Identification of Novel Transcriptional Regulators of Uncoupling Protein 1 Using a Modified CRISPR-Cas9 Methodology

Obesity is an epidemiological dilemma due to its associated comorbidities such as diabetes, cancer, and heart disease. Because traditional weight loss strategies such as diet and exercise are thus far ineffective at curtailing the growing prevalence of obesity, new therapeutic approaches are being sought after. In this regard, brown adipose tissue (BAT) has garnered attention due to its ability to burn fat as fuel during thermogenesis, which is carried out by the mitochondrial uncoupling protein 1 (UCP1). Since UCP1 is mainly regulated at the level of transcription, our laboratory sought to identify novel transcriptional regulators of UCP1 using a modified CRISPR-Cas9 methodology termed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) (Fujii and Fujita, 2015). enChIP takes advantage of dCas9, which lacks endonuclease activity while maintaining functional DNA binding activity, fused to a 3xFLAG tag that can be used for immunoprecipitation. For our study, we delivered five guide RNAs (gRNAs) targeting the Ucp1 promoter region in a mouse-derived mature brown adipocyte line that had been modified to endogenously express the dCas9-3XFLAG fusion protein. Following treatment with either vehicle or CL316,243, we immunoprecipitated the resulting Ucp1/dCas9/3XFLAG complex using an anti-FLAG antibody. We then utilized tandem liquid chromatography-mass spectrometry for an unbiased proteomic screen to evaluate proteins enriched by β3-adrenergic stimulation. Three biological replicates revealed 17 enriched proteins in mature adipocytes following β-adrenergic stimulation. These included histone variants and proteins involved in transcriptional regulation, such as H2A.Z and CRIP2, respectively. These identified proteins have the potential to be manipulated to regulate the expression of UCP1, thereby affecting the thermogenic function of BAT and systemic metabolism.