Cyclin D1 represses gluconeogenesis via inhibition of the transcriptional coactivator PGC1α.

Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1-mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

Time window expansion for HDX analysis of an intrinsically disordered protein.

Application of typical HDX methods to examine intrinsically disordered proteins (IDP), proteins that are natively unstructured and highly dynamic at physiological pH, is limited because of the rapid exchange of unprotected amide hydrogens with solvent. The exchange rates of these fast exchanging amides are usually faster than the shortest time scale (10 s) employed in typical automated HDX-MS experiments. Considering the functional importance of IDPs and their association with many diseases, it is valuable to develop methods that allow the study of solution dynamics of these proteins as well as the ability to probe the interaction of IDPs with their wide range of binding partners. Here, we report the application of time window expansion to the millisecond range by altering the on-exchange pH of the HDX experiment to study a well-characterized IDP; the activation domain of the nuclear receptor coactivator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). This method enabled mapping the regions of PGC-1α that are stabilized upon binding the ligand binding domain (LBD) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). We further demonstrate the method's applicability to other binding partners of the IDP PGC-1α and pave the way for characterizing many other biologically important ID proteins.

Disorder-to-order transition underlies the structural basis for the assembly of a transcriptionally active PGC-1α/ERRγ complex.

Peroxisome proliferator activated receptor (PPAR) γ coactivator-1α (PGC-1α) is a potent transcriptional coactivator of oxidative metabolism and is induced in response to a variety of environmental cues. It regulates a broad array of target genes by coactivating a whole host of transcription factors. The estrogen-related receptor (ERR) family of nuclear receptors are key PGC-1α partners in the regulation of mitochondrial and tissue-specific oxidative metabolic pathways; these receptors also demonstrate strong physical and functional interactions with this coactivator. Here we perform comprehensive biochemical, biophysical, and structural analyses of the complex formed between PGC-1α and ERRγ. PGC-1α activation domain (PGC-1α(2-220)) is intrinsically disordered with limited secondary and no defined tertiary structure. Complex formation with ERRγ induces significant changes in the conformational mobility of both partners, highlighted by significant stabilization of the ligand binding domain (ERRγLBD) as determined by HDX (hydrogen/deuterium exchange) and an observed disorder-to-order transition in PGC-1α(2-220). Small-angle X-ray scattering studies allow for modeling of the solution structure of the activation domain in the absence and presence of ERRγLBD, revealing a stable and compact binary complex. These data show that PGC-1α(2-220) undergoes a large-scale conformational change when binding to the ERRγLBD, leading to substantial compaction of the activation domain. This change results in stable positioning of the N-terminal part of the activation domain of PGC-1α, favorable for assembly of an active transcriptional complex. These data also provide structural insight into the versatile coactivation profile of PGC-1α and can readily be extended to understand other transcriptional coregulators.

Separation of the gluconeogenic and mitochondrial functions of PGC-1{alpha} through S6 kinase.

PGC-1α is a transcriptional coactivator that powerfully regulates many pathways linked to energy homeostasis. Specifically, PGC-1α controls mitochondrial biogenesis in most tissues but also initiates important tissue-specific functions, including fiber type switching in skeletal muscle and gluconeogenesis and fatty acid oxidation in the liver. We show here that S6 kinase, activated in the liver upon feeding, can phosphorylate PGC-1α directly on two sites within its arginine/serine-rich (RS) domain. This phosphorylation significantly attenuates the ability of PGC-1α to turn on genes of gluconeogenesis in cultured hepatocytes and in vivo, while leaving the functions of PGC-1α as an activator of mitochondrial and fatty acid oxidation genes completely intact. These phosphorylations interfere with the ability of PGC-1α to bind to HNF4α, a transcription factor required for gluconeogenesis, while leaving undisturbed the interactions of PGC-1α with ERRα and PPARα, factors important for mitochondrial biogenesis and fatty acid oxidation. These data illustrate that S6 kinase can modify PGC-1α and thus allow molecular dissection of its functions, providing metabolic flexibility needed for dietary adaptation.

PRDM16 controls a brown fat/skeletal muscle switch.

Brown fat can increase energy expenditure and protect against obesity through a specialized program of uncoupled respiration. Here we show by in vivo fate mapping that brown, but not white, fat cells arise from precursors that express Myf5, a gene previously thought to be expressed only in the myogenic lineage. We also demonstrate that the transcriptional regulator PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16) controls a bidirectional cell fate switch between skeletal myoblasts and brown fat cells. Loss of PRDM16 from brown fat precursors causes a loss of brown fat characteristics and promotes muscle differentiation. Conversely, ectopic expression of PRDM16 in myoblasts induces their differentiation into brown fat cells. PRDM16 stimulates brown adipogenesis by binding to PPAR-gamma (peroxisome-proliferator-activated receptor-gamma) and activating its transcriptional function. Finally, Prdm16-deficient brown fat displays an abnormal morphology, reduced thermogenic gene expression and elevated expression of muscle-specific genes. Taken together, these data indicate that PRDM16 specifies the brown fat lineage from a progenitor that expresses myoblast markers and is not involved in white adipogenesis.

Structure of the heterodimeric ecdysone receptor DNA-binding complex.

Ecdysteroids initiate molting and metamorphosis in insects via a heterodimeric receptor consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). The EcR-USP heterodimer preferentially mediates transcription through highly degenerate pseudo-palindromic response elements, resembling inverted repeats of 5'-AGGTCA-3' separated by 1 bp (IR-1). The requirement for a heterodimeric arrangement of EcR-USP subunits to bind to a symmetric DNA is unusual within the nuclear receptor superfamily. We describe the 2.24 A structure of the EcR-USP DNA-binding domain (DBD) heterodimer bound to an idealized IR-1 element. EcR and USP use similar surfaces, and rely on the deformed minor groove of the DNA to establish protein-protein contacts. As retinoid X receptor (RXR) is the mammalian homolog of USP, we also solved the 2.60 A crystal structure of the EcR-RXR DBD heterodimer on IR-1 and found the dimerization and DNA-binding interfaces to be the same as in the EcR-USP complex. Sequence alignments indicate that the EcR-RXR heterodimer is an important model for understanding how the FXR-RXR heterodimer binds to IR-1 sites.

Structural basis for bile acid binding and activation of the nuclear receptor FXR.

The nuclear receptor FXR is the sensor of physiological levels of enterohepatic bile acids, the end products of cholesterol catabolism. Here we report crystal structures of the FXR ligand binding domain in complex with coactivator peptide and two different bile acids. An unusual A/B ring juncture, a feature associated with bile acids and no other steroids, provides ligand discrimination and triggers a pi-cation switch that activates FXR. Helix 12, the activation function 2 of the receptor, adopts the agonist conformation and stabilizes coactivator peptide binding. FXR is able to interact simultaneously with two coactivator motifs, providing a mechanism for enhanced binding of coactivators through intermolecular contacts between their LXXLL sequences. These FXR complexes provide direct insights into the design of therapeutic bile acids for treatment of hyperlipidemia and cholestasis.

The active site of the SET domain is constructed on a knot.

The SET domain contains the catalytic center of lysine methyltransferases that target the N-terminal tails of histones and regulate chromatin function. Here we report the structure of the SET7/9 protein in the absence and presence of its cofactor product, S-adenosyl-L-homocysteine (AdoHcy). A knot within the SET domain helps form the methyltransferase active site, where AdoHcy binds and lysine methylation is likely to occur. A structure-guided comparison of sequences within the SET protein family suggests that the knot substructure and active site environment are conserved features of the SET domain.