Abstract
The Gal4/upstream activating sequence (UAS) system is a powerful genetic tool for the temporal and spatial expression of target genes. In this study, the dynamic activity of the Gal4/UAS system was monitored in zebrafish throughout the entire lifespan and during germline transmission, using an optimized Gal4/UAS, KalTA4/4xUAS, which is driven by two muscle-specific regulatory sequences. We found that UAS-linked gene expression was transcriptionally amplified by Gal4/UAS during early developmental stages and that the amplification effects tended to weaken during later stages and even disappear in subsequent generations. In the F2 generation, the transcription of a UAS-linked enhanced green fluorescent protein (EGFP) reporter was transcriptionally silent from 16 days post-fertilization (dpf) into adulthood, yet offspring of this generation showed reactivation of the EGFP reporter in some strains. We further show that the transcriptional silencing and reactivation of UAS-driven EGFP correlated with the DNA methylation levels of the UAS regulatory sequences. Notably, asymmetric DNA methylation of the 4xUAS occurred in oocytes and sperm. Moreover, the paternal and maternal 4xUAS sequences underwent different DNA methylation dynamics after fertilization. Our study suggests that the Gal4/UAS system may represent a powerful tool for tracing the DNA methylation dynamics of paternal and maternal loci during zebrafish development and that UAS-specific DNA methylation should be seriously considered when the Gal4/UAS system is applied in zebrafish.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Akitake CM, Macurak M, Halpern ME, Goll MG (2011) Transgenerational analysis of transcriptional silencing in zebrafish. Developmental Biology In Press, Corrected Proof
Andersen IS, Reiner AH, Aanes H, Alestrom P, Collas P (2012) Developmental features of DNA methylation during activation of the embryonic zebrafish genome. Genome Biol 13(7):R65. doi:10.1186/gb-2012-13-7-r65
Baier H, Scott EK (2009) Genetic and optical targeting of neural circuits and behavior—zebrafish in the spotlight. Curr Opin Neurobiol 19(5):553–560. doi:10.1016/j.conb.2009.08.001
Cedar H, Bergman Y (2012) Programming of DNA methylation patterns. Annu Rev Biochem 81:97–117. doi:10.1146/annurev-biochem-052610-091920
Distel M, Wullimann MF, Koster RW (2009) Optimized Gal4 genetics for permanent gene expression mapping in zebrafish. Proc Natl Acad Sci U S A 106(32):13365–13370. doi:10.1073/pnas.0903060106
Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34(1-2):1–15. doi:10.1002/gene.10150
Farthing CR, Ficz G, Ng RK, Chan CF, Andrews S, Dean W, Hemberger M, Reik W (2008) Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet 4(6):e1000116. doi:10.1371/journal.pgen.1000116
Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol 196(2):261–282. doi:10.1016/0022-2836(87)90689-9
Goll MG, Anderson R, Stainier DYR, Spradling AC, Halpern ME (2009) Transcriptional silencing and reactivation in transgenic zebrafish. Genetics 182(3):747–755. doi:10.1534/genetics.109.102079
Gong Z, Wan H, Tay TL, Wang H, Chen M, Yan T (2003) Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle. Biochem Biophys Res Commun 308(1):58–63
Halpern ME, Rhee J, Goll MG, Akitake CM, Parsons M, Leach SD (2008) Gal4/UAS transgenic tools and their application to zebrafish. Zebrafish 5(2):97–110. doi:10.1089/zeb.2008.0530
Hattori N, Imao Y, Nishino K, Hattori N, Ohgane J, Yagi S, Tanaka S, Shiota K (2007) Epigenetic regulation of Nanog gene in embryonic stem and trophoblast stem cells. Genes Cells Devot Mole Cell Mech 12(3):387–396. doi:10.1111/j.1365-2443.2007.01058.x
Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2(4):924–932. doi:10.1038/nprot.2007.132
Iyer M, Wu L, Carey M, Wang Y, Smallwood A, Gambhir SS (2001) Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters. Proc Natl Acad Sci U S A 98(25):14595–14600. doi:10.1073/pnas.251551098
Jiang L, Zhang J, Wang JJ, Wang L, Zhang L, Li G, Yang X, Ma X, Sun X, Cai J, Zhang J, Huang X, Yu M, Wang X, Liu F, Wu CI, He C, Zhang B, Ci W, Liu J (2013) Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos. Cell 153(4):773–784. doi:10.1016/j.cell.2013.04.041
Ju B, Chong SW, He J, Wang X, Xu Y, Wan H, Tong Y, Yan T, Korzh V, Gong Z (2003) Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter. Dev Dyn 227(1):14–26. doi:10.1002/dvdy.10273
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310. doi:10.1002/aja.1002030302
Lewandoski M (2001) Conditional control of gene expression in the mouse. Nat Rev Genet 2(10):743–755. doi:10.1038/35093537
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408. doi:10.1006/meth.2001.1262
Martin CC, McGowan R (1995) Parent-of-origin specific effects on the methylation of a transgene in the zebrafish, Danio rerio. Dev Genet 17(3):233–239. doi:10.1002/dvg.1020170308
Meissner A (2010) Epigenetic modifications in pluripotent and differentiated cells. Nat Biotechnol 28(10):1079–1088. doi:10.1038/nbt.1684
Mhanni AA, McGowan RA (2004) Global changes in genomic methylation levels during early development of the zebrafish embryo. Dev Genes Evol 214(8):412–417. doi:10.1007/s00427-004-0418-0
Pang S-C, Wang H-P, Li K-Y, Zhu Z-Y, Kang J, Sun Y-H (2014) Double transgenesis of humanized fat1 and fat2 genes promotes omega-3 polyunsaturated fatty acids synthesis in a zebrafish model. Mar Biotechnol 1–14. doi:10.1007/s10126-014-9577-9
Potok ME, Nix DA, Parnell TJ, Cairns BR (2013) Reprogramming the maternal zebrafish genome after fertilization to match the paternal methylation pattern. Cell 153(4):759–772. doi:10.1016/j.cell.2013.04.030
Schilling TF, Kimmel CB (1997) Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo. Development 124(15):2945–2960
Seisenberger S, Peat JR, Reik W (2013) Conceptual links between DNA methylation reprogramming in the early embryo and primordial germ cells. Curr Opin Cell Biol 25(3):281–288. doi:10.1016/j.ceb.2013.02.013
Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9(6):465–476. doi:10.1038/nrg2341
Thisse B, Heyer V, Lux A, Alunni V, Degrave A, Seiliez I, Kirchner J, Parkhill JP, Thisse C (2004) Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell Biol 77:505–519
Wei CY, Wang HP, Zhu ZY, Sun YH (2014) Transcriptional factors smad1 and smad9 act redundantly to mediate zebrafish ventral specification downstream of smad5. J Biol Chem 289(10):6604–6618. doi:10.1074/jbc.M114.549758
Xiong F, Wei ZQ, Zhu ZY, Sun YH (2013) Targeted expression in zebrafish primordial germ cells by Cre/loxP and Gal4/UAS systems. Mar Biotechnol (NY) 15(5):526–539. doi:10.1007/s10126-013-9505-4
Acknowledgments
We thank Dr. Yin Zhan for providing the mylpfa regulatory region fragment and Dr. Reinhard W. Kӧster for providing the 4XKGFP and the 4xKaloop constructs. We thank Ming Li and Kuoyu Li (CZRC) for fish husbandry. This work was supported by the China 863 High-Tech Program Grant 2011AA100404, the National Science Fund for Excellent Young Scholars of NSFC Grant 31222052, the China 973 Basic Research Program Grant 2012CB944504, and FEBL Grant 2011FBZ23 to Y. H. S.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplemental Fig. 1
(DOC 501 kb)
Supplemental Fig. 2
(DOC 680 kb)
Supplemental Fig. 3
(DOC 2243 kb)
Supplemental Fig. 4
(DOC 344 kb)
Supplemental Fig. 5
(DOC 606 kb)
Supplemental Fig. 6
(DOC 448 kb)
Supplemental Table S1
(DOC 31 kb)
Rights and permissions
About this article
Cite this article
Pang, SC., Wang, HP., Zhu, ZY. et al. Transcriptional Activity and DNA Methylation Dynamics of the Gal4/UAS System in Zebrafish. Mar Biotechnol 17, 593–603 (2015). https://doi.org/10.1007/s10126-015-9641-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10126-015-9641-0