No abstract

Vertebrate hemoglobin (Hb) and myoglobin (Mb) were among the first proteins whose structures and sequences were determined over 50 years ago. In the subsequent pregenomic period, numerous related proteins came to light in plants, invertebrates and bacteria, that shared the myoglobin fold, a signature sequence motif characteristic of a 3-on-3 α-helical sandwich. Concomitantly, eukaryote and bacterial globins with a truncated 2-on-2 α-helical fold were discovered. Genomic information over the last 20 years has dramatically expanded the list of known globins, demonstrating their existence in a limited number of archaeal genomes, a majority of bacterial genomes and an overwhelming majority of eukaryote genomes. In vertebrates, 6 additional globin types were identified, namely neuroglobin (Ngb), cytoglobin (Cygb), globin E (GbE), globin X (GbX), globin Y (GbY) and androglobin (Adgb). Furthermore, functions beyond the familiar oxygen transport and storage have been discovered within the vertebrate globin family, including NO metabolism, peroxidase activity, scavenging of free radicals, and signaling functions. The extension of the knowledge on globin functions suggests that the original roles of bacterial globins must have been enzymatic, involved in defense against NO toxicity, and perhaps also as sensors of O 2 , regulating taxis away or towards high O 2 concentrations. In this review, we aimed to discuss the evolution and remarkable functional diversity of vertebrate globins with particular focus on the variety of non-canonical expression sites of mammalian globins and their according impressive variability of atypical functions.

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that—in addition to the testes—ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.