A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

Optimal sampling interval for characterisation of the circadian rhythm of body temperature in homeothermic animals using periodogram and cosinor analysis.

Core body temperature (T c) is a critical aspect of homeostasis in birds and mammals and is increasingly used as a biomarker of the fitness of an animal to its environment. Periodogram and cosinor analysis can be used to estimate the characteristics of the circadian rhythm of T c from data obtained on loggers that have limited memory capacity and battery life. The sampling interval can be manipulated to maximise the recording period, but the impact of sampling interval on the output of periodogram or cosinor analysis is unknown. Some basic guidelines are available from signal analysis theory, but those guidelines have never been tested on T c data. We obtained data at 1-, 5- or 10-min intervals from nine avian or mammalian species, and re-sampled those data to simulate logging at up to 240-min intervals. The period of the rhythm was first analysed using the Lomb-Scargle periodogram, and the mesor, amplitude, acrophase and adjusted coefficient of determination (R 2) from the original and the re-sampled data were obtained using cosinor analysis. Sampling intervals longer than 60 min did not affect the average mesor, amplitude, acrophase or adjusted R 2, but did impact the estimation of the period of the rhythm. In most species, the period was not detectable when intervals longer than 120 min were used. In all individual profiles, a 30-min sampling interval modified the values of the mesor and amplitude by less than 0.1°C, and the adjusted R 2 by less than 0.1. At a 30-min interval, the acrophase was accurate to within 15 min for all species except mice. The adjusted R 2 increased as sampling frequency decreased. In most cases, a 30-min sampling interval provides a reliable estimate of the circadian T c rhythm using periodogram and cosinor analysis. Our findings will help biologists to select sampling intervals to fit their research goals.

Mediator subunit MDT-15 promotes expression of propionic acid breakdown genes to prevent embryonic lethality in Caenorhabditis elegans.

The micronutrient vitamin B12 is an essential cofactor for two enzymes: methionine synthase, which plays a key role in the one-carbon cycle; and methylmalonyl-CoA mutase, an enzyme in a pathway that breaks down branched-chain amino acids and odd-chain fatty acids. A second, vitamin B12-independent pathway that degrades propionic acid was recently described in Caenorhabditis elegans, the propionate shunt pathway. Activation of five shunt pathway genes in response to low vitamin B12 availability or high propionic acid levels is accomplished by a transcriptional regulatory mechanism involving two nuclear hormone receptors, NHR-10 and NHR-68. Here, we report that the C. elegans Mediator subunit mdt-15 is also essential for the activation of the propionate shunt pathway genes, likely by acting as a transcriptional coregulator for NHR-10. C. elegans mdt-15 mutants fed with a low vitamin B12 diet have transcriptomes resembling those of wild-type worms fed with a high vitamin B12 diet, with low expression of the shunt genes. Phenotypically, the embryonic lethality of mdt-15 mutants is specifically rescued by diets high in vitamin B12, but not by dietary polyunsaturated fatty acids, which rescue many other phenotypes of the mdt-15 mutants. Finally, NHR-10 binds to MDT-15 in yeast two-hybrid assays, and the transcriptomes of nhr-10 mutants share overlap with those of mdt-15 mutants. Our data show that MDT-15 is a key coregulator for an NHR regulating propionic acid detoxification, adding to roles played by NHR:MDT-15 partnerships in metabolic regulation and pinpointing vitamin B12 availability as a requirement for mdt-15 dependent embryonic development.

Diet-altered body temperature rhythms are associated with altered rhythms of clock gene expression in peripheral tissues in vivo.

Temperature rhythms can act as potent signals for the modulation of the amplitude and phase of clock gene expression in peripheral organs in vitro, but the relevance of the circadian rhythm of core body temperature (Tc) as a modulating signal in vivo has not yet been investigated. Using calorie restriction and cafeteria feeding, we induced a larger and a dampened Tc amplitude, respectively, in male Wistar rats, and investigated the circadian expression profile of the core clock genes Bmal1, Per2, Cry1, and Rev-erbα, the heat-responsive genes heat shock protein 90 (Hsp90) and cold-inducible RNA binding protein (Cirbp), and Pgc1α, Pparα/γ/δ, Glut1/4, and Chop10 in the liver, skeletal muscle, white adipose tissue (WAT), and adrenal glands. Diet-altered Tc rhythms differentially affected the profiles of clock genes, Hsp90, and Cirbp expression in peripheral tissues. Greater Tc amplitudes elicited by calorie restriction were associated with large amplitudes of Hsp90 and Cirbp expression in the liver and WAT, in which larger amplitudes of clock gene expression were also observed. The amplitudes of metabolic gene expression were greater in the WAT, but not in the liver, in calorie-restricted rats. Conversely, diet-altered Tc rhythms were not translated to distinct changes in the amplitude of Hsp90, Cirbp, or clock or metabolic genes in the skeletal muscle or adrenal glands. While it was not possible to disentangle the effects of diet and temperature in this model, taken together with previous in vitro studies, our study presents novel data consistent with the notion that the circadian Tc rhythm can modulate the amplitude of circadian gene expression in vivo. The different responses of Hsp90 and Cirbp in peripheral tissues may be linked to the tissue-specific responses of peripheral clocks to diet and/or body temperature rhythms, but the association with the amplitude of metabolic gene expression is limited to the WAT.

Daily temperature cycles prolong lifespan and have sex-specific effects on peripheral clock gene expression in Drosophila melanogaster.

Circadian rhythms optimize health by coordinating the timing of physiological processes to match predictable daily environmental challenges. The circadian rhythm of body temperature is thought to be an important modulator of molecular clocks in peripheral tissues, but how daily temperature cycles affect physiological function is unclear. Here, we examined the effect of constant temperature (Tcon, 25°C) and cycling temperature (Tcyc, 28°C:22°C during light:dark) paradigms on lifespan of Drosophila melanogaster, and the expression of clock genes, heat shock protein 83 (Hsp83), Frost (Fst) and senescence marker protein-30 (smp-30). Male and female D. melanogaster housed at Tcyc had longer median lifespans than those housed at Tcon. Tcyc induced robust Hsp83 rhythms and rescued the age-related decrease in smp-30 expression that was observed in flies at Tcon, potentially indicating an increased capacity to cope with age-related cellular stress. Ageing under Tcon led to a decrease in the amplitude of expression of all clock genes in the bodies of male flies, except for cyc, which was non-rhythmic, and for per and cry in female flies. Strikingly, housing under Tcyc conditions rescued the age-related decrease in amplitude of all clock genes, and generated rhythmicity in cyc expression, in the male flies, but not the female flies. The results suggest that ambient temperature rhythms modulate D. melanogaster lifespan, and that the amplitude of clock gene expression in peripheral body clocks may be a potential link between temperature rhythms and longevity in male D. melanogaster. Longevity due to Tcyc appeared predominantly independent of clock gene amplitude in female D. melanogaster.

MDT-15/MED15 permits longevity at low temperature via enhancing lipidostasis and proteostasis.

Low temperatures delay aging and promote longevity in many organisms. However, the metabolic and homeostatic aspects of low-temperature-induced longevity remain poorly understood. Here, we show that lipid homeostasis regulated by Caenorhabditis elegans Mediator 15 (MDT-15 or MED15), a transcriptional coregulator, is essential for low-temperature-induced longevity and proteostasis. We find that inhibition of mdt-15 prevents animals from living long at low temperatures. We show that MDT-15 up-regulates fat-7, a fatty acid desaturase that converts saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs), at low temperatures. We then demonstrate that maintaining a high UFA/SFA ratio is essential for proteostasis at low temperatures. We show that dietary supplementation with a monounsaturated fatty acid, oleic acid (OA), substantially mitigates the short life span and proteotoxicity in mdt-15(-) animals at low temperatures. Thus, lipidostasis regulated by MDT-15 appears to be a limiting factor for proteostasis and longevity at low temperatures. Our findings highlight the crucial roles of lipid regulation in maintaining normal organismal physiology under different environmental conditions.

Episodic Ultradian Events-Ultradian Rhythms.

In the fast lane of chronobiology, ultradian events are short-term rhythms that have been observed since the beginning of modern biology and were quantified about a century ago. They are ubiquitous in all biological systems and found in all organisms, from unicellular organisms to mammals, and from single cells to complex biological functions in multicellular animals. Since these events are aperiodic and last for a few minutes to a few hours, they are better classified as episodic ultradian events (EUEs). Their origin is unclear. However, they could have a molecular basis and could be controlled by hormonal inputs-in vertebrates, they originate from the activity of the central nervous system. EUEs are receiving increasing attention but their aperiodic nature requires specific sampling and analytic tools. While longer scale rhythms are adaptations to predictable changes in the environment, in theory, EUEs could contribute to adaptation by preparing organisms and biological functions for unpredictability.

NHR-49/HNF4 integrates regulation of fatty acid metabolism with a protective transcriptional response to oxidative stress and fasting.

Endogenous and exogenous stresses elicit transcriptional responses that limit damage and promote cell/organismal survival. Like its mammalian counterparts, hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor α (PPARα), Caenorhabditis elegans NHR-49 is a well-established regulator of lipid metabolism. Here, we reveal that NHR-49 is essential to activate a transcriptional response common to organic peroxide and fasting, which includes the pro-longevity gene fmo-2/flavin-containing monooxygenase. These NHR-49-dependent, stress-responsive genes are also upregulated in long-lived glp-1/notch receptor mutants, with two of them making critical contributions to the oxidative stress resistance of wild-type and long-lived glp-1 mutants worms. Similar to its role in lipid metabolism, NHR-49 requires the mediator subunit mdt-15 to promote stress-induced gene expression. However, NHR-49 acts independently from the transcription factor hlh-30/TFEB that also promotes fmo-2 expression. We show that activation of the p38 MAPK, PMK-1, which is important for adaptation to a variety of stresses, is also important for peroxide-induced expression of a subset of NHR-49-dependent genes that includes fmo-2. However, organic peroxide increases NHR-49 protein levels, by a posttranscriptional mechanism that does not require PMK-1 activation. Together, these findings establish a new role for the HNF4/PPARα-related NHR-49 as a stress-activated regulator of cytoprotective gene expression.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice.

Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

Gain-of-Function Alleles in Caenorhabditis elegans Nuclear Hormone Receptor nhr-49 Are Functionally Distinct.

Nuclear hormone receptors (NHRs) are transcription factors that regulate numerous physiological and developmental processes and represent important drug targets. NHR-49, an ortholog of Hepatocyte Nuclear Factor 4 (HNF4), has emerged as a key regulator of lipid metabolism and life span in the nematode worm Caenorhabditis elegans. However, many aspects of NHR-49 function remain poorly understood, including whether and how it regulates individual sets of target genes and whether its activity is modulated by a ligand. A recent study identified three gain-of-function (gof) missense mutations in nhr-49 (nhr-49(et7), nhr-49(et8), and nhr-49(et13), respectively). These substitutions all affect the ligand-binding domain (LBD), which is critical for ligand binding and protein interactions. Thus, these alleles provide an opportunity to test how three specific residues contribute to NHR-49 dependent gene regulation. We used computational and molecular methods to delineate how these mutations alter NHR-49 activity. We find that despite originating from a screen favoring the activation of specific NHR-49 targets, all three gof alleles cause broad upregulation of NHR-49 regulated genes. Interestingly, nhr-49(et7) and nhr-49(et8) exclusively affect nhr-49 dependent activation, whereas the nhr-49(et13) surprisingly affects both nhr-49 mediated activation and repression, implicating the affected residue as dually important. We also observed phenotypic non-equivalence of these alleles, as they unexpectedly caused a long, short, and normal life span, respectively. Mechanistically, the gof substitutions altered neither protein interactions with the repressive partner NHR-66 and the coactivator MDT-15 nor the subcellular localization or expression of NHR-49. However, in silico structural modeling revealed that NHR-49 likely interacts with small molecule ligands and that the missense mutations might alter ligand binding, providing a possible explanation for increased NHR-49 activity. In sum, our findings indicate that the three nhr-49 gof alleles are non-equivalent, and highlight the conserved V411 residue affected by et13 as critical for gene activation and repression alike.

Altered energy intake and the amplitude of the body temperature rhythm are associated with changes in phase, but not amplitude, of clock gene expression in the rat suprachiasmatic nucleus in vivo.

Circadian rhythms in mammals are driven by a central clock in the suprachiasmatic nucleus (SCN). In vitro, temperature cycles within the physiological range can act as potent entraining cues for biological clocks. We altered the body temperature (Tc) rhythm in rats by manipulating energy intake (EI) to determine whether EI-induced changes in Tc oscillations are associated with changes in SCN clock gene rhythms in vivo. Male Wistar rats (n = 16 per diet) were maintained on either an ad libitum diet (CON), a high energy cafeteria diet (CAF), or a calorie restricted diet (CR), and Tc was recorded every 30 min for 6-7 weeks. SCN tissue was harvested from rats at zeitgeber time (ZT) 0, ZT6, ZT12, or ZT18. Expression of the clock genes Bmal1, Per2, Cry1, and Rev-erbα, the heat shock transcription factor Hsf1, and the heat shock protein Hsp90aa1, were determined using qPCR. The circadian profile of gene expression for each gene was characterized using cosinor analysis. Compared to the CON rats, the amplitude of Tc was decreased in CAF rats by 0.1 °C (p < 0.001), and increased in CR rats by 0.3 °C (p < 0.001). The amplitude of Hsp90aa1 expression was lowest in CAF rats and highest in CR rats (p = 0.045), but the amplitude of all of the clock genes and Hsf1 were unaffected by diet (p > 0.25). Compared to CON, phase advances of the Tc, Bmal1, and Per2 rhythms were observed with CR feeding (p < 0.05), but CAF feeding elicited no significant changes in phase. The present results indicate that in vivo, the SCN is largely resistant to entrainment by EI-induced changes in the Tc rhythm, although some phase entrainment may occur.

YIP1 family member 4 (YIPF4) is a novel cellular binding partner of the papillomavirus E5 proteins.

E5 proteins are amongst the least understood of the Human Papillomavirus (HPV) encoded gene products. They are small, membrane-integrated proteins known to modulate a number of critical host pathways associated with pathogenesis including growth factor receptor signaling and immune evasion. Their role in the virus life cycle is less clear, indicating a role in the productive stages of the life cycle. However, a mechanism for this is currently lacking. Here we describe the identification of a novel binding partner of E5, YIPF4 using yeast two-hybrid analysis. YIPF4 is also a poorly characterized membrane spanning protein. Mutagenesis studies implicated the transmembrane regions of each protein as important for their interaction. Binding to YIPF4 was found for all E5 proteins tested suggesting that this interaction may mediate a conserved E5 function. In normal human keratinocytes YIPF4 expression was down-regulated upon differentiation and this reduction was partially rescued in cells harbouring HPV. Despite the conserved nature of the interaction with E5, siRNA mediated depletion of YIPF4 failed to impede two well-characterized functions of E5, namely EGFR trafficking or HLA class I presentation. Continued studies of YIPF4 are warranted to determine its role in the PV life cycle.

The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator.

The Mediator multiprotein complex ('Mediator') is an important transcriptional coregulator that is evolutionarily conserved throughout eukaryotes. Although some Mediator subunits are essential for the transcription of all protein-coding genes, others influence the expression of only subsets of genes and participate selectively in cellular signaling pathways. Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study of developmental and physiological regulation in vivo. In this nematode, unbiased genetic screens have revealed critical roles for Mediator components in core developmental pathways such as epidermal growth factor (EGF) and Wnt/ß-catenin signaling. More recently, important roles for C. elegans Mediator subunits have emerged in the regulation of lipid metabolism and of systemic stress responses, engaging conserved transcription factors such as nuclear hormone receptors (NHRs). We emphasize instances where similar functions for individual Mediator subunits exist in mammals, highlighting parallels between Mediator subunit action in nematode development and in human cancer biology. We also discuss a parallel between the association of the Mediator subunit MED12 with several human disorders and the role of its C. elegans ortholog mdt-12 as a regulatory hub that interacts with numerous signaling pathways.

The conserved Mediator subunit MDT-15 is required for oxidative stress responses in Caenorhabditis elegans.

Reactive oxygen species (ROS) play important signaling roles in metazoans, but also cause significant molecular damage. Animals tightly control ROS levels using sophisticated defense mechanisms, yet the transcriptional pathways that induce ROS defense remain incompletely understood. In the nematode Caenorhabditis elegans, the transcription factor SKN-1 is considered a master regulator for detoxification and oxidative stress responses. Here, we show that MDT-15, a subunit of the conserved Mediator complex, is also required for oxidative stress responses in nematodes. Specifically, mdt-15 is required to express SKN-1 targets upon chemical and genetic increase in SKN-1 activity. mdt-15 is also required to express genes in SKN-1-dependent and SKN-1-independent fashions downstream of insulin/IGF-1 signaling and for the longevity of daf-2/insulin receptor mutants. At the molecular level, MDT-15 binds SKN-1 through a region distinct from the classical transcription-factor-binding KIX-domain. Moreover, mdt-15 is essential for the transcriptional response to and survival on the organic peroxide tert-butyl-hydroperoxide (tBOOH), a largely SKN-1-independent response. The MDT-15 interacting nuclear hormone receptor, NHR-64, is specifically required for tBOOH but not arsenite resistance, but NHR-64 is dispensable for the transcriptional response to tBOOH. Hence, NHR-64 and MDT-15's mode of action remain elusive. Lastly, the role of MDT-15 in oxidative stress defense is functionally separable from its function in fatty acid metabolism, as exogenous polyunsaturated fatty acid complementation rescues developmental, but not stress sensitivity phenotypes of mdt-15 worms. Our findings reveal novel conserved players in the oxidative stress response and suggest a broad cytoprotective role for MDT-15.