Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function.
References
[1]
Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev Genet. 2014;15: 272–286. doi: 10.1038/nrg3682. pmid:24614317
[2]
Maston GA, Landt SG, Snyder M, Green MR. Characterization of enhancer function from genome-wide analyses. Annu Rev Genomics Hum Genet. 2012; 13:29–57. doi: 10.1146/annurev-genom-090711-163723. pmid:22703170
[3]
Bhatia S, Kleinjan DA. Disruption of long-range gene regulation in human genetic disease: a kaleidoscope of general principles, diverse mechanisms and unique phenotypic consequences. Hum Genet. 2014;133: 815–845. doi: 10.1007/s00439-014-1424-6. pmid:24496500
[4]
Spitz F, Furlong EEM. Transcription factors: from enhancer binding to developmental control. Nat Rev Genet. 2012;13: 613–626. doi: 10.1038/nrg3207. pmid:22868264
[5]
Phillips JB, Westerfield M. Zebrafish models in translational research: tipping the scales toward advancements in human health. Dis Model Mech. 2014;7: 739–743. doi: 10.1242/dmm.015545. pmid:24973743
[6]
Rinkwitz S1, Mourrain P, Becker TS. Zebrafish: an integrative system for neurogenomics and neurosciences. Progress in Neurobiology. 2011;93(2): 231–43. doi: 10.1016/j.pneurobio.2010.11.003. pmid:21130139
[7]
Gordon CT, Attanasio C, Bhatia S, Benko S, Ansari M, et al. Identification of Novel Craniofacial Regulatory Domains Located far Upstream of SOX9 and Disrupted in Pierre Robin Sequence. Hum Mutat. 2014;35: 1011–1020. doi: 10.1002/humu.22606. pmid:24934569
[8]
Kawakami K. Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Methods Cell Biol. 2004;77: 201–222. pmid:15602913 doi: 10.1016/s0091-679x(04)77011-9
[9]
Jeong Y, Leskow FC, El-Jaick K, Roessler E, Muenke M, et al. Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein. Nat Genet. 2008; 40: 1348–1353. doi: 10.1038/ng.230. pmid:18836447
[10]
Geng X, Speirs C, Lagutin O, Inbal A, Liu W, et al. Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly. Dev Cell. 2009;15(2): 236–47. doi: 10.1016/j.devcel.2008.07.003
[11]
Ando H, Kobayashi M, Tsubokawa T, Uyemura K, Furuta T, et al. Lhx2 mediates the activity of Six3 in zebrafish forebrain growth. Dev Biol.2005; 287: 456–468. pmid:16226737 doi: 10.1016/j.ydbio.2005.09.023
[12]
Anderson E, Peluso S, Lettice LA, Hill RE. Human limb abnormalities caused by disruption of hedgehog signaling. Trends Genet. 2012;28: 364–373. doi: 10.1016/j.tig.2012.03.012. pmid:22534646
[13]
Lettice LA, Hill AE, Devenney PS, Hill RE. Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly. Hum Mol Genet. 2008;17: 978–985. pmid:18156157 doi: 10.1093/hmg/ddm370
[14]
Bhatia S, Bengani H, Fish M, Brown A, Divizia MT, et al. Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia. Am J Hum Genet. 2013;93: 1126–1134. doi: 10.1016/j.ajhg.2013.10.028. pmid:24290376
[15]
Rahimov F, Marazita ML, Visel A, Cooper ME, Hitchler MJ, et al. Disruption of an AP-2alpha binding site in an IRF6 enhancer is associated with cleft lip. Nat Genet. 2008;40: 1341–1347. doi: 10.1038/ng.242. pmid:18836445
[16]
Dougherty M, Kamel G, Grimaldi M, Gfrerer L, Shubinets V, et al. Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis. Development. 2013;140: 76–81. doi: 10.1242/dev.080473. pmid:23154410
[17]
Gordon CT, Tan TY, Benko S, Fitzpatrick D, Lyonnet S, et al. Long-range regulation at the SOX9 locus in development and disease. J Med Genet. 2009;46: 649–656. doi: 10.1136/jmg.2009.068361. pmid:19473998
[18]
Benko S, Fantes JA, Amiel J, Kleinjan DJ, Thomas S, et al. Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence. Nat Genet. 2009;41: 359–364. doi: 10.1038/ng.329. pmid:19234473
[19]
Attanasio C, Nord AS, Zhu Y, Blow MJ, Li Z, et al. Fine tuning of craniofacial morphology by distant-acting enhancers. Science. 2013;342: 1241006. doi: 10.1126/science.1241006. pmid:24159046
[20]
Weedon MN, Cebola I, Patch AM, Flanagan SE, De Franco E, et al. Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis. Nat Genet. 2014;46: 61–64. doi: 10.1038/ng.2826. pmid:24212882
[21]
Patwardhan RP, Hiatt JB, Witten DM, Kim MJ, Smith RP, et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol. 2012;30: 265–270. doi: 10.1038/nbt.2136. pmid:22371081
[22]
Kwasnieski JC, Mogno I, Myers CA, Corbo JC, Cohen BA. Complex effects of nucleotide variants in a mammalian cis-regulatory element. Proc Natl Acad Sci U S A. 2012;109(47):19498–503. doi: 10.1073/pnas.1210678109. pmid:23129659
[23]
White MA, Myers CA, Corbo JC, Cohen BA. Massively parallel in vivo enhancer assay reveals that highly local features determine the cis-regulatory function of ChIP-seq peaks. Proc Natl Acad Sci U S A. 2013;110(29):11952–7. doi: 10.1073/pnas.1307449110. pmid:23818646
[24]
Roberts JA, Miguel-Escalada I, Slovik KJ, Walsh KT, Hadzhiev Y, et al. Targeted transgene integration overcomes variability of position effects in zebrafish. Development. 2014;141: 715–724. doi: 10.1242/dev.100347. pmid:24449846
[25]
Smith MCA, Till R, Brady K, Soultanas P, Thorpe H, et al. Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination. Nucleic Acids Res. 2004;32: 2607–2617. pmid:15141031 doi: 10.1093/nar/gkh538
[26]
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013;31: 227–229. doi: 10.1038/nbt.2501. pmid:23360964
[27]
Jao LE, Wente SR, Chen W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 2013; 110: 13904–13909. doi: 10.1073/pnas.1308335110. pmid:23918387
[28]
Kirchmaier S, H？ckendorf B, M？ller EK, Bornhorst D, Spitz F, et al. Efficient site-specific transgenesis and enhancer activity tests in medaka using PhiC31 integrase. Development. 2013;140: 4287–4295. doi: 10.1242/dev.096081. pmid:24048591
[29]
Kleinjan DA, Seawright A, Childs AJ, van Heyningen V. Conserved elements in Pax6 intron 7 involved in (auto)regulation and alternative transcription. Dev Biol. 2004;265: 462–477. pmid:14732405 doi: 10.1016/j.ydbio.2003.09.011
[30]
Kleinjan DA, van Heyningen V. Long-range control of gene expression: emerging mechanisms and disruption in disease. Am J Hum Genet. 2005; 76: 8–32. pmid:15549674 doi: 10.1086/426833
[31]
Kleinjan DJ, Coutinho P. Cis-ruption mechanisms: disruption of cis-regulatory control as a cause of human genetic disease. Brief Funct Genomic Proteomic. 2009;8: 317–332. doi: 10.1093/bfgp/elp022. pmid:19596743
[32]
Panne D. The enhanceosome. Curr Opin Struct Biol. 2008;18: 236–242. doi: 10.1016/j.sbi.2007.12.002. pmid:18206362
[33]
Schwarzer W, Spitz F. The architecture of gene expression: integrating dispersed cis-regulatory modules into coherent regulatory domains. Curr Opin Genet Dev. 2014;27C: 74–82. doi: 10.1016/j.gde.2014.03.014
[34]
Weirauch MT, Hughes TR. Conserved expression without conserved regulatory sequence: the more things change, the more they stay the same. Trends Genet. 2010;26: 66–74. doi: 10.1016/j.tig.2009.12.002. pmid:20083321
[35]
Lee AP, Kerk SY, Tan YY, Brenner S, Venkatesh B. Ancient vertebrate conserved noncoding elements have been evolving rapidly in teleost fishes. Mol Biol Evol. 2011;28: 1205–1215. doi: 10.1093/molbev/msq304. pmid:21081479
[36]
Taylor JS, Braasch I, Frickey T, Meyer A, Van de Peer Y. Genome duplication, a trait shared by 22000 species of ray-finned fish. Genome Res. 2003;13: 382–390. pmid:12618368 doi: 10.1101/gr.640303
[37]
Fisher S. et al. Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nature Protocols. 2006b;1(3):1297–1305. pmid:17406414 doi: 10.1038/nprot.2006.230
[38]
Frazer KA et al. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 2004;32 (Web Server issue):W273–9 doi: 10.1093/nar/gkh458
[39]
Ovcharenko I et al. ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Research 32(Web Server Issue). 2004: W280–W286. pmid:15215395 doi: 10.1093/nar/gkh355
[40]
Westerfield M. The Zebrafish Book: A guide for the laboratory use of zebrafish Danio rerio. University of Oregon Press, Eugene, OR. 2007
[41]
Kimmel CB et al. Stages of embryonic development of the zebrafish. Dev Dyn. 1995;203: 253–310. pmid:8589427 doi: 10.1002/aja.1002030302
[42]
Ishibashi M, Mechaly AS, Becker TS, Rinkwitz S. Using zebrafish transgenesis to test human genomic sequences for specific enhancer activity. Methods. 2013;62: 216–225. doi: 10.1016/j.ymeth.2013.03.018. pmid:23542551
[43]
Thisse C, Thisse B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc. 2008;3: 59–69. doi: 10.1038/nprot.2007.514. pmid:18193022
