Abstract Elucidating new control mechanisms for cholesterol homeostasis that affect atherosclerosis will advance our understanding of fundamental physiological processes and may suggest new opportunities for intervention in human metabolic disease. We propose to define the mechanism of action and physiological function of two new LXR-dependent regulatory circuits mediated by two novel E3 ubiquitin ligases. Molecular mechanisms mediating crosstalk between the SREBP-2 and LXR pathways are poorly understood. Using an RNA sequencing strategy to identify new LXR targets, we have uncovered two uncharacterized genes–Rnf145 and Rnf186–that may mediate critical pathways for crosstalk between LXR and SREBP signaling. Rnf145 and Rnf186 encode RING domain-containing proteins predicted to be E3 ubiquitin ligases; however, their ubiquitination targets and biological function are unknown. Our preliminary data have revealed that both RNF145 and RNF186 are previously unrecognized modulators of SREBP-2 signaling. Manipulation of Rnf145 and Rnf186 expression in mouse liver regulates SREBP-2-dependent gene expression and plasma cholesterol levels by distinct but complementary mechanisms. Based on these findings, we propose a series of molecular, cell biological and physiological studies to define the functions of RNF145 and RNF186 in cholesterol homeostasis and their contribution to LXR-SREBP crosstalk. Aim 1 is to elucidate the function and mechanism of action of RNF145 in lipid metabolism. Aim 2 is to elucidate the function and mechanism of action of RNF186 in lipid metabolism. Aim 3 is to determine the physiological importance of RNF145 and RNF186 for LXR- SREBP crosstalk and atherosclerosis.