Project Summary/Abstract Obesity and its metabolic sequelae are rapidly increasing in the United States and worldwide, leading to high morbidity and mortality in type 2 diabetes, cardiovascular disease, and certain cancer. Under the current pandemic, obesity has been recognized as a key risk factor for severe COVID-19. Central to these pathologies is adipose tissue. There are functionally distinct types of adipose tissue. White adipose tissue is the primary site of the triglyceride storehouse. In contrast, thermogenic fat, which consists of classical brown adipose tissue (BAT) and inducible beige/brite adipocytes, concentrates on thermogenic energy expenditure. It has been recently reported that people with BAT have a significantly lower prevalence of cardiometabolic diseases, highlighting the metabolic benefits and therapeutic potential of BAT in humans. To make the therapeutics possible, improved knowledge of the regulation of thermogenic adipocytes is urgently needed. The thermogenic function of brown and beige adipocytes are coordinately regulated by specific transcriptional and epigenetic regulators. While transcription of the thermogenic gene uncoupling protein 1 (Ucp1) in response to beta-adrenergic stimulation has been broadly studied, little is known about how histone positioning and chromatin folding influences the expression of Ucp1 and other thermogenic genes. Using an unbiased CRISPR-based screen, we identified the histone variant H2A.Z and the LIM domaining-containing zinc-finger protein Crip2 as trans-acting factors recruited to the Ucp1 promoter/enhancer region by beta3-adrenergic receptor stimulation. Importantly, deletion of H2A.Z or Crip2 in mature brown adipocytes not only impeded Ucp1 transcription, but also reduced the expression of multiple thermogenic genes and led to impaired cellular thermogenesis. This proposal will determine the signaling events mediating the activation of Crip2 and H2A.Z deposition and the impact of Crip2 or H2A.Z deficiency in the cellular thermogenesis and bioenergetic profiles of thermogenic adipocytes murine and human origins. Since histone variants play an important role in determining chromatin remodeling, we will examine how Crip2-H2A.Z interaction influences chromatin architecture, thereby regulating thermogenic transcription and cellular respiration. To establish the physiological significance of Crip2 and H2A.Z in metabolic regulation, we will generate brown fat-specific Crip2 or H2A.Z knockout mice and thoroughly characterize their metabolic phenotypes. Completing the proposed studies will increase fundamental knowledge on the role of chromatin remodeling in the regulation of thermogenic program and pave ways to establish new therapeutic approaches for combating metabolic diseases.