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1. HIF signaling in the prothoracic gland regulates growth and development in hypoxia but not normoxia in Drosophila

   https://doi.org/10.1242/jeb.247697  

   Campbell, Jacob B; Shingleton, Alexander W; Greenlee, Kendra J; Gray, Alyx E; Smith, Hunter C; Callier, Viviane; Lundquist, Taylor; Harrison, Jon F (September 2024, Journal of Experimental Biology)

   ABSTRACT The developmental regulation of body size is a fundamental life-history characteristic that in most animals is tied to the transition from juvenile to adult form. In holometabolous insects, this transition is ostensibly initiated at the attainment of a critical weight in the final larval instar. It has been hypothesized that the size-sensing mechanism used to determine attainment of critical weight exploits oxygen limitation as a larvae grows beyond the oxygen-delivery capacity of its fixed tracheal system; that is, developmentally induced cellular hypoxia initiates the synthesis of the molting hormone ecdysone by the prothoracic gland. We tested this hypothesis in Drosophila by assaying cellular hypoxia throughout the third larval instar at 21 and 10 kPa O2, using the activity of the HIF (hypoxia inducible factor)-signaling pathway as a measure of hypoxia. While HIF signaling was elevated at low levels of environmental O2, it did not markedly increase during development at either oxygen level, and was only suppressed by hyperoxia after feeding had ceased. Further, changes in HIF signaling in the prothoracic gland alone did not alter body size or developmental time in a way that would be expected if cellular hypoxia in the prothoracic gland was part of the critical weight mechanism. Our data do show, however, that reduced HIF signaling in the prothoracic gland decreases survival and retards development at 10 kPa O2, suggesting that prothoracic HIF signaling is a necessary part of the beneficial plasticity mechanism that controls growth and development in response to low oxygen level. 

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2. The Effects of Hypoxia on Heart and Brain Tissue of the African Naked Mole-Ra

   Applegate, Daniel (March 2025, University of Illinois Chicago)

   SUMMARY This thesis examines the critical issues hypoxia causes for mammals and highlights the intricate adaptive mechanisms that sustain cellular equilibrium under 02-deficient conditions. Mitochondrial oxidative phosphorylation is emphasized as the primary, efficient method for energy production in aerobic scenarios, contrasting with the less efficient glycolysis pathway. The Hypoxia-Inducible Factor (HIF) pathway is identified as a crucial mediator for cellular adaptation during low 02 conditions, influencing gene expression to facilitate adaptive responses in various physiological contexts. The dissertation presents the naked mole-rat as a model organism with exceptional adaptations to hypoxia and metabolic traits that enable survival in low-02 environments. This resilience is demonstrated through brain slice models that maintain synaptic function under extreme 02 deprivation. The dissertation aims to expand the understanding of hypoxia tolerance in naked mole- rats, focusing on their cardiac and neural responses, excitotoxicity, and comparative analysis across species. These insights are anticipated to have broader implications for health and disease management related to hypoxic conditions. The cardiac responses of naked mole-rats to hypoxia are investigated by comparing them with C57BL/6 mice. Results reveal that naked mole-rats possess remarkable tolerance to hypoxia, maintain breathing and heart rate under severe conditions, and recover swiftly upon reoxygenation, unlike mice. The discussion elaborates on the functional recovery of isolated hearts and metabolic adaptations in naked mole-rats, such as fructose metabolism and increased GLUT5 transporter expression. It notes the stabilization of HIF-1α protein in their hearts, potentially reducing oxidative stress. Neural adaptations of naked mole-rats to hypoxia are demonstrated using EEG x recordings, lactate measurements, and NMDA infusions into the caudate. The study evaluates brain activity during wakefulness, sleep, hypoxia, and recovery and investigates lactate's role in brain metabolism and neuroprotection. Mexican free-tailed bats and neonate mice were included in this study to examine hypoxia tolerance across a broader range of species. 

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3. NCBI GEO Accession ID GSE289863; The cistrome response to hypoxia in human umbilical vein endothelial cells

   Singh, A; Pastukh, V; Roberts, JT; Turpin, ZM; Fazler, ZS; Daly, GT; Benoit, JM; Gillespie, MN; Bass, HW (June 2026, Gene Expression Omnibus GEO)

   The primary goal of this study was to achieve a comprehensive, high-resolution understanding of how human umbilical vein endothelial cells (HUVECs) remodel their chromatin landscape in response to hypoxic stress. While it is well established that hypoxia-inducible factors (HIFs) play a pivotal role in activating hypoxia-responsive genes, the discovery that only a small fraction of known hypoxia response elements (HREs) is bound by HIFs under low-oxygen conditions suggests the involvement of additional, complementary regulatory factors. To investigate these putative co-regulatory elements, we adapted and applied a recently developed technique, MOA-seq (MNase-defined cistrome-Occupancy Analysis), which excels at identifying genome-wide, protein-occupied loci (MOA footprints) with approximately 30-bp resolution. By profiling small MNase-sensitive footprints, MOA-seq offers an orthogonal and more precise view of transcription factor (TF) occupancy compared to traditional methods like ChIP-seq or ATAC-seq. Through a time-course analysis (1, 3, and 24 hours of hypoxia), we generated a "cistrome atlas" of MOA footprints for more than 5,000 candidate regulatory regions undergoing hypoxia-induced changes. Our approach not only recapitulated canonical hypoxia-regulated pathways but also uncovered novel sites and TF motifs that do not co-localize with HIF-binding motifs. These high-resolution footprints revealed new potential avenues for understanding how endothelial cells respond to low-oxygen environments, beyond the classic HIF-centric paradigm. Critically, such a broad scope of regulation underscores the complexity of vascular remodeling, oxidative stress responses, and angiogenic signaling processes vital to both normal physiological adaptation and the development of pathologies such as cancer, atherosclerosis, and other hypoxia-linked conditions. By integrating MOA-seq TF occupancy profiling with differential gene expression analyses, we are expanding the toolkit for dissecting gene regulatory networks over time. This insight has far-reaching implications: the newly uncovered regulatory elements may serve as potential therapeutic targets or biomarkers for diseases where hypoxia is a key driver. Moreover, the single-assay simplicity of MOA-seq provides a scalable strategy for profiling diverse cellular contexts, adding value to fundamental research and translational applications. 

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4. NCBI BioProject Accession ID PRJNA1217882; The cistrome response to hypoxia in human umbilical vein endothelial cells

   Singh, A; Pastukh, V; Roberts, JT; Turpin, ZM; Fazler, ZS; Daly, GT; Benoit, JM; Gillespie, MN; Bass, HW (August 2026, NCBI BioProject)

   The primary goal of this study was to achieve a comprehensive, high-resolution understanding of how human umbilical vein endothelial cells (HUVECs) remodel their chromatin landscape in response to hypoxic stress. While it is well established that hypoxia-inducible factors (HIFs) play a pivotal role in activating hypoxia-responsive genes, the discovery that only a small fraction of known hypoxia response elements (HREs) is bound by HIFs under low-oxygen conditions suggests the involvement of additional, complementary regulatory factors. To investigate these putative co-regulatory elements, we adapted and applied a recently developed technique, MOA-seq (MNase-defined cistrome-Occupancy Analysis), which excels at identifying genome-wide, protein-occupied loci (MOA footprints) with approximately 30-bp resolution. By profiling small MNase-sensitive footprints, MOA-seq offers an orthogonal and more precise view of transcription factor (TF) occupancy compared to traditional methods like ChIP-seq or ATAC-seq. Through a time-course analysis (1, 3, and 24 hours of hypoxia), we generated a "cistrome atlas" of MOA footprints for more than 5,000 candidate regulatory regions undergoing hypoxia-induced changes. Our approach not only recapitulated canonical hypoxia-regulated pathways but also uncovered novel sites and TF motifs that do not co-localize with HIF-binding motifs. These high-resolution footprints revealed new potential avenues for understanding how endothelial cells respond to low-oxygen environments, beyond the classic HIF-centric paradigm. Critically, such a broad scope of regulation underscores the complexity of vascular remodeling, oxidative stress responses, and angiogenic signaling processes vital to both normal physiological adaptation and the development of pathologies such as cancer, atherosclerosis, and other hypoxia-linked conditions. By integrating MOA-seq TF occupancy profiling with differential gene expression analyses, we are expanding the toolkit for dissecting gene regulatory networks over time. This insight has far-reaching implications: the newly uncovered regulatory elements may serve as potential therapeutic targets or biomarkers for diseases where hypoxia is a key driver. Moreover, the single-assay simplicity of MOA-seq provides a scalable strategy for profiling diverse cellular contexts, adding value to fundamental research and translational applications. 

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5. BAP1 is a novel regulator of HIF-1α

   https://doi.org/10.1073/pnas.2217840120  

   Bononi, Angela; Wang, Qian; Zolondick, Alicia A.; Bai, Fang; Steele-Tanji, Mika; Suarez, Joelle S.; Pastorino, Sandra; Sipes, Abigail; Signorato, Valentina; Ferro, Angelica; et al (January 2023, Proceedings of the National Academy of Sciences)

   BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1β forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations. 

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