A Role for The CRL4-WDTC1 E3 Ligase in the Suppression of Adipogenesis

Groh, Beezly Sultana

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March 19, 2019

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Groh, Beezly Sultana
- Affiliation: School of Medicine, Department of Biochemistry and Biophysics

Abstract

Excess lipid accumulation in fat tissues underlies obesity and a myriad of associated pathologies that range from type 2 diabetes to cancer. Lipid synthesis in adipocytes, the primary cells of fat tissues, is regulated via extracellular and intracellular mechanisms. WDTC1 encodes an evolutionarily conserved suppressor of lipid accumulation in multicellular organisms. Decreased WDTC1 expression is associated with obesity in mice and humans. Yet, the molecular mechanism underlying its anti-obesity function remains elusive. WDTC1 is a candidate DWD protein (DDB1 binding WD40 repeat protein) that potentially functions as a substrate specificity factor for a cullin 4 E3 ligase (CRL4) complex. I hypothesized that WDTC1 mediates its anti-adipogenic effect through targeting substrates for ubiquitylation by the CRL4 complex. In this dissertation, I aim to understand the molecular function of WDTC1 in adipogenesis. I demonstrate that WDTC1 is indeed a CRL4 substrate receptor and its interaction with a CRL4 complex is central to its function. Using 3T3-L1 cell culture model of adipogenesis, I show that WDTC1 mutations disrupting its interaction with CRL4 impair the suppression of lipid accumulation and increase adipogenic gene expression. Rescue experiments demonstrate that the WDTC1 knockdown phenotypes can be rescued by ectopic expression of wild-type but not CRL4 binding mutants, underscoring the critical importance of the CRL4-WDTC1 interaction to the observed adipogenic suppression. In addition, I found that Cul4a knockout mice exhibit defects analogous to those reported in Wdtc1+/- mice, such as adipocyte hypertrophy and poor metabolic parameters. Mechanistically, I examined whether WDTC1 plays a role in transcriptional control. I provide evidence that the CRL4-WDTC1 complex promotes histone H2AK119 monoubiquitylation, an epigenetic modification that is associated with transcriptional silencing. Hence, I propose that CRL4-WDTC1 E3 ligase may mediate its anti-adipogenic effect, at least in part, by repressing a subset of proadipogenic genes through histone H2AK119 monoubiquitylation. I also describe proteomic screens in 3T3-L1 cells to identify WDTC1 interacting proteins via mass spectrometry. Collectively, this work reveals a function of the CRL4-WDTC1 complex in adipogenesis and provides a potential mechanism by which WDTC1 suppresses lipid accumulation in adipocytes.

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