Summary Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4FH. O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, as they are during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus.

Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5 cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2␣ and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxiainducible factor 2␣ expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant capbinding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins.

Hypoxia is an aspect of the tumor microenvironment that is linked to radiation and chemotherapy resistance, metastasis, and poor prognosis. The ability of hypoxic tumor cells to achieve these cancer hallmarks is, in part, due to changes in their gene expression profiles. Cancer cells have a high demand for protein synthesis, and translational control is subsequently deregulated. Various mechanisms of translation initiation are active to improve the translation efficiency of select transcripts to drive cancer progression. This review will focus on a noncanonical cap-dependent translation initiation mechanism that utilizes the eIF4E homolog eIF4E2, a hypoxia-activated cap-binding protein that is implicated in hypoxic cancer cell migration, invasion, and tumor growth in mouse xenografts. A historical perspective about eIF4E2 and its various aliases will be provided followed by an evaluation of potential therapeutic strategies. The recent successes of disabling canonical translation and eIF4E with drugs should highlight the novel therapeutic potential of targeting the homologous eIF4E2 in the treatment of hypoxic solid tumors.

The ability to determine the genetic etiology of intellectual disability (ID) and neurodevelopmental disorders (NDD) has improved immensely over the last decade. One prevailing metric from these studies is the large percentage of genes encoding epigenetic regulators, including many members of the ATP-dependent chromatin remodeling enzyme family. Chromatin remodeling proteins can be subdivided into five classes that include SWI/SNF, ISWI, CHD, INO80, and ATRX. These proteins utilize the energy from ATP hydrolysis to alter nucleosome positioning and are implicated in many cellular processes. As such, defining their precise roles and contributions to brain development and disease pathogenesis has proven to be complex. In this review, we illustrate that complexity by reviewing the roles of ATRX on genome stability, replication, and transcriptional regulation and how these mechanisms provide key insight into the phenotype of ATR-X patients.

Multicellular organisms balance oxygen delivery and toxicity by having oxygen pass through several barriers before cellular delivery. In human cell culture, these physiologic barriers are removed, exposing cells to higher oxygen levels. Human cells cultured in ambient air may appear normal, but this is difficult to assess without a comparison at physiologic oxygen. Here, we examined the effects of culturing human cells throughout the spectrum of oxygen availability on oxidative damage to macromolecules, viability, proliferation, the antioxidant and DNA damage responses, metabolism, and mitochondrial fusion and morphology. We surveyed 4 human cell lines cultured for 3 d at 7 oxygen conditions between 1 and 21% O2. We show that oxygen levels and cellular benefit are not inversely proportional, but the benefit peaks within the physioxic range. Normoxic cells are in a perpetual state of responding to damaged macromolecules and mitochondrial networks relative to physioxic cells, which could compromise an investigation. These data contribute to the concept of an optimal oxygen availability for cell culture in the physioxic range where the oxygen is not too high to reduce oxidative damage, and not too low for efficient oxidative metabolism, but just right: the Goldiloxygen zone.—Timpano, S., Guild, B. D., Specker, E. J., Melanson, G., Medeiros, P. J., Sproul, S. L. J., Uniacke, J. Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage. FASEB J. 33, 5716–5728 (2019). http://www.fasebj.org

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked mGTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4F), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the mGTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Chromatin remodeling proteins utilize the energy from ATP hydrolysis to mobilize nucleosomes often creating accessibility for transcription factors within gene regulatory elements. Aberrant chromatin remodeling has diverse effects on neuroprogenitor homeostasis altering progenitor competence, proliferation, survival, or cell fate. Previous work has shown that inactivation of the ISWI genes, Smarca5 (encoding Snf2h) and Smarca1 (encoding Snf2l) have dramatic effects on brain development. Smarca5 conditional knockout mice have reduced progenitor expansion and severe forebrain hypoplasia, with a similar effect on the postnatal growth of the cerebellum. In contrast, Smarca1 mutants exhibited enlarged forebrains with delayed progenitor differentiation and increased neuronal output. Here, we utilized cerebellar granule neuron precursor (GNP) cultures from Smarca1 mutant mice (Ex6DEL) to explore the requirement for Snf2l on progenitor homeostasis. The Ex6DEL GNPs showed delayed differentiation upon plating that was not attributed to changes in the Sonic Hedgehog pathway but was associated with overexpression of numerous positive effectors of proliferation, including targets of Wnt activation. Transcriptome analysis identified increased expression of Fosb and Fosl2 while ATACseq experiments identified a large increase in chromatin accessibility at promoters many enriched for Fos/Jun binding sites. Nonetheless, the elevated proliferation index was transient and the Ex6DEL cultures initiated differentiation with a high concordance in gene expression changes to the wild type cultures. Genes specific to Ex6DEL differentiation were associated with an increased activation of the ERK signaling pathway. Taken together, this data provides the first indication of how Smarca1 mutations alter progenitor cell homeostasis and contribute to changes in brain size.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m 7 GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4F H ), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m 7 GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.