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Cited by 44Genomics and Chromatin DynamicsDevelopmental Biology and Gene RegulationCRISPR and Genetic Engineering

Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-enhancer redundancy

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Hörnblad, A., Bastide, S., Langenfeld, K., Langa, F., Spitz, F.·2021·Nature Communications
DOI 10.1038/s41467-020-20714-yPMC PMC7815712

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Abstract

Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.

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  • Facilitators, as described here, may be relatively common elements in multipartite enhancers.

    “Review of diverse, albeit incomplete, previous analyses of SEs16,17,19,20,22 suggest that facilitators, as described here, may be relatively common elements in multipartite enhancers.”

    From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [19]

    The Results section (‘The functional subunits of CE64 are interdependent’; page unavailable) describes two subunits that are both required for enhancer function but cannot direct the relevant expression alone. That supports CE64 as an example of interdependent elements in a multipartite enhancer. The paper does not establish that such facilitator-like elements are relatively common across multipartite enhancers. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • A number of groups have dissected super-enhancers, yielding various conclusions, from additive to super-additive, redundant, synergistic, and hierarchical cooperation.

    “A number of groups have dissected SEs, yielding various conclusions, from additive16 to super-additive,22 redundant,19 synergistic,21 and hierarchical20 cooperation.”

    From Super-enhancers include classical enhancers and facilitators to fully activate gene expression (2023) · cites this paper as [19]

  • Homologous enhancers in a pair of distantly related species can retain their function in the non-host organism despite differences in DNA sequence.

    “Consistent with this, homologous enhancers in a pair of distantly related species can retain their function in the non-host organism despite differences in DNA sequence [101, 110, 111].”

    From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [111]

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References 71 · Cited by 44

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  1. [1]Lettice LA Development of five digits is controlled by a bipartite long-range cis-regulator Development 2014 141 1715 1725 10.1242/dev.095430 24715461 PMC3978833 · DOI
  2. [2]Benko S Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence Nat. Genet. 2009 41 359 364 10.1038/ng.329 19234473 · DOI
  3. [3]Bagheri-Fam S Long-range upstream and downstream enhancers control distinct subsets of the complex spatiotemporal Sox9 expression pattern Dev. Biol. 2006 291 382 397 10.1016/j.ydbio.2005.11.013 16458883 · DOI
  4. [4]McBride DJ Buckle A van Heyningen V Kleinjan DA DNaseI hypersensitivity and ultraconservation reveal novel, interdependent long-range enhancers at the complex Pax6 cis-regulatory region PLoS ONE 2011 6 e28616 10.1371/journal.pone.0028616 22220192 PMC3248410 · DOI
  5. [5]Montavon T A regulatory archipelago controls Hox genes transcription in digits Cell 2011 147 1132 1145 10.1016/j.cell.2011.10.023 22118467 · DOI · Paper page
  6. [6]Marinić M Aktas T Ruf S Spitz F An Integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape Dev. Cell 2013 24 530 542 10.1016/j.devcel.2013.01.025 23453598 · DOI
  7. [7]Pennacchio LA In vivo enhancer analysis of human conserved non-coding sequences Nature 2006 444 499 502 10.1038/nature05295 17086198 · DOI · Paper page
  8. [8]Visel A ChIP-seq accurately predicts tissue-specific activity of enhancers Nature 2009 457 854 858 10.1038/nature07730 19212405 PMC2745234 · DOI · Paper page
  9. [9]Barolo S Shadow enhancers: frequently asked questions about distributed cis-regulatory information and enhancer redundancy BioEssays 2011 34 135 141 10.1002/bies.201100121 22083793 PMC3517143 · DOI · Paper page
  10. [10]Ahituv N Deletion of ultraconserved elements yields viable mice PLoS Biol. 2007 5 e234 10.1371/journal.pbio.0050234 17803355 PMC1964772 · DOI
  11. [12]Xiong N Kang C Raulet DH Redundant and unique roles of two enhancer elements in the TCRγ locus in gene regulation and γδ T cell development Immunity 2002 16 453 463 10.1016/S1074-7613(02)00285-6 11911829 · DOI
  12. [13]Wiersma EJ Ronai D Berru M Tsui FWL Shulman MJ Role of the intronic elements in the endogenous immunoglobulin heavy chain locus. Either the matrix attachment regions or the core enhancer is sufficient to maintain expression J. Biol. Chem. 1999 274 4858 4862 10.1074/jbc.274.8.4858 9988726 · DOI
  13. [14]Danielian PS Echelard Y Vassileva G McMahon AP A 5.5-kb enhancer is both necessary and sufficient for regulation of Wnt-1 transcription in vivo Dev. Biol. 1997 192 300 309 10.1006/dbio.1997.8762 9441669 · DOI
  14. [15]Fiering S Targeted deletion of 5’HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus Genes Dev. 1995 9 2203 2213 10.1101/gad.9.18.2203 7557375 · DOI
  15. [16]Yanagisawa H Clouthier DE Richardson JA Charite J Olson EN Targeted deletion of a branchial arch-specific enhancer reveals a role of dHAND in craniofacial development Development 2003 130 1069 1078 10.1242/dev.00337 12571099 · DOI
  16. [17]Meyers EN Lewandoski M Martin GR An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination Nat. Genet. 1998 18 136 141 10.1038/ng0298-136 9462741 · DOI
  17. [18]Frank DU An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome Development 2002 129 4591 4603 12223415 PMC1876665
  18. [19]Macatee TL Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development Development 2003 130 6361 6374 10.1242/dev.00850 14623825 PMC1876660 · DOI
  19. [20]Moon AM Moon AM Capecchi MR Capecchi MR Fgf8 is required for outgrowth and patterning of the limbs Nat. Genet. 2000 26 455 459 10.1038/82601 11101845 PMC2001274 · DOI
  20. [21]Lewandoski M Sun X Martin GR Fgf8 signalling from the AER is essential for normal limb development Nat. Genet. 2000 26 460 463 10.1038/82609 11101846 · DOI · Paper page
  21. [22]Perantoni AO Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development Development 2005 132 3859 3871 10.1242/dev.01945 16049111 · DOI
  22. [23]Crossley PH Martinez S Martin GR Midbrain development induced by FGF8 in the chick embryo Nature 1996 380 66 68 10.1038/380066a0 8598907 · DOI
  23. [24]Irving C Mason I Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression Development 2000 127 177 186 10654611
  24. [25]Martinez S Crossley PH Cobos I Rubenstein JL Martin GR FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression Development 1999 126 1189 1200 10021338
  25. [26]Chi CL Martinez S Wurst W Martin GR The isthmic organizer signal FGF8 is required for cell survival in the prospective midbrain and cerebellum Development 2003 130 2633 2644 10.1242/dev.00487 12736208 · DOI
  26. [27]Sato T Joyner AL The duration of Fgf8 isthmic organizer expression is key to patterning different tectal-isthmo-cerebellum structures Development 2009 136 3617 3626 10.1242/dev.041210 19793884 PMC2761110 · DOI
  27. [28]Basson MA Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development Development 2008 135 889 898 10.1242/dev.011569 18216176 PMC2555978 · DOI
  28. [29]Komisarczuk AZ Kawakami K Becker TS Cis-regulation and chromosomal rearrangement of the fgf8 locus after the teleost/tetrapod split Dev. Biol. 2009 336 301 312 10.1016/j.ydbio.2009.09.029 19782672 · DOI
  29. [30]Chambers D Medhurst AD Walsh FS Price J Mason I Differential display of genes expressed at the midbrain-hindbrain junction identifies sprouty2: An FGF8-inducible member of a family of intracellular FGF antagonists Mol. Cell. Neurosci. 2000 15 22 35 10.1006/mcne.1999.0801 10662503 · DOI
  30. [31]Li C Scott DA Hatch E Tian X Mansour SL Dusp6 (Mkp3) is a negative feedback regulator of FGF-stimulated ERK signaling during mouse development Development 2007 134 167 176 10.1242/dev.02701 17164422 PMC2424197 · DOI
  31. [32]Inoue F Parvin MS Yamasu K Transcription of fgf8 is regulated by activating and repressive cis-elements at the midbrain–hindbrain boundary in zebrafish embryos Dev. Biol. 2008 316 471 486 10.1016/j.ydbio.2008.01.013 18280464 · DOI
  32. [33]McMahon AP Bradley A The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain Cell 1990 62 1073 1085 10.1016/0092-8674(90)90385-R 2205396 · DOI
  33. [34]McMahon AP Joyner AL Bradley A McMahon JA The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum Cell 1992 69 581 595 10.1016/0092-8674(92)90222-X 1534034 · DOI
  34. [35]Davis CA Noble-Topham SE Rossant J Joyner AL Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain Genes Dev. 1988 2 361 371 10.1101/gad.2.3.361 2454212 · DOI
  35. [36]Davis CA Joyner AL Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development Genes Dev. 1988 2 1736 1744 10.1101/gad.2.12b.1736 2907320 · DOI
  36. [37]Liu A Joyner AL EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region Development 2001 128 181 191 11124114
  37. [38]Frankel N Phenotypic robustness conferred by apparently redundant transcriptional enhancers Nature 2010 466 490 493 10.1038/nature09158 20512118 PMC2909378 · DOI · Paper page
  38. [39]Perry MW Boettiger AN Bothma JP Levine M Shadow enhancers foster robustness of Drosophila gastrulation Curr. Biol. 2010 20 1562 1567 10.1016/j.cub.2010.07.043 20797865 PMC4257487 · DOI · Paper page
  39. [40]Osterwalder M Enhancer redundancy provides phenotypic robustness in mammalian development Nature 2018 554 239 243 10.1038/nature25461 29420474 PMC5808607 · DOI · Paper page
  40. [41]Shin HY Hierarchy within the mammary STAT5-driven Wap super-enhancer Nat. Genet. 2016 48 904 90 10.1038/ng.3606 27376239 PMC4963296 · DOI · Paper page
  41. [43]Schneider I Shubin NH The origin of the tetrapod limb: from expeditions to enhancers Trends Genet. 2013 29 419 426 10.1016/j.tig.2013.01.012 23434323 · DOI
  42. [44]Gehrke AR Shubin NH Cis-regulatory programs in the development and evolution of vertebrate paired appendages Semin. Cell Dev. Biol. 2016 57 31 39 10.1016/j.semcdb.2016.01.015 26783722 PMC5360378 · DOI
  43. [45]Inoue F Genomic organization, alternative splicing, and multiple regulatory regions of the zebrafish fgf8 gene Dev. Growth Differ. 2006 48 447 462 10.1111/j.1440-169X.2006.00882.x 16961592 · DOI
  44. [46]Ye W Distinct regulators control the expression of the mid-hindbrain organizer signal FGF8 Nat. Neurosci. 2001 4 1175 1181 10.1038/nn761 11704761 · DOI
  45. [47]Schwarz M Alvarez-Bolado G Urbánek P Busslinger M Gruss P Conserved biological function between Pax-2 and Pax-5 in midbrain and cerebellum development: evidence from targeted mutations Proc. Natl Acad. Sci. USA 1997 94 14518 14523 10.1073/pnas.94.26.14518 9405645 PMC25040 · DOI
  46. [48]Sidow A A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant Nat. Genet. 1999 23 104 107 10.1038/12709 10471509 · DOI
  47. [49]Kano H Kurahashi H Toda T Genetically regulated epigenetic transcriptional activation of retrotransposon insertion confers mouse dactylaplasia phenotype Proc. Natl Acad. Sci. USA 2007 104 19034 19039 10.1073/pnas.0705483104 17984064 PMC2141903 · DOI
  48. [50]Rajagopal N High-throughput mapping of regulatory DNA Nat. Biotechnol. 2016 34 167 174 10.1038/nbt.3468 26807528 PMC5108523 · DOI · Paper page
  49. [53]Sanjana NE High-resolution interrogation of functional elements in the noncoding genome Science 2016 353 1545 1549 10.1126/science.aaf7613 27708104 PMC5144102 · DOI
  50. [55]Diao Y A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells Nat. Methods 2017 14 629 635 10.1038/nmeth.4264 28417999 PMC5490986 · DOI · Paper page
  51. [56]Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat. Biotechnol. 34, 192–198 (2016). 26751173
  52. [57]Will AJ Composition and dosage of a multipartite enhancer cluster control developmental expression of Ihh (Indian hedgehog) Nat. Genet. 2017 49 1539 1545 10.1038/ng.3939 28846100 PMC5617800 · DOI
  53. [58]Symmons O The Shh topological domain facilitates the action of remote enhancers by reducing the effects of genomic distances Dev. Cell 2016 39 529 543 10.1016/j.devcel.2016.10.015 27867070 PMC5142843 · DOI · Paper page
  54. [59]Pongubala J Atchison ML PU.1 can participate in an active enhancer complex without its transcriptional activation domain Proc. Natl Acad. Sci. USA 1997 94 127 132 10.1073/pnas.94.1.127 8990172 PMC19254 · DOI · Paper page
  55. [60]Lettice LA Devenney P De Angelis C Hill RE The conserved sonic hedgehog limb enhancer consists of discrete functional elements that regulate precise spatial expression Cell Rep. 2017 20 1396 1408 10.1016/j.celrep.2017.07.037 28793263 PMC5561167 · DOI · Paper page
  56. [61]Wang H One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering Cell 2013 153 910 918 10.1016/j.cell.2013.04.025 23643243 PMC3969854 · DOI · Paper page
  57. [62]Schedl A A method for the generation of Yac transgenic mice by pronuclear microinjection Nucl. Acids Res. 1993 21 4783 4787 10.1093/nar/21.20.4783 8233827 PMC331506 · DOI
  58. [63]Sharpe J Optical projection tomography as a tool for 3D microscopy and gene expression studies Science 2002 296 541 545 10.1126/science.1068206 11964482 · DOI
  59. [64]Cheddad A Svensson C Sharpe J Georgsson F Ahlgren U Image processing assisted algorithms for optical projection tomography IEEE Trans. Med Imaging 2012 31 1 15 10.1109/TMI.2011.2161590 21768046 · DOI
  60. [65]Limaye, A. Drishti: a volume exploration and presentation tool. in (ed. Stock, S. R.) Vol. 8506, 85060X–85060X–9 (SPIE, 2012).
  61. [66]Crossley PH Martin GR The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo Development 1995 121 439 451 7768185
  62. [67]Bailey TL Johnson J Grant CE Noble WS The MEME Suite Nucl. Acids Res. 2015 43 W39 W49 10.1093/nar/gkv416 25953851 PMC4489269 · DOI

Authors
Hörnblad, A., Bastide, S., Langenfeld, K., Langa, F., & Spitz, F.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • European Molecular Biology Laboratory (DE)
Corresponding author
Spitz, F.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-01-19
Volume
12
Issue
1
Pages
439-439
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-20714-y
PMC
PMC7815712
OpenAlex
W3121272729
Cited by
44
References
71
Citations by year
2026: 3 · 2025: 8 · 2024: 5 · 2023: 8 · 2022: 9 · 2021: 6 · 2020: 5
FWCI
2.20
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
  • KTH Publication Database DiVA (KTH Royal Institute of Technology) (submittedVersion, other-oa)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Deutsches Krebsforschungszentrum
  • European Molecular Biology Laboratory
  • Deutsche Forschungsgemeinschaft (1331/3-1, SP 1331/3-1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
FGF8, enhancer redundancy, enhancer function, apical ectodermal ridge, midbrain-hindbrain boundary, limb development, developmental gene regulation, enhancer evolution
MeSH terms
Animals, Ectoderm, Embryo, Mammalian, Enhancer Elements, Genetic, Extremities, Feasibility Studies, Female, Genetic Engineering, Male, Mesencephalon, Mice, Transgenic, Rhombencephalon, Gene Expression Regulation, Developmental, Embryonic Development, Mice, Fibroblast Growth Factor 8, Gene Regulatory Networks, CRISPR-Cas Systems

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