Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis

Abstract

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.

Androglobin, a chimeric mammalian globin, is required for male fertility

“…Moreover, a recent study reported the consistent presence of Adgb in the ciliomes of three distinct evolutionary ancestral taxa, further suggesting a conserved function related to microtubular organisation and likely flagella formation (Sigg et al 2017). In line with this study, our recent in vitro investigations have demonstrated that Adgb is transcriptionally regulated by FoxJ1, a master regulator of ciliogenesis (Koay et al 2021).…”

“…Decreased mRNA expression levels in infertile versus fertile men (Platts et al 2007) suggest a potential role of Adgb in spermatogenesis. Gene regulation and expression studies further suggest an association of Adgb with ciliogenesis including flagellum formation (Koay et al 2021). However, the in vivo function of Adgb remains unexplored.…”

Composition and function of the C1b/C1f region in the ciliary central apparatus

“…Thus, we speculate that the Adgb and FAP42 have similar position within C1b/C1f supercomplex. Importantly, in mammals androglobin is expressed at higher level in cells assembling motile cilia and its expression is co-regulated by FOXJ1 24 , 45 .…”

Spatial Transcriptome Uncovers the Mouse Lung Architectures and Functions

“…Jund has been reported to be involved in multiple mechanisms of cell fate regulation in the lung, including mediating transcriptional activation of angiotensinogen ( Abdul-Hafez et al, 2009 ), inducing interleukin-6 ( Eickelberg et al, 1999 ), and participating in lung carcinogenesis ( Ruiz et al, 2021 ). Foxj1 affects the differentiation and functional performance of ciliated cells distributed throughout the airway tree, including the trachea, lobar bronchi, and terminal bronchiole ( You et al, 2004 ; Koay et al, 2021 ). Hcfc1 drives cell-cycle gene expression as a transcriptional regulator, depending on heat-shock proteins 90 ( Antonova et al, 2019 ).…”