carbon dioxide output (VCO₂), respiratory exchange ratio, food and water intake. It can monitor mice for a long time in their natural active state.

Experimental Methods 

Place the mice in metabolic cages in a room with controlled temperature and humidity. The room temperature is set to 4°C or 30°C, the relative humidity is 50%, and the light/dark cycle is 12 hours. Before monitoring, the mice need to be acclimatized in the metabolic cages for 24 hours. After acclimatization, use the animal metabolic monitoring system to measure the oxygen consumption and thermogenesis data of the mice to evaluate the energy metabolism status of the mice under different conditions, and thus explore the impact of related genes or enhancers on the energy metabolism of mice. For example, when studying the impact of Ucp1 - En4 on the whole - body energy metabolism of mice, observe whether the energy metabolism indicators of mice change after inhibiting Ucp1 - En4 through this system.

Reference

Su, D., Jiang, T., Song, Y. et al. Identification of a distal enhancer of Ucp1 essential for thermogenesis and mitochondrial function in brown fat. *Commun Biol* 8, 31 (2025). https://doi.org/10.1038/s42003 - 025 - 07468 - 3

We are committed to making your research easier, more accurate, and more efficient and helping you build confidence in your data! We have provided services for a large number of customers and have rich experiences in offering customized, professional solutions according to your needs.