In Situ Saturating Mutagenesis Screening Identifies a Functional Genomic Locus that Regulates Ucp1 Expression

Abstract

A better understanding of the molecular mechanisms that control the UCP1 expression in brown and beige adipocytes is essential for us to modulate adipose cell fate and promote thermogenesis, which may provide a therapeutic view for the treatment of obesity and obesity-related diseases. To systematically identify cis-element(s) that transcriptionally regulates Ucp1, we here took advantage of the high-throughput CRIPSR-Cas9 screening system, and performed an in situ saturating mutagenesis screen, by using a customized sgRNA library targeting the ~ 20 kb genomic region near Ucp1. Through the screening, we have identified several genomic loci that may contain key regulatory element for Ucp1 expression in cultured brown and white adipocytes in vitro, and in inguinal white adipose tissue in vivo. Our study highlights a broadly useful approach for studying cis-regulatory elements in a high-throughput manner.

Keywords: Brown and beige adipocytes; In situ saturating mutagenesis screen; UCP1; sgRNA-9768.

Fig. 1

In situ saturating mutagenesis screening identifies a functional genomic locus that regulates Ucp1 expression. a Schematic view of Ucp1 sgRNA library design for in situ cis-element screen with the Ucp1-GFP reporter cell line. b Workflow of the screening strategy. c Scatterplot displaying enriched or depleted sgRNAs in UCP1_pos group as compared to UCP1_all group. TSS, transcriptional start site. d Ucp1 mRNA expression analyses in brown adipocytes treated with individual sgRNAs as indicated. n = 4. e Representative images of GFP intensity in brown adipocytes treated with individual sgRNAs. Scale bar = 50 µm. f UCP1 protein expression analyses in white adipocytes under basal level or upon rosiglitazone stimulation, treated with or without indicated sgRNAs. g Oxygen consumption analysis in brown adipocytes under basal level treated with sgRNA -9768 or control vectors. n = 5. h Schematic illustration of the CRISPR-Cas9 KI mice and AAV constructs used in this study (left). In vivo luminescence analysis after 2 weeks’ injection of AAV vectors (right). i Genome editing activity in inguinal white adipose tissues from mice treated with sgRNA − 9768 or control vectors. j UCP1 protein expression analyses in inguinal white adipose tissues from control or sgRNA − 9768-treated mice after exposure to cold (4 °C) for 2 days. n = 7