Project3-ProjectSummary Inadditiontothewell-studiedcircadianrhythm,aclusterofgenesthatcycleatthesecond(12hperiod)harmonic ofcircadianrhythmicitywasdiscoveredinseveralperipheralmousetissuesinvivo.Genesexhibitingapparent 12h rhythmicity were enriched in endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways,whichareuniversallyconservedadaptiveresponsestocopewithaccumulationofunfoldedproteinin the ER. Despite these initial findings, the exact prevalence of the 12h rhythm in vivo, its relationship with the circadianclock,howthe12hrhythmisestablishedatthemolecularlevelanditspreciserolesinregulatingboth physiology and pathology still remain elusive. We recently developed a novel mathematical approach to decomposetime-seriesgeneexpressiondataandrevealhiddenoscillationsseparatefromthe24hrhythmicity. Weunexpectedlydiscoveredthat,inadditiontotheUPRgenes,the12hrhythmicityismuchmoreprevalentthan wasinitiallythoughtandiswidelyfoundinmetabolicgenesinmouseliver.Wefurtheruncoveredprevalent12h oscillationsinlivermetabolisminvivo.Thefactthatthe12hrhythmicityremainsintactintheabsenceoffunctional 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,whichcoordinatesoscillationsofERstressanddynamicbioenergeticmetabolismtoensuresystemic homeostasis,and2)thatthehepatic12hclockistranscriptionallyregulatedbySRC-3andXBP1s.InAim1the transcriptionalregulationofthe12hrhythmofgeneexpressionandmetabolismwillbeinvestigatedwithafocus ontheinterplaybetweenUPRTFXBP1andcoactivatorSRC-3usingChIP-Seq,RNA-Seq,metabolomicsand mathematical modeling approaches. In Aim 2, whether XBP1s/SRC-3 dependent 12-hour clock dysregulation contributestochronicERstress-inducedNAFLDwillbedetermined.InAim3,whetherXBP1s/SRC-3dependent 12-hourclockdysregulationcontributestonutritionalchallenge-inducedNAFLDwillbedetermined.