Regulation of Chromatin Accessibility by hypoxia and HIF

Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) coordinate a transcriptional programme required for survival and adaptation to hypoxia. The effects of hypoxia and HIF on the chromatin accessibility landscape are still unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility enriched at hypoxia transcriptionally responsive genes. These changes are predominantly HIF dependent, reversible upon reoxygenation and partially mimicked by chemical HIF stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that indeed, HIF stabilisation is necessary and sufficient to alter chromatin accessibility in hypoxia, with implications for our understanding of gene expression regulation by hypoxia and HIF.

RATIO OF TOTAL SLOW AND SUPER SLOW CEREBRAL BIOELECTRICAL ACTIVITY IN CHILDREN WITH HYPOXIA

The aim of the study is to identify changes in the total slow and super slow bioelectric activity of the cerebral hemispheres in 8–11-year-old children under reduced oxygen partial pressure in the inhaled air and in norm. Materials and Methods. The authors examined 82 8–11-year-old children. EEG registration was carried out on a complex Russian device "Telepat-103" with computer processing of the results. Slow potentials were recorded using a special computer complex for studying constant potential level (CPL) and cerebral energy consumption "Neuroenergon". Hypoxia was created by a special medical device "Hypoxicator" (Trade Medical). The choice of the oxygen content in the test hypoxic mixture (14 % O2) was based on the results of a three-stage hypoxic test carried out before the complex examination. Results. Hypoxia increases the index and amplitude of delta oscillations and decreases the number of alpha waves in 8–11-year-old children. The results obtained indicate that it is more important to provide the brain with a sufficient amount of oxygen corresponding to its needs than simply to increase the brain blood supply during hypoxia. The study also indicates the contribution of the body's sensitivity to hypoxic response. Under hypoxia, an increase in the constant potential level in the studied brain areas is observed in 8–11-year-old children. An increase in electrical activity during normobaric hypoxia is noted at low baseline values of constant potential level. An increase in the slow electrical brain activity occurs due to the change of brain energy supply according to the biochemical indicators of energy metabolism. Short-term hypoxia is accompanied by a simultaneous increase in slow-wave activity due to delta activity and CPL in 8–11-year-old children. Conclusion. Thus, we can say that a picture of functional brain activity with simultaneous inhibitory phenomena in the cortex is formed. It may reflect the development of a special state of the central nervous system. Key words: constant potential level, electroencephalography, hypoxia, adaptation to hypoxia. Цель работы – выявление изменений суммарной медленной и сверхмедленной биоэлектрической активности полушарий головного мозга у детей в возрасте от 8 до 11 лет в условиях нормального и сниженного парциального давления кислорода во вдыхаемом воздухе. Материалы и методы. Обследовано 82 ребенка 8–11-летнего возраста. Регистрация ЭЭГ осуществлялась на комплексной отечественной установке «Телепат-103» с компьютерной обработкой результатов. Медленные потенциалы фиксировались с помощью специального компьютерного комплекса для исследования уровня постоянных потенциалов (УПП) и энергозатрат головного мозга «Нейроэнергон». Гипоксические условия создавались аппаратом «Гипоксикатор» фирмы Trade Medical. Выбор содержания кислорода в тестирующей гипоксической смеси (14 % О2) основывался на результатах проведенного до комплексного обследования трехступенчатого гипоксического теста. Результаты. У детей от 8 до 11 лет действие гипоксии на головной мозг приводит к возрастанию индекса и амплитуды дельта-колебаний и убыванию числа альфа-волн. Полученные результаты говорят о том, что большое значение имеет не столько усиление кровоснабжения головного мозга при гипоксии, сколько обеспечение мозга достаточным, соответствующим его потребностям, количеством кислорода, а также о роли чувствительности организма к гипоксии. При воздействии гипоксии у детей этого возраста наблюдается возрастание уровня постоянных потенциалов в исследуемых областях головного мозга. При низких фоновых значениях уровня постоянных потенциалов отмечается возрастание электрической активности при нормобарической гипоксии. Возрастание медленной электрической активности головного мозга происходит из-за того, что изменяется энергообеспечение головного мозга по биохимическим показателям энергетического обмена. Действие кратковременной гипоксии сопровождается одновременным возрастанием медленноволновой активности за счет дельта-активности и УПП у детей от 8 до 11 лет. Выводы. Таким образом, можно говорить о том, что формируется своеобразная картина функциональной активности с одновременными тормозными явлениями в коре, что может являться отражением развития особого состояния центральной нервной системы. Ключевые слова: уровень постоянных потенциалов, электроэнцефалография, гипоксия, адаптация к гипоксии.

Understanding p300-transcription factor interactions using sequence variation and hybridization

The hypoxic response is central to cell function and plays a significant role in the growth and survival of solid tumours. HIF-1 regulates the hypoxic response by activating over 100 genes responsible for adaptation to hypoxia, making it a potential target for anticancer drug discovery. Although there is significant structural and mechanistic understanding of the interaction between HIF-1α and p300 alongside negative regulators of HIF-1α such as CITED2, there remains a need to further understand the sequence determinants of binding. In this work we use a combination of protein expression, chemical synthesis, fluorescence anisotropy and isothermal titration calorimetry for HIF-1α sequence variants and a HIF-1α- CITED hybrid sequence which we term CITIF. We show the HIF-1α sequence is highly tolerant to sequence variation through reduced enthalpic and less unfavourable entropic contributions, These data imply backbone as opposed to side chain interactions and ligand folding control the binding interaction and that sequence variations are tolerated as a result of adopting a more disordered bound interaction or fuzzy complex.

Characterization of the pVHL Interactome in Human Testis Using High-Throughput Library Screening

Functional impairment of the von Hippel-Lindau (pVHL) tumor suppressor is causative of a familiar increased risk to develop cancer. As E3 substrate recognition particle, pVHL marks for degradation the hypoxia inducible factor 1α (HIF-1α) in normoxic conditions, thus acting as a key regulator of both acute and chronic cell adaptation to hypoxia. Further evidence showed pVHL to also play relevant roles in microtubules stabilization, participate in the formation of the extracellular matrix, as well as to regulate cell senescence and apoptosis. Male mice model carrying VHL gene conditional knockout present significative abnormalities in testis development paired with defects in spermatogenesis and infertility, indicating that pVHL exerts testis-specific roles, at least in mice. Here, we describe 55 novel interactors of the human pVHL obtained by testis-tissue library screening. We show that pVHL interacts with multiple human proteins directly involved in spermatogenesis and reproductive metabolism, suggesting that, in addition to its role in cancer formation, pVHL may be pivotal in the correct gonads development also in human.

BNIP3 Mediates the Different Adaptive Responses of Fibroblast-like Synovial Cells to Hypoxia in Patients With Osteoarthritis and Rheumatoid Arthritis

Background: Hypoxia is one of the important characteristics of synovial microenvironment in rheumatoid arthritis (RA), and it is very important in the process of synovial hyperplasia. Fibroblast-like synovial cells (FLSs) are relatively affected by hypoxia injury in cell survival, while FLSs from patients with RA (RA-FLSs) are particularly resistant to hypoxia-induced cell death. The purpose of this study was to evaluate whether FLSs in patients with osteoarthritis (OA) and RA-FLSs have the same adaptation to hypoxia. Methods: CCK-8, flow cytometry and BrdU were used to detect the proliferation of OA-FLSs and RA-FLSs under different oxygen concentrations. Apoptosis was detected by AV/PI, TUNEL and Western blot, mitophagy was observed by electron microscope and Western blot, mitochondrial state was detected by reactive oxygen species (ROS) and mitochondrial membrane potential by flow cytometry, BNIP3 and HIF-1α were detected by Western blot and RT-qPCR. The silencing of BNIP3 is achieved by stealth RNA system technology. Results: After hypoxia, the survival rate of OA-FLSs was reduced, and the proliferation activity of RA-FLSs was further increased. Hypoxia induced increased apoptosis and inhibited autophagy of OA-FLSs, but not in RA-FLSs. Hypoxia treatment led to a more lasting adaptive response. RA-FLSs showed a more significant increase in gene expression regulated by HIF-1α transcription. Interestingly, they showed higher BNIP3 expression than OA-FLSs, and showed stronger mitophagy and proliferation activities. The BNIP3 siRNA experiment in RA-FLSs confirmed the potential role of BNIP3 in the survival of FLSs. The inhibition of BNIP3 resulted in the decrease of cell proliferation and the decrease of mitophagy and the increase of apoptosis. Conclusion: In summary, RA-FLSs maintained redox balance through mitophagy to promote cell survival under hypoxia. The mitophagy of OA-FLSs was too little to maintain the redox balance of mitochondria, leading to apoptosis. The difference of mitophagy between OA-FLSs and RA-FLSs under hypoxia is mediated by the expression of BNIP3.

Mitochondrial Redox Metabolism: The Epicenter of Metabolism during Cancer Progression

Mitochondrial redox metabolism is the central component in the cellular metabolic landscape, where anabolic and catabolic pathways are reprogrammed to maintain optimum redox homeostasis. During different stages of cancer, the mitochondrial redox status plays an active role in navigating cancer cells’ progression and regulating metabolic adaptation according to the constraints of each stage. Mitochondrial reactive oxygen species (ROS) accumulation induces malignant transformation. Once vigorous cell proliferation renders the core of the solid tumor hypoxic, the mitochondrial electron transport chain mediates ROS signaling for bringing about cellular adaptation to hypoxia. Highly aggressive cells are selected in this process, which are capable of progressing through the enhanced oxidative stress encountered during different stages of metastasis for distant colonization. Mitochondrial oxidative metabolism is suppressed to lower ROS generation, and the overall cellular metabolism is reprogrammed to maintain the optimum NADPH level in the mitochondria required for redox homeostasis. After reaching the distant organ, the intrinsic metabolic limitations of that organ dictate the success of colonization and flexibility of the mitochondrial metabolism of cancer cells plays a pivotal role in their adaptation to the new environment.

Comprehensive Analysis of 13C6 Glucose Fate in the Hypoxia-Tolerant Blind Mole Rat Skin Fibroblasts

The bioenergetics of the vast majority of terrestrial mammals evolved to consuming glucose (Glc) for energy production under regular atmosphere (about 21% oxygen). However, some vertebrate species, such as aquatic turtles, seals, naked mole rat, and blind mole rat, Spalax, have adjusted their homeostasis to continuous function under severe hypoxic environment. The exploration of hypoxia-tolerant species metabolic strategies provides a better understanding of the adaptation to hypoxia. In this study, we compared Glc homeostasis in primary Spalax and rat skin cells under normoxic and hypoxic conditions. We used the targeted-metabolomics approach, utilizing liquid chromatography and mass spectrometry (LC-MS) to track the fate of heavy Glc carbons (13C6 Glc), as well as other methodologies to assist the interpretation of the metabolic landscape, such as bioenergetics profiling, Western blotting, and gene expression analysis. The metabolic profile was recorded under steady-state (after 24 h) of the experiment. Glc-originated carbons were unequally distributed between the cytosolic and mitochondrial domains in Spalax cells compared to the rat. The cytosolic domain is dominant apparently due to the hypoxia-inducible factor-1 alpha (HIF-1α) mastering, since its level is higher under normoxia and hypoxia in Spalax cells. Consumed Glc in Spalax cells is utilized for the pentose phosphate pathway maintaining the NADPH pool, and is finally harbored as glutathione (GSH) and UDP-GlcNAc. The cytosolic domain in Spalax cells works in the semi-uncoupled mode that limits the consumed Glc-derived carbons flux to the tricarboxylic acid (TCA) cycle and reduces pyruvate delivery; however, it maintains the NAD+ pool via lactate dehydrogenase upregulation. Both normoxic and hypoxic mitochondrial homeostasis of Glc-originated carbons in Spalax are characterized by their massive cataplerotic flux along with the axis αKG→Glu→Pro→hydroxyproline (HPro). The product of collagen degradation, HPro, as well as free Pro are apparently involved in the bioenergetics of Spalax under both normoxia and hypoxia. The upregulation of 2-hydroxyglutarate production detected in Spalax cells may be involved in modulating the levels of HIF-1α. Collectively, these data suggest that Spalax cells utilize similar metabolic frame for both normoxia and hypoxia, where glucose metabolism is switched from oxidative pathways (conversion of pyruvate to Acetyl-CoA and further TCA cycle processes) to (i) pentose phosphate pathway, (ii) lactate production, and (iii) cataplerotic pathways leading to hexosamine, GSH, and HPro production.

Mechanisms and triggers of adaptation to hypoxia

It is believed that hypoxia-induced factor (HIF1) is the key mediator of oxygen metabolism. It was first identified as a transcription factor activated in cells and tissues by lowering the partial pressure of oxygen (O2). The HIF1 activator spectrum includes both external factors hypoxia, psycho-emotional stress and in ternal factors and varies from hormones to iron chelators. This review is dedicated to the molecular mechanisms of HIF1 activation, some of its natural activators HIF1, the potential for which is due to the low level of toxicity and the reduced likelihood of undesirable side effects. In turn, this opens up new options to treat diseases associated with local and general ischemia and hypoxia, the possibilities of their prophylactic use for researchers and clinicians in order to reduce the degree of damage in the event of an unforeseen condition of acute injurious to organs and tissues by hypoxia and reperfusion after it.