Androglobin (ADGB) is a highly conserved and recently identified member of the globin superfamily. Although previous studies revealed a link to ciliogenesis and an involvement in murine spermatogenesis, its physiological function remains mostly unknown. Apart from FOXJ1-dependent regulation, the transcriptional landscape of the ADGB gene remains unexplored. We, therefore, aimed to obtain further insights into regulatory mechanisms governing ADGB expression. To this end, changes in ADGB promoter activity were examined using luciferase reporter gene assays in the presence of a set of more than 475 different exogenous transcription factors. MYBL2 and PITX2 resulted in the most pronounced increase in ADGB promoter-dependent luciferase activity. Subsequent truncation strategies of the ADGB promoter fragment narrowed down the potential MYBL2 and PITX2 binding sites within the proximal ADGB promoter. Furthermore, MYBL2 binding sites on the ADGB promoter were further validated via a guide RNA-mediated interference strategy using reporter assays. Chromatin immunoprecipitation (ChIP)-qPCR experiments illustrated enrichment of the endogenous ADGB promoter region upon MYBL2 and PITX2 overexpression. Consistently, ectopic MYBL2 expression induced endogenous ADGB mRNA levels. Collectively, our data indicate that ADGB is strongly regulated at the transcriptional level and might have functions beyond ciliogenesis.

Androglobin (Adgb) is the most distinctive member of the globin superfamily. Its characteristic globin domain is permuted, interrupted by a calmodulin-binding motif, and embedded within a large multidomain protein of ∼1500 amino acids that also contains a calpain protease domain. Initially described as testis-specific in mammals, Adgb is also expressed in ciliated epithelia of the female reproductive tract, lung, and brain, and knockout studies reveal its pivotal role during spermatogenesis. To trace its evolutionary origin, we performed comprehensive phylogenetic analysis across diverse eukaryotic taxa. Adgb is present in all major flagellated eukaryotic lineages but absent from nonflagellated clades. Orthology analysis indicates Adgb has been maintained as a predominantly single-copy gene across >1 billion years of evolution—a pattern contrasting sharply with other globins that underwent repeated duplication and functional diversification. Analysis of publicly available transcriptomes from early-branching metazoans confirmed robust Adgb expression in ciliated cell types and provides evidence for its regulation by the ancient ciliogenic transcription factor cRFXa in the choanoflagellate Salpingoeca rosetta. RNA in situ hybridization validated these findings, and comparative analyses suggest that ancestral Adgb homologs lacked the permuted globin domain found in metazoans. Collectively, our results demonstrate that Adgb exemplifies a rare evolutionary trajectory where structural innovation (domain permutation and fusion) enabled functional specialization while being an integral part of the nonredundant ciliary machinery. Adgb thus illustrates how constraint and innovation combine to shape the long-term fate of a protein.