We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

We have previously reported that human cytomegalovirus (HCMV) infection induces large-scale changes to host cell glycolytic, nucleic acid, and phospholipid metabolism. Here we explore the viral mechanisms involved in fatty acid biosynthetic activation. Our results indicate that HCMV targets ACC1, the rate-limiting enzyme of fatty acid biosynthesis, through multiple mechanisms. HCMV infection was found to activate ACC1 expression, increasing the abundance of both ACC1 mRNA and protein. Viral gene expression but not viral DNA replication was found to be necessary for HCMV-mediated induction of ACC1 levels. HCMV infection was also found to increase the proteolytic processing of SREBP-2, a transcription factor whose proteolytic cleavage is known to activate a variety of phospholipid metabolic genes. Processing of SREBP-2 was found to be dependent on mTOR activity; pharmaceutical inhibition of mTOR blocked HCMV-induced SREBP-2 processing and prevented the induction of fatty acid biosynthesis and ACC1 expression. Independent of the increases in ACC1 expression, HCMV infection also induced ACC1's enzymatic activity. Inhibition of ACC1 through either RNA interference (RNAi) or inhibitor treatment was found to attenuate HCMV replication, and HCMV replication was sensitive to ACC1 inhibition even at the later stages of infection, suggesting a late role for fatty acid biosynthesis during HCMV replication. These findings indicate that HCMV infection actively modulates numerous functional aspects of a key metabolic regulatory enzyme that is important for high-titer viral replication.For decades, numerous reports have indicated that infection with a wide variety of evolutionarily divergent viruses results in a general activation of host cell metabolism (7,15,16,26,29,39,42). This metabolic activation can be therapeutically useful; for example, increased or divergent nucleotide metabolism is often clinically targeted to treat various viral infections, such as hepatitis B virus, HIV, human cytomegalovirus (HCMV), and herpes simplex virus (3,13,18,28). Despite the successes of these antiviral strategies, relatively little is known about the specific metabolic activities induced by viral infection and the mechanisms responsible for their activation. Given the viral reliance on the host cell metabolic network for the production of viral progeny, elucidating the mechanisms of viral metabolic manipulation will likely highlight novel avenues for therapeutic development.HCMV is a widespread opportunistic pathogen that can cause severe disease in various immunosuppressed populations, including the elderly, cancer patients receiving immunosuppressive chemotherapy, transplant recipients, and AIDS patients (17,38). Additionally, congenital HCMV infection occurs in 1 to 2% of all live births (3) and can result in multiple organ system abnormalities, with central nervous system damage occurring in the majority of symptomatic newborns (11,38).HCMV is a large, double-stranded DNA virus that contains an ϳ240-kb genome encoding over 200 open re...

2-Hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The D-(R)-enantiomer (D-2-HG) is an oncometabolite generated from ␣-ketoglutarate (␣-KG) by mutant isocitrate dehydrogenase, whereas L-(S)-2-HG is generated by lactate dehydrogenase and malate dehydrogenase in response to hypoxia. Because acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate ␣-KG levels further stimulated this elevation. Studies with isolated lactate dehydrogenase-1 and malate dehydrogenase-2 revealed that generation of 2-HG by both enzymes was stimulated severalfold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial L-2-HG removal enzyme L-2-HG dehydrogenase and to stimulate the reverse reaction of isocitrate dehydrogenase (carboxylation of ␣-KG to isocitrate). Furthermore, because acidic pH is known to stabilize hypoxia-inducible factor (HIF) and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably overexpressing L-2-HG dehydrogenase exhibited a blunted HIF response to acid. Together, these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.The field of cancer biology has long been rapt by the notion of a cancer-specific metabolic phenotype, perhaps most famously embodied in the "Warburg effect," wherein glycolytic metabolism predominates in cancer cells despite O 2 availability and largely intact mitochondrial respiratory function (1). A prominent feature of the cancer metabolic phenotype (reviewed in Ref. 2) is an elevated level of the small metabolic acid 2-hydroxyglutarate (2-HG), 2 derived from the TCA cycle intermediate ␣-ketoglutarate (␣-KG) (3). The D-(R)-enantiomer of 2-HG (D-2-HG) was shown to be generated by mutant forms of isocitrate dehydrogenase (ICDH) that are associated with a variety of cancers including aggressive gliomas (4). In addition, more recently the L-(S)-enantiomer (L-2-HG) was shown to be generated under hypoxic conditions by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) (5, 6). We have also reported elevated D/L-2-HG levels in the heart following ischemic preconditioning (7).In addition to synthesis, 2-HG levels are regulated by a pair of dehydrogenases that convert 2-HG back to ␣-KG (i.e. L-2-HGDH and D-2-HGDH). Mutations in these enzymes manifest as the hydroxyglutaric acidurias, devastating inherited metabolic diseases with symptoms including epilepsy and cerebellar ataxia (8 -11). However, the importance of these dehydrogenases in regulating 2-HG levels in other settings is not clear.The downstream signaling roles ...

SUMMARY Metabolic reprogramming is critical to oncogenesis, but the emergence and function of this profound reorganization remain poorly understood. Here we find that cooperating oncogenic mutations drive large-scale metabolic reprogramming, which is both intrinsic to cancer cells and obligatory for the transition to malignancy. This involves synergistic regulation of several genes encoding metabolic enzymes, including the lactate dehydrogenases LDHA and LDHB and mitochondrial glutamic pyruvate transaminase 2 (GPT2). Notably, GPT2 engages activated glycolysis to drive the utilization of glutamine as a carbon source for TCA cycle anaplerosis in colon cancer cells. Our data indicate that the Warburg effect supports oncogenesis via GPT2-mediated coupling of pyruvate production to glutamine catabolism. Although critical to the cancer phenotype, GPT2 activity is dispensable in cells that are not fully transformed, thus pinpointing a metabolic vulnerability specifically associated with cancer cell progression to malignancy.

Human Cytomegalovirus (HCMV) infection induces several metabolic activities that are essential for viral replication. Despite the important role that this metabolic modulation plays during infection, the viral mechanisms involved are largely unclear. We find that the HCMV UL38 protein is responsible for many aspects of HCMV-mediated metabolic activation, with UL38 being necessary and sufficient to drive glycolytic activation and induce the catabolism of specific amino acids. UL38’s metabolic reprogramming role is dependent on its interaction with TSC2, a tumor suppressor that inhibits mTOR signaling. Further, shRNA-mediated knockdown of TSC2 recapitulates the metabolic phenotypes associated with UL38 expression. Notably, we find that in many cases the metabolic flux activation associated with UL38 expression is largely independent of mTOR activity, as broad spectrum mTOR inhibition does not impact UL38-mediated induction of glycolysis, glutamine consumption, or the secretion of proline or alanine. In contrast, the induction of metabolite concentrations observed with UL38 expression are largely dependent on active mTOR. Collectively, our results indicate that the HCMV UL38 protein induces a pro-viral metabolic environment via inhibition of TSC2.