Project 3 - Project Summary In addition to the well-studied circadian rhythm, a cluster of genes that cycle at the second (12h period) harmonic of circadian rhythmicity was discovered in several peripheral mouse tissues in vivo. Genes exhibiting apparent 12h rhythmicity were enriched in endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways, which are universally conserved adaptive responses to cope with accumulation of unfolded protein in the ER. Despite these initial findings, the exact prevalence of the 12h rhythm in vivo, its relationship with the circadian clock, how the 12h rhythm is established at the molecular level and its precise roles in regulating both physiology and pathology still remain elusive. We recently developed a novel mathematical approach to decompose time-series gene expression data and reveal hidden oscillations separate from the 24h rhythmicity. We unexpectedly discovered that, in addition to the UPR genes, the 12h rhythmicity is much more prevalent than was initially thought and is widely found in metabolic genes in mouse liver. We further uncovered prevalent 12h oscillations in liver metabolism in vivo. The fact that the 12h rhythmicity remains intact in the absence of functional 24h circadian clock suggests that the 12h rhythm is established and maintained by an independent clock component distinct from the 24h circadian clock. The objective of this proposal is to use the liver as a model organ to test the hypothesis that: 1) there is an equally important molecular clock establishing the 12h period rhythmicity, which coordinates oscillations of ER stress and dynamic bioenergetic metabolism to ensure systemic homeostasis, and 2) that the hepatic 12h clock is transcriptionally regulated by SRC-3 and XBP1s. In Aim 1 the transcriptional regulation of the 12h rhythm of gene expression and metabolism will be investigated with a focus on the interplay between UPR TF XBP1 and coactivator SRC-3 using ChIP-Seq, RNA-Seq, metabolomics and mathematical modeling approaches. In Aim 2, whether XBP1s/SRC-3 dependent 12-hour clock dysregulation contributes to chronic ER stress-induced NAFLD will be determined. In Aim 3, whether XBP1s/SRC-3 dependent 12-hour clock dysregulation contributes to nutritional challenge-induced NAFLD will be determined.