A hormone sends instant messages to the genome.

Bridging a gap between transcriptomics and the study of cis-acting elements (cistromics), Hah et al. (2011) apply a next-generation sequencing technique to gain an unprecedented view of the changes in RNA synthesis that occur following estrogen receptor activation in human breast cancer cells.

Hepatic p53 is regulated by transcription factor FOXO1 and acutely controls glycogen homeostasis.

The tumor suppressor p53 is involved in the adaptation of hepatic metabolism to nutrient availability. Acute deletion of p53 in the mouse liver affects hepatic glucose and triglyceride metabolism. However, long-term adaptations upon the loss of hepatic p53 and its transcriptional regulators are unknown. Here we show that short-term, but not chronic, liver-specific deletion of p53 in mice reduces liver glycogen levels, and we implicate the transcription factor forkhead box O1 protein (FOXO1) in the regulation of p53 and its target genes. We demonstrate that acute p53 deletion prevents glycogen accumulation upon refeeding, whereas a chronic loss of p53 associates with a compensational activation of the glycogen synthesis pathway. Moreover, we identify fasting-activated FOXO1 as a repressor of p53 transcription in hepatocytes. We show that this repression is relieved by inactivation of FOXO1 by insulin, which likely mediates the upregulation of p53 expression upon refeeding. Strikingly, we find that high-fat diet-induced insulin resistance with persistent FOXO1 activation not only blunted the regulation of p53 but also the induction of p53 target genes like p21 during fasting, indicating overlapping effects of both FOXO1 and p53 on target gene expression in a context-dependent manner. Thus, we conclude that p53 acutely controls glycogen storage in the liver and is linked to insulin signaling via FOXO1, which has important implications for our understanding of the hepatic adaptation to nutrient availability.

Complementary omics strategies to dissect p53 signaling networks under nutrient stress.

Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

ALYREF, a novel factor involved in breast carcinogenesis, acts through transcriptional and post-transcriptional mechanisms selectively regulating the short NEAT1 isoform.

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1, as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1.

Anti-diabetic rosiglitazone remodels the adipocyte transcriptome by redistributing transcription to PPARγ-driven enhancers.

Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We addressed this issue by studying the direct effects of rosi on gene transcription using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 min and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (enhancer RNAs [eRNAs]). Up-regulated eRNAs occurred almost exclusively at PPARγ-binding sites, to which rosi treatment recruited coactivators, including MED1, p300, and CBP. In contrast, transcriptional repression by rosi involved a loss of coactivators from eRNA sites devoid of PPARγ and enriched for other transcription factors, including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of anti-diabetic drugs.

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

ß,ß-Dimethylacrylshikonin Induces Apoptosis in Melanoma Cell Lines by NOXA Upregulation.

Melanoma is the most aggressive form of skin cancer, with high metastasis rates and poor prognosis. Survival rates and possible therapies depend on the state of the tumor and its mutational profile. BRAF and NRAS are the most frequent driver mutations. Currently, there is no efficient therapy for NRAS-mutated or late-stage melanoma. In this study, the therapeutic potential of ß,ß-dimethylacrylshikonin (DMAS) was investigated on melanoma. The influence of DMAS was determined in five different melanoma cell lines with different mutational profiles. The effects of this compound on cell viability, apoptosis, and gene and protein expression were examined. The results obtained were validated in vivo. DMAS significantly reduced the viability of several melanoma cell lines in a concentration- and time-dependent manner. Furthermore, DMAS induced caspase-3-dependent apoptosis via NOXA upregulation, as confirmed by NOXA knockdown experiments. This is the first time that NOXA-dependent apoptosis was shown with respect to a shikonin derivative and melanoma. Additionally, tumor regression and necrosis under DMAS treatment were demonstrated in vivo. Importantly, BRAF as well as NRAS-mutated metastatic human melanoma cell lines were treated successfully in vitro and in vivo. Taken together, DMAS showed promising results and is worthy of further study.

Downregulation of p53 drives autophagy during human trophoblast differentiation.

The placental barrier is crucial for the supply of nutrients and oxygen to the developing fetus and is maintained by differentiation and fusion of mononucleated cytotrophoblasts into the syncytiotrophoblast, a process only partially understood. Here transcriptome and pathway analyses during differentiation and fusion of cultured trophoblasts yielded p53 signaling as negative upstream regulator and indicated an upregulation of autophagy-related genes. We further showed p53 mRNA and protein levels decreased during trophoblast differentiation. Reciprocally, autophagic flux increased and cytoplasmic LC3B-GFP puncta became more abundant, indicating enhanced autophagic activity. In line, in human first trimester placenta p53 protein mainly localized to the cytotrophoblast, while autophagy marker LC3B as well as late autophagic compartments were predominantly detectable in the syncytiotrophoblast. Importantly, ectopic overexpression of p53 reduced levels of LC3B-II, supporting a negative regulatory role on autophagy in differentiating trophoblasts. This was also shown in primary trophoblasts and human first trimester placental explants, where pharmacological stabilization of p53 decreased LC3B-II levels. In summary our data suggest that differentiation-dependent downregulation of p53 is a prerequisite for activating autophagy in the syncytiotrophoblast.

Lysosomal acid lipase regulates fatty acid channeling in brown adipose tissue to maintain thermogenesis.

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.

APMAP interacts with lysyl oxidase-like proteins, and disruption of Apmap leads to beneficial visceral adipose tissue expansion.

Adipocyte plasma membrane-associated protein (APMAP) has been described as an adipogenic factor in 3T3-L1 cells with unknown biochemical function; we therefore aimed to investigate the physiologic function of APMAP in vivo We generated Apmap-knockout mice and challenged them with an obesogenic diet to investigate their metabolic phenotype. We identified a novel truncated adipocyte-specific isoform of APMAP in mice that is produced by alternative transcription. Mice lacking the full-length APMAP protein, the only isoform that is expressed in humans, have an improved metabolic phenotype upon diet-induced obesity, indicated by enhanced insulin sensitivity, preserved glucose tolerance, increased respiratory exchange ratio, decreased inflammatory marker gene expression, and reduced adipocyte size. At the molecular level, APMAP interacts with the extracellular collagen cross-linking matrix proteins lysyl oxidase-like 1 and 3. On a high-fat diet, the expression of lysyl oxidase-like 1 and 3 is strongly decreased in Apmap-knockout mice, paralleled by reduced expression of profibrotic collagens and total collagen content in epididymal white adipose tissue, indicating decreased fibrotic potential. Together, our data suggest that APMAP is a novel regulator of extracellular matrix components, and establish that APMAP is a potential target to mitigate obesity-associated insulin resistance.-Pessentheiner, A. R., Huber, K., Pelzmann, H. J., Prokesch, A., Radner, F. P. W., Wolinski, H., Lindroos-Christensen, J., Hoefler, G., Rülicke, T., Birner-Gruenberger, R., Bilban, M., Bogner-Strauss, J. G. APMAP interacts with lysyl oxidase-like proteins, and disruption of Apmap leads to beneficial visceral adipose tissue expansion.

Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.

The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic adaptation to starvation. Because of its tumor suppressor function, p53 stabilization by starvation could have implications for both metabolic and oncological diseases of the liver.-Prokesch, A., Graef, F. A., Madl, T., Kahlhofer, J., Heidenreich, S., Schumann, A., Moyschewitz, E., Pristoynik, P., Blaschitz, A., Knauer, M., Muenzner, M., Bogner-Strauss, J. G., Dohr, G., Schulz, T. J., Schupp, M. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.

p53 Functions in Adipose Tissue Metabolism and Homeostasis.

As a tumor suppressor and the most frequently mutated gene in cancer, p53 is among the best-described molecules in medical research. As cancer is in most cases an age-related disease, it seems paradoxical that p53 is so strongly conserved from early multicellular organisms to humans. A function not directly related to tumor suppression, such as the regulation of metabolism in nontransformed cells, could explain this selective pressure. While this role of p53 in cellular metabolism is gradually emerging, it is imperative to dissect the tissue- and cell-specific actions of p53 and its downstream signaling pathways. In this review, we focus on studies reporting p53's impact on adipocyte development, function, and maintenance, as well as the causes and consequences of altered p53 levels in white and brown adipose tissue (AT) with respect to systemic energy homeostasis. While whole body p53 knockout mice gain less weight and fat mass under a high-fat diet owing to increased energy expenditure, modifying p53 expression specifically in adipocytes yields more refined insights: (1) p53 is a negative regulator of in vitro adipogenesis; (2) p53 levels in white AT are increased in diet-induced and genetic obesity mouse models and in obese humans; (3) functionally, elevated p53 in white AT increases senescence and chronic inflammation, aggravating systemic insulin resistance; (4) p53 is not required for normal development of brown AT; and (5) when p53 is activated in brown AT in mice fed a high-fat diet, it increases brown AT temperature and brown AT marker gene expression, thereby contributing to reduced fat mass accumulation. In addition, p53 is increasingly being recognized as crucial player in nutrient sensing pathways. Hence, despite existence of contradictory findings and a varying density of evidence, several functions of p53 in adipocytes and ATs have been emerging, positioning p53 as an essential regulatory hub in ATs. Future studies need to make use of more sophisticated in vivo model systems and should identify an AT-specific set of p53 target genes and downstream pathways upon different (nutrient) challenges to identify novel therapeutic targets to curb metabolic diseases.

p53 as a Dichotomous Regulator of Liver Disease: The Dose Makes the Medicine.

Lifestyle-related disorders, such as the metabolic syndrome, have become a primary risk factor for the development of liver pathologies that can progress from hepatic steatosis, hepatic insulin resistance, steatohepatitis, fibrosis and cirrhosis, to the most severe condition of hepatocellular carcinoma (HCC). While the prevalence of liver pathologies is steadily increasing in modern societies, there are currently no approved drugs other than chemotherapeutic intervention in late stage HCC. Hence, there is a pressing need to identify and investigate causative molecular pathways that can yield new therapeutic avenues. The transcription factor p53 is well established as a tumor suppressor and has recently been described as a central metabolic player both in physiological and pathological settings. Given that liver is a dynamic tissue with direct exposition to ingested nutrients, hepatic p53, by integrating cellular stress response, metabolism and cell cycle regulation, has emerged as an important regulator of liver homeostasis and dysfunction. The underlying evidence is reviewed herein, with a focus on clinical data and animal studies that highlight a direct influence of p53 activity on different stages of liver diseases. Based on current literature showing that activation of p53 signaling can either attenuate or fuel liver disease, we herein discuss the hypothesis that, while hyper-activation or loss of function can cause disease, moderate induction of hepatic p53 within physiological margins could be beneficial in the prevention and treatment of liver pathologies. Hence, stimuli that lead to a moderate and temporary p53 activation could present new therapeutic approaches through several entry points in the cascade from hepatic steatosis to HCC.

Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-α in a gestational age-dependent manner.

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation.

NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer-specific survival in patients with aggressive B-cell lymphomas.

NR4A1 (Nur77) and NR4A3 (Nor-1) function as tumor suppressor genes as demonstrated by the rapid development of acute myeloid leukemia in the NR4A1 and NR4A3 knockout mouse. The aim of our study was to investigate NR4A1 and NR4A3 expression and function in lymphoid malignancies. We found a vastly reduced expression of NR4A1 and NR4A3 in chronic lymphocytic B-cell leukemia (71%), in follicular lymphoma (FL, 70%), and in diffuse large B-cell lymphoma (DLBCL, 74%). In aggressive lymphomas (DLBCL and FL grade 3), low NR4A1 expression was significantly associated with a non-germinal center B-cell subtype and with poor overall survival. To investigate the function of NR4A1 in lymphomas, we overexpressed NR4A1 in several lymphoma cell lines. Overexpression of NR4A1 led to a higher proportion of lymphoma cells undergoing apoptosis. To test the tumor suppressor function of NR4A1 in vivo, the stable lentiviral-transduced SuDHL4 lymphoma cell line harboring an inducible NR4A1 construct was further investigated in xenografts. Induction of NR4A1 abrogated tumor growth in the NSG mice, in contrast to vector controls, which formed massive tumors. Our data suggest that NR4A1 has proapoptotic functions in aggressive lymphoma cells and define NR4A1 as a novel gene with tumor suppressor properties involved in lymphomagenesis.

NAT8L (N-acetyltransferase 8-like) accelerates lipid turnover and increases energy expenditure in brown adipocytes.

NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.

Characterization of rat serum amyloid A4 (SAA4): a novel member of the SAA superfamily.

The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold during inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5'- and 3'RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/ß and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.

Lipid-associated macrophages between aggravation and alleviation of metabolic diseases.

Lipid-associated macrophages (LAMs) are phagocytic cells with lipid-handling capacity identified in various metabolic derangements. During disease development, they locate to atherosclerotic plaques, adipose tissue (AT) of individuals with obesity, liver lesions in steatosis and steatohepatitis, and the intestinal lamina propria. LAMs can also emerge in the metabolically demanding microenvironment of certain tumors. In this review, we discuss major questions regarding LAM recruitment, differentiation, and self-renewal, and, ultimately, their acute and chronic functional impact on the development of metabolic diseases. Further studies need to clarify whether and under which circumstances LAMs drive disease progression or resolution and how their phenotype can be modulated to ameliorate metabolic disorders.

Disordered regions mediate the interaction of p53 and MRE11.

The genome is frequently targeted by genotoxic agents, resulting in the formation of DNA scars. However, cells employ diverse repair mechanisms to restore DNA integrity. Among these processes, the Mre11-Rad50-Nbs1 complex detects double-strand breaks (DSBs) and recruits DNA damage response proteins such as ataxia-telangiectasia-mutated (ATM) kinase to DNA damage sites. ATM phosphorylates the transactivation domain (TAD) of the p53 tumor suppressor, which in turn regulates DNA repair, growth arrest, apoptosis, and senescence following DNA damage. The disordered glycine-arginine-rich (GAR) domain of double-strand break protein MRE11 (MRE11GAR) and its methylation are important for DSB repair, and localization to Promyelocytic leukemia nuclear bodies (PML-NBs). There is preliminary evidence that p53, PML protein, and MRE11 might co-localize and interact at DSB sites. To uncover the molecular details of these interactions, we aimed to identify the domains mediating the p53-MRE11 interaction and to elucidate the regulation of the p53-MRE11 interaction by post-translational modifications (PTMs) through a combination of biophysical techniques. We discovered that, in vitro, p53 binds directly to MRE11GAR mainly through p53TAD2 and that phosphorylation further enhances this interaction. Furthermore, we found that MRE11GAR methylation still allows for binding to p53. Overall, we demonstrated that p53 and MRE11 interaction is facilitated by disordered regions. We provide for the first time insight into the molecular details of the p53-MRE11 complex formation and elucidate potential regulatory mechanisms that will promote our understanding of the DNA damage response. Our findings suggest that PTMs regulate the p53-MRE11 interaction and subsequently their colocalization to PML-NBs upon DNA damage.

E2F transcription factor-1 modulates expression of glutamine metabolic genes in mouse embryonic fibroblasts and uterine sarcoma cells.

Metabolic reprogramming is considered as a hallmark of cancer and is clinically exploited as a novel target for therapy. The E2F transcription factor-1 (E2F1) regulates various cellular processes, including proliferative and metabolic pathways, and acts, depending on the cellular and molecular context, as an oncogene or tumor suppressor. The latter is evident by the observation that E2f1-knockout mice develop spontaneous tumors, including uterine sarcomas. This dual role warrants a detailed investigation of how E2F1 loss impacts metabolic pathways related to cancer progression. Our data indicate that E2F1 binds to the promoter of several glutamine metabolism-related genes. Interestingly, the expression of genes in the glutamine metabolic pathway were increased in mouse embryonic fibroblasts (MEFs) lacking E2F1. In addition, we confirm that E2f1-/- MEFs are more efficient in metabolizing glutamine and producing glutamine-derived precursors for proliferation. Mechanistically, we observe a co-occupancy of E2F1 and MYC on glutamine metabolic promoters, increased MYC binding after E2F1 depletion and that silencing of MYC decreased the expression of glutamine-related genes in E2f1-/- MEFs. Analyses of transcriptomic profiles in 29 different human cancers identified uterine sarcoma that showed a negative correlation between E2F1 and glutamine metabolic genes. CRISPR/Cas9 knockout of E2F1 in the uterine sarcoma cell line SK-UT-1 confirmed elevated glutamine metabolic gene expression, increased proliferation and increased MYC binding to glutamine-related promoters upon E2F1 loss. Together, our data suggest a crucial role of E2F1 in energy metabolism and metabolic adaptation in uterine sarcoma cells.