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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.

In the quest to unravel its functional significance, neuroglobin (Ngb), a brain-specific neuroprotective protein, has recently been proposed as an actor in neurodevelopment. As neural stem cells (NSCs) are fundamental during brain development, the present study aimed at investigating the role of Ngb in the growth and proliferation of NSCs by comparing an Ngb-floxed (Ngb-)NSC line, equivalent to the wild-type cellular situation, with an in-house created Ngb knockout (Ngb-)NSC line. Ngb-NSCs were characterized by an increased growth and proliferation capacity in vitro, supported by RNA sequencing and western blot results reporting the downregulation of Cdkn1a and the upregulation of Cdk6, both enhancing the cell cycle. Based on additional gene ontology enrichment and pathway analyses, we hypothesize that the loss of Ngb affects multiple cellular signaling pathways with the most important being the Akt-Tp53 axis.

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