We identified a p53 target gene, phosphate-activated mitochondrial glutaminase (GLS2), a key enzyme in conversion of glutamine to glutamate, and thereby a regulator of glutathione (GSH) synthesis and energy production. GLS2 expression is induced in response to DNA damage or oxidative stress in a p53-dependent manner, and p53 associates with the GLS2 promoter. Elevated GLS2 facilitates glutamine metabolism and lowers intracellular reactive oxygen species (ROS) levels, resulting in an overall decrease in DNA oxidation as determined by measurement of 8-OH-dG content in both normal and stressed cells. Further, siRNA down-regulation of either GLS2 or p53 compromises the GSH-dependent antioxidant system and increases intracellular ROS levels. High ROS levels following GLS2 knockdown also coincide with stimulation of p53-induced cell death. We propose that GLS2 control of intracellular ROS levels and the apoptotic response facilitates the ability of p53 to protect cells from accumulation of genomic damage and allows cells to survive after mild and repairable genotoxic stress. Indeed, overexpression of GLS2 reduces the growth of tumor cells and colony formation. Further, compared with normal tissue, GLS2 expression is reduced in liver tumors. Thus, our results provide evidence for a unique metabolic role for p53, linking glutamine metabolism, energy, and ROS homeostasis, which may contribute to p53 tumor suppressor function.glutathione antioxidant | glutaminolysis | tumor suppression | apoptosis

SUMMARY Multipotent progenitors confirm their T cell-lineage identity in the DN2 pro-T cell stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomic analysis revealed that Bcl11b associates with multiple cofactors, and that its direct action was needed to recruit these cofactors to selective target sites. These sites of Bcl11b-dependent cofactor recruitment were enriched near functionally regulated target genes, and deletion of individual cofactors relieved repression of many Bcl11b-repressed genes. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via Id2 and Zbtb16 (encoding PLZF), which were directly repressed by Bcl11b and controlled distinct alternative programs. Thus, this study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials.

The p53 tumor suppressor protein regulates many genes that can determine different cellular outcomes such as growth arrest or cell death. Promoter-selective transactivation by p53, although critical for the different cellular outcomes, is not well understood. We report here that the human cellular apoptosis susceptibility protein (hCAS/CSE1L) associates with a subset of p53 target promoters, including PIG3, in a p53-autonomous manner. Downregulation of hCAS/CSE1L decreases transcription from those p53 target promoters to which it preferentially binds and reduces apoptosis. In addition, hCAS/CSE1L silencing leads to increased methylation of histone H3 lysine 27 within the PIG3 gene. hCAS/CSE1L was previously shown to function as a nucleo-cytoplasmic transport factor, as does its closely related yeast homologue Cse1, which can also associate with chromatin and serve as a barrier protein that prevents spreading of heterochromatin. Thus, human CAS/CSE1L can bind select genes with significant functional consequences for p53-mediated transcription and determine cellular outcome.

Transcription factors normally regulate gene expression through their action at sites where they bind to DNA. However, the balance of activating and repressive functions that a transcription factor can mediate is not completely understood. Here, we showed that the transcription factor PU.1 regulated gene expression in early T cell development both by recruiting partner transcription factors to its own binding sites and by depleting them from the binding sites that they preferred when PU.1 was absent. The removal of partner factors Satb1 and Runx1 occurred primarily from sites where PU.1 itself did not bind. Genes linked to sites of partner factor "theft" were enriched for genes that PU.1 represses despite lack of binding, both in a model cell line system and in normal T cell development. Thus, system-level competitive recruitment dynamics permit PU.1 to affect gene expression both through its own target sites and through action at a distance.

PurposeIn a 10-week proof-of-concept study (LINC 1), the potent oral 11β-hydroxylase inhibitor osilodrostat (LCI699) normalized urinary free cortisol (UFC) in 11/12 patients with Cushing’s disease. The current 22-week study (LINC 2; NCT01331239) further evaluated osilodrostat in patients with Cushing’s disease.MethodsPhase II, open-label, prospective study of two patient cohorts. Follow-up cohort: 4/12 patients previously enrolled in LINC 1, offered re-enrollment if baseline mean UFC was above ULN. Expansion cohort: 15 newly enrolled patients with baseline UFC > 1.5 × ULN. In the follow-up cohort, patients initiated osilodrostat twice daily at the penultimate efficacious/tolerable dose in LINC 1; dose was adjusted as needed. In the expansion cohort, osilodrostat was initiated at 4 mg/day (10 mg/day if baseline UFC > 3 × ULN), with dose escalated every 2 weeks to 10, 20, 40, and 60 mg/day until UFC ≤ ULN. Main efficacy endpoint was the proportion of responders (UFC ≤ ULN or ≥50 % decrease from baseline) at weeks 10 and 22.ResultsOverall response rate was 89.5 % (n/N = 17/19) at 10 weeks and 78.9 % (n/N = 15/19) at 22 weeks; at week 22, all responding patients had UFC ≤ ULN. The most common AEs observed during osilodrostat treatment were nausea, diarrhea, asthenia, and adrenal insufficiency (n = 6 for each). New or worsening hirsutism (n = 2) and/or acne (n = 3) were reported among four female patients, all of whom had increased testosterone levels.ConclusionsOsilodrostat treatment reduced UFC in all patients; 78.9 % (n/N = 15/19) had normal UFC at week 22. Treatment with osilodrostat was generally well tolerated.Electronic supplementary materialThe online version of this article (doi:10.1007/s11102-015-0692-z) contains supplementary material, which is available to authorized users.

We investigated the role of the intrinsic mevalonate cascade in the neuronal cell death (NCD) induced by the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in rat primary cortical neurons cultured from the brains of 17-d-old fetal SD rats. HMG-CoA reductase inhibitors induced NCD [HMG-CoA reductase inhibitor-induced NCD (H-NCD)] in time- and dose-dependent manners. The apoptotic characteristics were revealed by the formation of the DNA ladder and by the electron microscopical observation. During the progression of H-NCD, p53 was induced followed by the expression of Bax. Although the mevalonate completely inhibited H-NCD, the cholesterol did not. Thus, we examined two major metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), using a novel liposome system for uptake into the cells. GGPP, not FPP, prohibited H-NCD with inhibition of the induction of p53 and Bax. The inhibition of HMG-CoA reductase decreased the amount of membrane-associated Rho small GTPase families, but not Ras small GTPase, and GGPP restored the blockage by HMG-CoA reductase inhibitor in the translocation or redistribution of Rho small GTPase families to membrane. These data indicated that (1) the inhibition of the intrinsic mevalonate cascade induces the apoptotic NCD with the induction of p53 followed by that of Bax, (2) the inhibition of HMG-CoA reductase concomitantly causes blockage of the translocation or redistribution of Rho small GTPase families, not Ras small GTPase, to membrane, and (3) GGPP, not FPP, is one of the essential metabolites in the mevalonate cascade for protecting neurons from H-NCD.

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The zinc finger transcription factor, Bcl11b, is expressed in T cells and group 2 innate lymphoid cells (ILC2s) among hematopoietic cells. In early T-lineage cells, Bcl11b directly binds and represses the gene encoding the E protein antagonist, Id2, preventing pro-T cells from adopting innate-like fates. In contrast, ILC2s co-express both Bcl11b and Id2. To address this contradiction, we have directly compared Bcl11b action mechanisms in pro-T cells and ILC2s. We found that Bcl11b binding to regions across the genome shows distinct cell type–specific motif preferences. Bcl11b occupies functionally different sites in lineage-specific patterns and controls totally different sets of target genes in these cell types. In addition, Bcl11b bears cell type–specific post-translational modifications and organizes different cell type–specific protein complexes. However, both cell types use the same distal enhancer region to control timing of Bcl11b activation. Therefore, although pro-T cells and ILC2s both need Bcl11b for optimal development and function, Bcl11b works substantially differently in these two cell types.