Since traditional weight loss strategies have been unsuccessful at slowing the increasing rate of obesity, alternative strategies are in high demand. Brown adipose tissue (BAT) is a thermogenic tissue that efficiently expends energy. Thermogenesis in BAT occurs through the action of uncoupling protein 1 (UCP1). Identifying novel transcriptional regulators of UCP1 may lead to potential targets to increase BAT activity since its expression is under tight transcriptional control. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) is a modified CRISPR/Cas9 methodology that identifies novel transcriptional regulators. EnChIP uses dCas9 (lacks endonuclease activity) and guide RNAs (gRNAs) targeting a specific promoter region. The DNA-bound dCas9 can be immunoprecipitated, and the associated promoter region/transcriptional machinery can be sent for proteomic analysis to identify novel transcriptional regulators. Following β-adrenergic stimulation, which activates UCP1 gene transcription in brown adipocytes, we performed enChIP by using five gRNAs targeting the UCP1 promoter. EnChIP analysis of the UCP1 promoter revealed cysteine-rich protein 2 (CRIP2) as a putative transcriptional regulator of UCP1, which was validated by ChIP-PCR. We demonstrated that knocking down CRIP2 in brown adipocytes significantly decreased β-adrenergic-induced UCP1 expression and heat production. Mechanistically, we demonstrated that following β-adrenergic stimulation, CRIP2 was phosphorylated and translocated from the cytosol to the nucleus, where it interacted with peroxisomal proliferator-activated receptor gamma (PPARγ). Taken together, using the unbiased CRISPR-based enChIP approach, we identify CRIP2 as a novel transcriptional regulator that modulates UCP1 expression in response to β-adrenergic stimulation. These findings pave the foundation for developing new anti-obesity and antidiabetes therapies.
J. Darcy: None. C. Wang: None. M. Lundh: Employee; Self; Gubra. F. Shamsi: None. M. Lynes: None. T. d'Aprigny: None. B. Emanuelli: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.
National Institutes of Health (R01DK077097, R01DK102898, T32DK007260)