Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100291
ISSN1083-351X
AutoresTeng Wei Koay, Carina Osterhof, Ilaria M.C. Orlando, Anna Keppner, Daniel Andre, Schayan Yousefian, María Suárez Alonso, Miguel Correia, Robert Markworth, Johannes Schödel, Thomas Hankeln, David Hoogewijs,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoAndroglobin (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 (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. Globins are small globular metallo-proteins consisting of about 150 amino acids, which comprise eight α-helical segments in a characteristic 3-over-3 α-helical sandwich structure. This conserved "globin fold" identifies them as members of a large protein superfamily. Globins contain a heme prosthetic group, by which they can reversibly bind gaseous ligands such as O2, CO, and NO. Historically, the familiar vertebrate O2-binding hemoglobin (HB), a tetramer of α- and β-globins, and the monomeric myoglobin (MB) were among the first proteins whose sequences and structures were determined already over 50 years ago. Genomic analyses have considerably altered and extended our view of the globin family in mammals, leading to the discovery of novel globin types such as neuroglobin (NGB) and cytoglobin (CYGB), which are expressed in nerve and fibroblast-like cells, respectively (1Burmester T. Weich B. Reinhardt S. Hankeln T. A vertebrate globin expressed in the brain.Nature. 2000; 407: 520-523Crossref PubMed Scopus (877) Google Scholar, 2Burmester T. Ebner B. Weich B. Hankeln T. Cytoglobin: A novel globin type ubiquitously expressed in vertebrate tissues.Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (428) Google Scholar). Both globin types perform yet-to-be-illuminated functions, which possibly reside in antioxidant defense, reactive oxygen species signaling, or even lipid metabolism (3Burmester T. Hankeln T. Function and evolution of vertebrate globins.Acta Physiol. (Oxf.). 2014; 211: 501-514Crossref PubMed Scopus (131) Google Scholar, 4Keppner A. Maric D. Correia M. Koay T.W. Orlando I.M.C. Vinogradov S.N. Hoogewijs D. Lessons from the post-genomic era: Globin diversity beyond oxygen binding and transport.Redox Biol. 2020; 37: 101687Crossref PubMed Scopus (14) Google Scholar). Recently, a novel family of large, chimeric proteins containing a globin-like domain was discovered and termed androglobin (ADGB) based on its preferential expression in mammalian testis tissue (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar). ADGB is a chimeric protein of about 1500 amino acids, which contains an embedded globin domain. This globin domain is permutated with respect to its characteristic alpha helices and interrupted by a calmodulin-binding motif. Nevertheless, the globin domain appears to be able to bind oxygen in vitro (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar). The N-terminal domain of ADGB shows high sequence similarity to the human protease calpain 7, although functionally important amino acid residues are mutated. ADGB was shown to be highly conserved throughout the metazoan tree of life, and orthologous copies of the ADGB gene could be found from humans and other vertebrates down to very basal taxa such as the cnidarian Nematostella vectensis, the placozoan Trichoplax adherens, and even the choanoflagellate Monosiga brevicollis (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar), which suggests an elementary and possibly conserved function in metazoans. ADGB is predominantly expressed in later stages of spermatogenesis in mammalian testes and, to a much lower extent, in the lung and brain tissue (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar). An important role of ADGB in spermatogenesis was supported by analysis of published microarray data revealing that endogenous levels of human ADGB mRNA were lower in the testes of infertile men versus their healthy counterparts. An in vitro cell culture study suggested that ADGB could act as an oncogene in glioma formation and an ADGB knockdown could inhibit the growth of glioma cell lines (6Huang B. Lu Y.S. Li X. Zhu Z.C. Li K. Liu J.W. Zheng J. Hu Z.L. Androglobin knockdown inhibits growth of glioma cell lines.Int. J. Clin. Exp. Pathol. 2014; 7: 2179-2184PubMed Google Scholar). Overall, studies on ADGB expression patterns and gene regulation were scarce, and the functional role of ADGB has remained elusive. Since the expressional profile of a gene, specifically addressing the organs and their cell types, can provide a valuable hint at its possible function (as illustrated e.g., by the specific presence of HB in erythrocytes), we revisited the expression patterns of ADGB using an integrative approach of bioinformatical data mining. In particular, novel RNA-Seq datasets from bulk and single-cell experiments were analyzed with the aim to recognize common patterns with functional implications. The data yielded valuable insight into the properties of ADGB-expressing cell types, which led us to characterize in detail the gene-regulatory landscape determining ADGB expression. We comprehensively mined epigenomic databases for accessible chromatin and promoter/enhancer-associated histone marks, identified transcription factors binding to the ADGB locus using reporter gene assays and chromatin immunoprecipitation (ChIP) experiments, and further characterized several functional distal enhancers in the ADGB locus. Finally, we performed ADGB overexpression in vitro to elicit a cellular phenotype. These different lines of experimental evidence converged and convincingly pointed out that the cellular function of ADGB is associated with the presence of motile cilia. The wealth of gene expression data, which have been produced since the initial description of ADGB in 2012, enabled us to define a much more detailed expression profile of the gene in mammalian tissues and cell types. As such, the bulk RNA-Seq data of the Human Protein Atlas (7Uhlen M. Fagerberg L. Hallstrom B.M. Lindskog C. Oksvold P. Mardinoglu A. Sivertsson A. Kampf C. Sjostedt E. Asplund A. Olsson I. Edlund K. Lundberg E. Navani S. Szigyarto C.A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (6478) Google Scholar) revealed the fallopian tube of the female reproductive tract as a novel expression site of ADGB mRNA (Fig. 1A). Transcript levels were even higher than in the lung, which was initially described as the second highest ADGB-expressing human organ (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar). To study this further, and noticing a shortage of data from healthy human samples, we evaluated bulk RNA-Seq data from the female reproductive tract of cattle. The bovine data sets confirmed Adgb expression in the oviducts, showing the highest amount of Adgb expression of all cattle organs analyzed, and in endometrial tissue (Fig. 1B). Human endometrial data appeared largely devoid of ADGB RNA, but sequencing data of separate stromal and epithelial fractions (8Altmae S. Koel M. Vosa U. Adler P. Suhorutsenko M. Laisk-Podar T. Kukushkina V. Saare M. Velthut-Meikas A. Krjutskov K. Aghajanova L. Lalitkumar P.G. Gemzell-Danielsson K. Giudice L. Simon C. et al.Meta-signature of human endometrial receptivity: A meta-analysis and validation study of transcriptomic biomarkers.Sci. Rep. 2017; 7: 10077Crossref PubMed Scopus (100) Google Scholar) revealed restriction of ADGB expression to the epithelial fraction only (Fig. S1). Both human and cattle RNA-Seq data revealed a high interindividual variability in expression intensity, which suggested a temporal and/or spatial restriction of Adgb expression in the female reproductive tract. To study the gene expression footprint of the hypothetical Adgb-expressing cell type involved, we subdivided the bovine endometrial samples into 2 groups, "Adgb-high" (TPM >20, n = 4) and "Adgb-low" (TPM <5, n = 4), and performed differential gene expression analysis to infer genes associated with either high or low levels of Adgb. Subsequent overrepresentation analysis (Table 1, Supplemental File 1) revealed that genes associated with high amounts of Adgb were connected to GO-terms such as "cilium and axoneme assembly", "dynein-dependent microtubular transport", "microtubular movement" and, interestingly, the "sperm flagellum." An independent clustering approach to identify genes with an Adgb-type expression pattern using an additional data set of human fallopian tube samples (9Schuster H. Peper J.K. Bosmuller H.C. Rohle K. Backert L. Bilich T. Ney B. Loffler M.W. Kowalewski D.J. Trautwein N. Rabsteyn A. Engler T. Braun S. Haen S.P. Walz J.S. et al.The immunopeptidomic landscape of ovarian carcinomas.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E9942-E9951Crossref PubMed Scopus (84) Google Scholar) generated a smaller subset of genes, which were even more strongly associated with cilia-related processes such as "cilium movement", "determination of left-right-symmetry" and the "differentiation of lung epithelial cells" (Table 2, Supplemental File 2). Among these approximately 100 Adgb-associated genes, we found Foxj1, the master transcription factor of ciliogenesis (10Yu X. Ng C.P. Habacher H. Roy S. Foxj1 transcription factors are master regulators of the motile ciliogenic program.Nat. Genet. 2008; 40: 1445-1453Crossref PubMed Scopus (274) Google Scholar), and Dnah5, a protein known for its specific localization to motile cilia of the respiratory tract (11Whitfield M. Thomas L. Bequignon E. Schmitt A. Stouvenel L. Montantin G. Tissier S. Duquesnoy P. Copin B. Chantot S. Dastot F. Faucon C. Barbotin A.L. Loyens A. Siffroi J.P. et al.Mutations in DNAH17, encoding a sperm-specific axonemal outer dynein arm heavy chain, cause isolated male infertility due to asthenozoospermia.Am. J. Hum. Genet. 2019; 105: 198-212Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The tissue with the highest amount of Adgb expression, however, was inconsistent between the samples of the two species (Fig. 1, A and B). A possible explanation could be that the samples were at different stages of the menstrual cycle, given that ciliogenesis is estrogen-dependent (12Comer M.T. Leese H.J. Southgate J. Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium.Hum. Reprod. 1998; 13: 3114-3120Crossref PubMed Scopus (68) Google Scholar, 13Haider S. Gamperl M. Burkard T.R. Kunihs V. Kaindl U. Junttila S. Fiala C. Schmidt K. Mendjan S. Knofler M. Latos P.A. Estrogen signaling drives ciliogenesis in human endometrial organoids.Endocrinology. 2019; 160: 2282-2297Crossref PubMed Scopus (31) Google Scholar). Additionally, depending on the part of the oviduct that was dissected, the ratio of epithelial cells to connective tissue and thus the overall number of ciliated cells may vary between samples (14Barton B.E. Herrera G.G. Anamthathmakula P. Rock J.K. Willie A. Harris E.A. Takemaru K.I. Winuthayanon W. Roles of steroid hormones in oviductal function.Reproduction. 2020; 159: R125-R137Crossref PubMed Scopus (23) Google Scholar). Reproductive aging (i.e., menopause), which decreases the number of ciliated cells (15Ferenczy A. Richart R.M. Agate Jr., F.J. Purkerson M.L. Dempsey E.W. Scanning electron microscopy of the human fallopian tube.Science. 1972; 175: 783-784Crossref PubMed Scopus (34) Google Scholar), may also have contributed to the observed Adgb expression differences.Table 1GO term enrichment analysis of genes coregulated with Adgb in differential gene expression analysis of bovine endometrial samplesGene setDescriptionEnrichment ratioFDRBiological process GO:0000226Microtubule cytoskeleton organization8.370 GO:0060271Cilium assembly17.660 GO:0007018Microtubule-based movement16.960 GO:0035082Axoneme assembly52.000 GO:0003341Cilium movement45.500 GO:0070286Axonemal dynein complex assembly58.500 GO:0007368Determination of left/right symmetry19.312.56E-09 GO:0030317Flagellated sperm motility15.366.96E-05 GO:0001539Cilium or flagellum-dependent cell motility29.251.11E-04 GO:0042073Intraciliary transport24.382.97E-04 GO:0044458Motile cilium assembly27.000.0022Cellular component GO:0015630Microtubule cytoskeleton6.160 GO:0005929Cilium14.600 GO:0031514Motile cilium27.160 GO:0005930Axoneme43.150 GO:0036126Sperm flagellum26.810 GO:00977299+2 Motile cilium25.746.18E-15 GO:0097223Sperm part17.103.49E-14 GO:0005815Microtubule organizing center6.195.86E-12 GO:0044447Axoneme part56.578.32E-12 GO:0005858Axonemal dynein complex68.951.69E-09 GO:0005875Microtubule-associated complex14.457.82E-09Molecular function GO:0003774Motor activity15.805.35E-06 GO:0003777Microtubule motor activity21.231.81E-05 GO:0015631Tubulin binding7.114.41E-05 GO:1990939ATP-dependent microtubule motor activity30.790.0021 GO:0008017Microtubule binding6.730.0061 GO:0008092Cytoskeletal protein binding3.230.0087 GO:0045504Dynein heavy chain binding37.530.0087 GO:0045503Dynein light chain binding37.530.0087All GO terms show a strong connection to the motile cilium. The associated gene list and the full list of enriched terms are provided in Supplemental File 1. Open table in a new tab Table 2Enriched GO terms in genes showing correlation with ADGB expression in human fallopian tube samplesGene setDescriptionEnrichment ratioFDRBiological process GO:0007017Microtubule-based process6.021.28E-04 GO:0035082Axoneme assembly28.761.96E-04 GO:0003341Cilium movement28.301.96E-04 GO:0007018Microtubule-based movement9.715.76E-04 GO:0001578Microtubule bundle formation19.718.64E-04 GO:0060271Cilium assembly6.700.0088 GO:0000226Microtubule cytoskeleton organization5.410.0146 GO:0007368Determination of left/right symmetry12.710.0439 GO:0060487Lung epithelial cell differentiation41.770.0439 GO:0009855Determination of bilateral symmetry11.890.0452Cellular component GO:0005929Cilium12.870 GO:0005930Axoneme30.263.82E-10 GO:0097014Ciliary plasm303.82E-10 GO:0031514Motile cilium19.382.28E-08 GO:0005858Axonemal dynein complex76.672.73E-05 GO:0005875Microtubule-associated complex16.322.86E-05 GO:0005874Microtubule6.010.0109 GO:00977299+2 Motile cilium14.680.0109 GO:0036157Outer dynein arm76.670.0191 GO:0070160Tight junction10.950.0284Molecular function GO:1990939ATP-dependent microtubule motor activity45.476.49E-06 GO:0045503Dynein light chain binding66.689.42E-06 GO:0003777Microtubule motor activity24.101.06E-04 GO:0003774Motor activity14.820.0014 GO:0051959Dynein light intermediate chain binding34.490.0228 GO:0045505Dynein intermediate chain binding33.340.0228All GO terms reveal a strong connection to the motile cilium. The associated gene list and the full list of enriched terms are provided in Supplemental File 2. Open table in a new tab All GO terms show a strong connection to the motile cilium. The associated gene list and the full list of enriched terms are provided in Supplemental File 1. All GO terms reveal a strong connection to the motile cilium. The associated gene list and the full list of enriched terms are provided in Supplemental File 2. Experimentally, we confirmed the fallopian tube and the endometrium as novel expression sites via RT-qPCR analysis in cattle. In addition, we also determined the amount of Dnah5 and Foxj1 mRNA in these samples (Fig. 1C). Foxj1 transcript levels showed a positive correlation with Adgb expression in the endometria and oviducts (R2 = 0.73 and R2 = 0.72, respectively). The association between Dnah5 and Adgb in the endometrium was even stronger (R2 = 0.93). Though not as prominent, there was also a significant positive correlation between expression of Adgb and Dnah5 in the oviducts (R2 = 0.74). Immunohistochemistry analysis further confirmed the localization of Adgb protein in the epithelia in the bovine endometrium and specifically in multiciliated cells in the oviduct (Fig. S2). We previously reported the lung to show the second highest Adgb mRNA expression, after the testes (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar). Bulk RNA-Seq data from this tissue, however, led to inconclusive results with high interindividual variability and overall low levels of expression, or, as in some human samples, no expression at all (Fig. 1). Therefore, we considered analyzing available single-cell RNA-Seq data obtained from the murine lung (16Montoro D.T. Haber A.L. Biton M. Vinarsky V. Lin B. Birket S.E. Yuan F. Chen S. Leung H.M. Villoria J. Rogel N. Burgin G. Tsankov A.M. Waghray A. Slyper M. et al.A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.Nature. 2018; 560: 319-324Crossref PubMed Scopus (476) Google Scholar). To prove that this method was sensitive enough to detect Adgb mRNA expression, we first reanalyzed single-cell RNA-Seq data from murine testis (17Lukassen S. Bosch E. Ekici A.B. Winterpacht A. Characterization of germ cell differentiation in the male mouse through single-cell RNA sequencing.Sci. Rep. 2018; 8: 6521Crossref PubMed Scopus (42) Google Scholar). We could show that, in accordance with Hoogewijs et al. (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar), Adgb mRNA expression was restricted to later stages of spermatogenesis, where round spermatids differentiate into elongating spermatids and form the flagellum, a motile microtubular structure very similar to a motile cilium. Fully differentiated condensed spermatids, however, did not express Adgb mRNA anymore (Fig. S3). Following this proof of principle, we performed clustering analysis on single-cell RNA-Seq data sets from epithelial fractions of murine lungs (dataset from Montoro et al. (16Montoro D.T. Haber A.L. Biton M. Vinarsky V. Lin B. Birket S.E. Yuan F. Chen S. Leung H.M. Villoria J. Rogel N. Burgin G. Tsankov A.M. Waghray A. Slyper M. et al.A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.Nature. 2018; 560: 319-324Crossref PubMed Scopus (476) Google Scholar)). This revealed a distinct entity of lung cells expressing Adgb. Using known cell-type markers from literature, and in accordance with our original report, we identified these cells as being multiciliated (Fig. 2). As we had observed in the correlation analysis on fallopian tube samples, Adgb expression correlated well with Dnah5 and Foxj1, although the overall number of Adgb-positive cells was lower. An additional round of clustering of these ciliated cells revealed no subtypes with noticeable differences in Adgb expression, so that we assume that Adgb-negative ciliated cells are due to dropout artifacts because of rather low endogenous levels of Adgb mRNA (Fig. S4). No Adgb expression was observed in progenitors of multiciliated cells, such as basal cells (Fig. 2). Cell subcluster 4 (Fig. S4) showed slightly lower levels of both, Adgb and Foxj1, but a higher amount of expression of the basal cell marker Aqp3 (16Montoro D.T. Haber A.L. Biton M. Vinarsky V. Lin B. Birket S.E. Yuan F. Chen S. Leung H.M. Villoria J. Rogel N. Burgin G. Tsankov A.M. Waghray A. Slyper M. et al.A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.Nature. 2018; 560: 319-324Crossref PubMed Scopus (476) Google Scholar). This could indicate that Adgb expression rises during differentiation and is rather associated with later stages of ciliogenesis or with a maintenance function in cells with already established cilia. Cells with multiple motile cilia are not only found in the airways and the reproductive tract, but also in the ventricles of the brain, where they maintain proper circulation of cerebrospinal fluid (reviewed in (18Spassky N. Meunier A. The development and functions of multiciliated epithelia.Nat. Rev. Mol. Cell Biol. 2017; 18: 423-436Crossref PubMed Scopus (168) Google Scholar)). To obtain further evidence for a functional association of Adgb and motile cilia, and looking to explain the previously reported low expression in brain tissue (5Hoogewijs D. Ebner B. Germani F. Hoffmann F.G. Fabrizius A. Moens L. Burmester T. Dewilde S. Storz J.F. Vinogradov S.N. Hankeln T. Androglobin: A chimeric globin in metazoans that is preferentially expressed in mammalian testes.Mol. Biol. Evol. 2012; 29: 1105-1114Crossref PubMed Scopus (79) Google Scholar), we reanalyzed single-cell RNA-Seq data from mouse brains enriched for ependymal cells and their neuronal progenitors (19Campbell J.N. Macosko E.Z. Fenselau H. Pers T.H. Lyubetskaya A. Tenen D. Goldman M. Verstegen A.M. Resch J.M. McCarroll S.A. Rosen E.D. Lowell B.B. Tsai L.T. A molecular census of arcuate hypothalamus and median eminence cell types.Nat. Neurosci. 2017; 20: 484-496Crossref PubMed Scopus (375) Google Scholar). As expected, we could specifically detect Adgb mRNA expression in fully mature ependymal cells, although only in a small proportion of cells (Fig. 3). In addition, a subpopulation of tanycytes (designated as "2") showed a moderate amount of Adgb positive cells. GO term analysis of genes overrepresented in ependymocytes and tanycytes "2" again showed a high amount of cilia-associated genes (Fig. 3D). Further analysis revealed that Adgb-positive tanycytes belong to the α-subtype, whereas β-tanycytes were Adgb-negative. Although not multiciliated such as lung epithelial cells or ependymocytes, α-tanycytes can be biciliated with the motile 9 + 2 microtubule conformation, whereas β-tanycytes only form 9 + 0 immotile cilia, if any (20Mirzadeh Z. Kusne Y. Duran-Moreno M. Cabrales E. Gil-Perotin S. Ortiz C. Chen B. Garcia-Verdugo J.M. Sanai N. Alvarez-Buylla A. Bi- and uniciliated ependymal cells define continuous floor-plate-derived tanycytic territories.Nat. Commun. 2017; 8: 13759Crossref PubMed Scopus (49) Google Scholar). Altogether, these data point at an association of Adgb with cilia formation and/or function and a possible regulation by Foxj1. Gene expression is determined to a great extent by epigenetics and regulatory elements at promoters. As information on this for ADGB is scarce, we first inspected data derived from the ENCODE consortium. ENCODE data illustrate that the upstream region surrounding the ADGB first exon displays strong DNase hypersensitivity, enrichment of the promoter histone mark H3K4me3, and substantial transcription factor occupancy, all indicating chromatin accessibility and suggesting putative promoter activity (Fig. S5). Furthermore, chromatin segmentation states coupled to HMM motifs suggest promoter activity of this region in six different cell lines. Additional analysis of epigenetic modifications typical of active chromatin regions showed that H3K4me3 was also enriched at this region in multiple additional cell lines. (Fig. S5). This epigenetic profile reflecting open chromatin is in striking contrast to the rather limited, cell-type-specific expression of ADGB (see Discussion). To experimentally explore the basal activity of the putative human ADGB promoter, several potential promoter fragments (431 bp, 1031 bp, and 1981 bp long and starting at −33 bp upstream of the transcriptional start site—TSS) were cloned in a pGL3-luciferase basic vector (Fig. 4A). Reporter gene assays were performed in three cell lines able to form cilia (21Gerdes J.M. Liu Y. Zaghloul N.A. Leitch C.C. Lawson S.S. Kato M. Beachy P.A. Beales P.L. DeMartino G.N. Fisher S. Badano J.L. Katsanis N. Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response.Nat. Genet. 2007; 39: 1350-1360Crossref PubMed Scopus (320) Google Scholar, 22Kowal T.J. Falk M.M. Primary cilia found on HeLa and other cancer cells.Cell Biol. Int. 2015; 39: 1341-1347Crossref PubMed Scopus (19) Google Scholar, 23Tang Z. Lin M.G. Stowe T.R. Chen S. Zhu M. Stearns T. Franco B. Zhong Q. Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites.Nature. 2013; 502: 254-257Crossref PubMed Scopus (264) Google Scholar) and displaying reasonable mRNA expression levels of FOXJ1 and RFX2. Following transfection in HeLa and MCF-7 cells, moderate but consistent basal promoter activity could be observed (Fig. 4A). No substantial changes were seen in HEK293 cells. Based on screening of ENCODE-integrated ChIP-sequencing data for candidate promoter regulating factors, these vectors were cotransfected in HeLa cells and consistently increased ADGB promoter-dependent luciferase activity (Fig. S6A). Additional cotransfection experiments with increasing amounts of GATA-3 encoding plasmids indicated GATA-3-dependent regulation of the ADGB promoter in a dose-dependent way (Fig. S6B). Next, we employed CRISPR activation (CRISPRa) technology to activate transcription at the ADGB promoter. CRISPRa is based on a fusion of catalytically inactive Cas9 (dCas9) with the activation domains of three potent transcription factors, VP64, p65, and Rta (dCas9-VPR), which is targeted to a specific genom
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