Abstract
Androglobin (ADGB), the most recently identified member of the mammalian globin family, is a chimeric protein with an unusual, embedded globin domain that is circularly permutated and exhibits hallmarks of a hexacoordinated heme-binding scheme. Whereas abundant expression of ADGB was initially found to be mainly restricted to cells in the postmeiotic stages of spermatogenesis, more recent RNA-Seq-based expression analysis data revealed that ADGB is detectable in cells carrying motile cilia or flagella. This very tight regulation of ADGB gene expression urges the need for alternative techniques to study endogenous expression in classical mammalian cell models, which do not express ADGB. We describe here the use of CRISPR activation (CRISPRa) technology to induce endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells from its promoter and illustrate how this method can be employed to validate putative regulatory DNA elements of ADGB in promoter and enhancer regions.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Keppner A, Maric D, Correia M, Koay TW, Orlando IMC, Vinogradov SN, Hoogewijs D (2020) Lessons from the post-genomic era: globin diversity beyond oxygen binding and transport. Redox Biol 37:101687. https://doi.org/10.1016/j.redox.2020.101687
Hoogewijs D, Ebner B, Germani F, Hoffmann FG, Fabrizius A, Moens L, Burmester T, Dewilde S, Storz JF, Vinogradov SN, Hankeln T (2012) Androglobin: a chimeric globin in metazoans that is preferentially expressed in Mammalian testes. Mol Biol Evol 29(4):1105–1114. https://doi.org/10.1093/molbev/msr246
Keppner A, Correia M, Santambrogio S, Koay TW, Maric D, Osterhof C, Winter DV, Clerc A, Stumpe M, Chalmel F, Dewilde S, Odermatt A, Kressler D, Hankeln T, Wenger RH, Hoogewijs D (2022) Androglobin, a chimeric mammalian globin, is required for male fertility. eLife 11:e72374. https://doi.org/10.7554/eLife.72374
Koay TW, Osterhof C, Orlando IMC, Keppner A, Andre D, Yousefian S, Suarez Alonso M, Correia M, Markworth R, Schodel J, Hankeln T, Hoogewijs D (2021) Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis. J Biol Chem 296:100291. https://doi.org/10.1016/j.jbc.2021.100291
Bracke A, Hoogewijs D, Dewilde S (2018) Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda. Anal Biochem 543:62–70. https://doi.org/10.1016/j.ab.2017.11.027
Hardison RC (2012) Evolution of hemoglobin and its genes. Cold Spring Harb Perspect Med 2(12):a011627. https://doi.org/10.1101/cshperspect.a011627
Hay D, Hughes JR, Babbs C, Davies JOJ, Graham BJ, Hanssen L, Kassouf MT, Marieke Oudelaar AM, Sharpe JA, Suciu MC, Telenius J, Williams R, Rode C, Li PS, Pennacchio LA, Sloane-Stanley JA, Ayyub H, Butler S, Sauka-Spengler T, Gibbons RJ, Smith AJH, Wood WG, Higgs DR (2016) Genetic dissection of the alpha-globin super-enhancer in vivo. Nat Genet 48(8):895–903. https://doi.org/10.1038/ng.3605
Higgs DR, Engel JD, Stamatoyannopoulos G (2012) Thalassaemia. Lancet 379(9813):373–383. https://doi.org/10.1016/S0140-6736(11)60283-3
Antoniani C, Meneghini V, Lattanzi A, Felix T, Romano O, Magrin E, Weber L, Pavani G, El Hoss S, Kurita R, Nakamura Y, Cradick TJ, Lundberg AS, Porteus M, Amendola M, El Nemer W, Cavazzana M, Mavilio F, Miccio A (2018) Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human beta-globin locus. Blood 131(17):1960–1973. https://doi.org/10.1182/blood-2017-10-811505
Schörg A, Santambrogio S, Platt JL, Schödel J, Lindenmeyer MT, Cohen CD, Schrödter K, Mole DR, Wenger RH, Hoogewijs D (2015) Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping. Nucleic Acids Res 43(12):5810–5823. https://doi.org/10.1093/nar/gkv506
Storti F, Santambrogio S, Crowther LM, Otto T, Abreu-Rodriguez I, Kaufmann M, Hu CJ, Dame C, Fandrey J, Wenger RH, Hoogewijs D (2014) A novel distal upstream hypoxia response element regulating oxygen-dependent erythropoietin gene expression. Haematologica 99(4):e45–e48. https://doi.org/10.3324/haematol.2013.102707
Bicker A, Dietrich D, Gleixner E, Kristiansen G, Gorr TA, Hankeln T (2014) Extensive transcriptional complexity during hypoxia-regulated expression of the myoglobin gene in cancer. Hum Mol Genet 23(2):479–490. https://doi.org/10.1093/hmg/ddt438
Tam KT, Chan PK, Zhang W, Law PP, Tian Z, Fung Chan GC, Philipsen S, Festenstein R, Tan-Un KC (2017) Identification of a novel distal regulatory element of the human Neuroglobin gene by the chromosome conformation capture approach. Nucleic Acids Res 45(1):115–126. https://doi.org/10.1093/nar/gkw820
Randi EB, Vervaet B, Tsachaki M, Porto E, Vermeylen S, Lindenmeyer MT, Thuy LTT, Cohen CD, Devuyst O, Kistler AD, Szabo C, Kawada N, Hankeln T, Odermatt A, Dewilde S, Wenger RH, Hoogewijs D (2020) The Antioxidative role of Cytoglobin in podocytes: implications for a role in chronic kidney disease. Antioxid Redox Signal 32(16):1155–1171. https://doi.org/10.1089/ars.2019.7868
De Backer J, Lin A, Berghe WV, Bogaerts A, Hoogewijs D (2022) Cytoglobin inhibits non-thermal plasma-induced apoptosis in melanoma cells through regulation of the NRF2-mediated antioxidant response. Redox Biol 55:102399. https://doi.org/10.1016/j.redox.2022.102399
De Backer J, Maric D, Zuhra K, Bogaerts A, Szabo C, Vanden Berghe W, Hoogewijs D (2022) Cytoglobin silencing promotes melanoma malignancy but sensitizes for ferroptosis and pyroptosis therapy response. Antioxidants 11(8):1548. https://doi.org/10.3390/antiox11081548
Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, E PRI, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12(4):326–328. https://doi.org/10.1038/nmeth.3312
Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536):583–588. https://doi.org/10.1038/nature14136
De Backer J, Maric D, Bosman M, Dewilde S, Hoogewijs D (2021) A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma. Sci Rep 11(1):10879. https://doi.org/10.1038/s41598-021-90284-6
Hoogewijs D, Houthoofd K, Matthijssens F, Vandesompele J, Vanfleteren JR (2008) Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Mol Biol 9:9. https://doi.org/10.1186/1471-2199-9-9
Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 10(10):973–976. https://doi.org/10.1038/nmeth.2600
Acknowledgments
This work was supported by the Swiss National Science Foundation (grants 31003A_173000 and 310030_207460).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Koay, T.W., Schödel, J., Hoogewijs, D. (2023). CRISPR Activator Approaches to Study Endogenous Androglobin Gene Regulation. In: Weinert, E.E. (eds) Oxygen Sensing. Methods in Molecular Biology, vol 2648. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3080-8_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3080-8_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3079-2
Online ISBN: 978-1-0716-3080-8
eBook Packages: Springer Protocols