Examining the Involvement of Bhlhe40 Transcription Factor in Peroxisome Biology and Oxidative Metabolism

Bhlhe40 (also known as Stra13, Dec1, Sharp2, or BHLHB2) is a transcription factor highly expression inskeletal muscle (SKM), where it regulates the activation of myogenic stem cells and protect SKM fromreactive oxidative species (ROS) induced damage. Recently, our group has demonstrated that Bhlhe40 canrepress the myogenic regulatory factors (MRFs, including Myf5, MyoD, Myogenin and Mrf4) activatedPGC-1α, a key regulator of oxidative metabolism, expression in SKM, which can be relieved when thecoactivator P/CAF is supplied in over-dose. Bhlhe40 and MRFs directly bind to PGC-1α promoter at theE1- and E2- boxes respectively, which reduces the interaction between MyoD and its key coactivator P/CAFand thus leading to repression of PGC-1α expression. SKM is a major metabolic organ that can adjust thebalance between glycolytic and oxidative metabolism to meet the energy requirement under variousphysiological conditions, and this adaptation relies much on the expression and activity of PGC-1α. Ourlatest discovery found that PGC-1α directly interacted with Bhlhe40 through their N-terminal domains andthey co-occupied PGC-1α targeted gene promoters/enhancers simultaneously, which in turn repressedPGC-1α transactivational activity on these cis-elements. Combining our previous studies, it suggests thatBhlhe40 regulates PGC-1α at both transcriptional and activity levels, therefore, its level and activity in thecells should have profound influence on cellular oxidative metabolism. It has been shown that PGC-1α playcritical roles in the regulation of the mitochondrial and peroxisomal biogenesis and function throughcoactivating the activity of various DNA-binding transcription factors, such as PPARγ, ERRα, and NRF1.As mitochondria and peroxisomes are the major sites for oxidative metabolism, their biogenesis andmetabolic activity should be intimately regulated by the action of Bhlhe40. Our preliminary results indicatethat perturbation of the Bhlhe40 expression level or blocking its repressive effect on PGC-1α in the myogeniccells has significant effect on the biology of both peroxisome and mitochondrion; however, the influence onperoxisome biology is more obvious than on that of mitochondria. Currently, we have already set upBhlhe40 over-expressed (Bhlhe40-OV) and knockdown (Bhlhe40-KD) stable clones of C2C12 myoblasts.The stable clones of VBH135 (a fusion protein of VP16 activator and Bhlhe40 N-terminal 1-135 amino acids)over-expressed cells (VBH135-OV) have also been established. In the near future, we will acquire Bhlhe40knockout (Bhlhe40-KO) mice from the Knockout mouse project (KOMP) Repository. Using thesematerials, we like to delineate the roles of Bhlhe40 in peroxisome biology and oxidative metabolism in SKMby achieving the following specific aims:1. Identifying genes regulated by Bhlhe40 in SKM.2. Examining the homeostasis of peroxisomes in VBH135-OV, Bhlhe40-KD, and Bhlhe40-OV cells.3. Examining the catabolic and anabolic functions of peroxisomes in VBH135-OV, Bhlhe40-KD, andBhlhe40-OV cells.4. Verifying the roles of Bhlhe40 targeted genes in peroxisome biology.5. Examining the biogenesis and functions of peroxisomes in sedentary and exercised Bhlhe40-KOmice.6. Verifying the oxidative metabolism-promoting effect of VBH135 protein in obese and type IIdiabetes mice.