Molecular Mechanisms And Functions Of NADPH In Regulating Epigenetic

Objective:Metabolic activities are the basis for living systems,which provide essential nutrients and signaling molecules for cell growth.Interestingly,recent studies have exhibited that both metabolites and metabolic enzymes may be involved in cell fate determination through their non-metabolic effects.Accordingly,there is limited understanding of metabolic processes.NADPH provides reducing equivalents for reductive biosynthesis and antioxidant defence.And NADPH level is delicately regulated to maintain stable.Importantly,increasing evidence have suggested that NADPH may be another rate-limiting step for cell growth and proliferation.However,it remians unclear about the underlying mechanisms.In addition,malic enzymes play important roles in regulating cell growth and senescence.But it remians poorly understand how these metabolic pathways function to determine cell fate.In this study,we explore the potential mechanisms by which malic enzymes regulate cell fate.More importantly,we identify a novel function of NADPH in regulating epigenetic state.Methods:To explore the effect of malic enzymes in regulating adipocyte differentiation and gene transcription,expression levels of malic enzymes were altered in preadipocyte line 3T3-L1 and other tumor cell lines.And histone acetylation levels were detected.To explore the mechanism by which malic enzymes affect histone acetylation levels,enzymatic activities of HAT and HDAC in malic enzyme-knockdown cells were detected.Meanwhile,HDAC expression was inhibited by a series of inhibitors and small interfering RNAs to determine the specific HDAC isoforms that mediate the effect of malic enzymes to regulate histone acetylation.To explore the potential effect of malic enzymes in regulating chromatin remodeling,ChIP and ChIP-seq experiments were performed to detect the peaks of acetylated histones when altered malic enzyme expression.To determine whether malic enzyme regulates histone acetylation levels through HDAC3,we counted the intersection of the peaks of HDAC3 and peaks of acetylated histones when altered malic enzyme expression.To explore the potential mechanism of NADPH in inhibiting HDAC3 activity,NADPH levels were detected in both cells and animals with reduced malic enzyme expression.And further detected the effect of NADPH on HDCA3 enzyme activity.Importantly,we investigated the direct binding between HDAC3 and NADPH,as well as NAD+,NADH and NADP+ through SPR,DARTS and CETSA.To further determine the effect of intracellular NADPH content on HDAC3 enzyme activity,intracellular NADPH levels were increased by X-tremeGENE HP DNA transfection reagent as well as by overexpressing G6PD.On the contrary,intracellular NADPH levels were decreased by TPNOX system.And we detected the corresponding changes in HDAC3 activity as well as histone acetylation levels.Furthermore,through co-immunoprecipitation,molecular docking and molecular dynamics simulation,the molecular mechanism of NADPH in inhibiting HDAC3 enzymatic activity was explained from molecular and structural perspectives.Finally,by supplementing NADPH in malic-knockdown cells,we explored whether malic enzymes regulate gene expression through NADPH.We further verified the universality of this mechanism that NADPH regulates histone acetylation through HD AC3 by detecting in a variety of tumor cell lines as well as altering the expression of different metabolic enzymes that supply NADPH.Results:1.When expression of malic enzymes was inhibited,the acetylation level of histones was significantly down-regulated,and gene transcription level was down-regulated in both cell and animals.2.Malic enzyme regulates histone acetylation levels by inhibiting HDAC3 activity,thereby regulating gene expression.3.Malic enzymes regulate chromatin modulation by inhibiting HDAC3 activity.4.NADPH can directly bind and inhibit HDAC3 activity.5.Malic enzymes inhibit HDAC3 activity by generating NADPH,and eventually affect histone acetylation.6.NADPH competes with Ins(1,4,5,6)P4 to bind HDAC3,and inhibits the binding of HDAC3 to Ncor-DAD and Ins(1,4,5,6)P4,thereby inhibiting the enzymatic activity of HDAC3,which ultimately affects histone acetylation levels.7.It may be a general rule for NADPH to regulate histone acetylation through HDAC3.Conclusion:NADPH inhibits HDAC3 activity by binding to histone deacetylase HDAC3,and eventually affects histone acetylation levels.This study suggests that NADPH can function as a signaling molecule to participate in the regulation of epigenetic status.While HDAC3 acts as an intracellular NADPH sensor,thereby regulating histone acetylation and gene transcription.