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Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors

Nature Genetics volume 48, pages 292–298 (2016)Cite this article

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Abstract

Genes are regulated by transcription factors that bind to regions of genomic DNA called enhancers. Considerable effort is focused on identifying transcription factor binding sites, with the goal of predicting gene expression from DNA sequence. Despite this effort, general, predictive models of enhancer function are currently lacking. Here we combine quantitative models of enhancer function with manipulations using engineered transcription factors to examine the extent to which enhancer function can be controlled in a quantitatively predictable manner. Our models, which incorporate few free parameters, can accurately predict the contributions of ectopic transcription factor inputs. These models allow the predictable 'tuning' of enhancers, providing a framework for the quantitative control of enhancers with engineered transcription factors.

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Figure 1: A logistic model predicts the observed graded repression of the eve 3 + 7 enhancer.
Figure 2: A logistic model predicts the outcome of the balance between activation and repression at the eve 4 + 6 enhancer.
Figure 3: TALEAs can overcome ventral repression of the rho enhancer.
Figure 4: A balance of transcriptional activators and repressors in the eve 2 enhancer.

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Acknowledgements

We thank M. Levine, J. Bothma, S. Barolo, J. Jaynes, N. Luscombe, the entire Stern laboratory and the members of the Janelia Transcriptional Imaging Consortium for discussions and advice throughout the project. We thank C. Standley, J. Cande, E. Preger–Ben Noon, J. Shaevitz and especially R. Mann for critically reviewing our manuscript. We thank D. Papatsenko and H. Janssens for supplying the dorsoventral rho and tll mutant data, respectively.

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  1. Justin Crocker and Garth R Ilsley: These authors contributed equally to this work.

Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Justin Crocker & David L Stern

  2. Okinawa Institute of Science and Technology Graduate University, Onna, Japan

    Garth R Ilsley

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Contributions

J.C. and G.R.I. conceived the study, designed and executed the experiments, and analyzed the data, with mentorship from D.L.S. J.C., G.R.I. and D.L.S. wrote the manuscript.

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Correspondence to Justin Crocker or Garth R Ilsley.

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Integrated supplementary information

Supplementary Figure 1 eve stripe 3+7 TALE-GFP does not alter eve expression, and different levels of TALER expression do not alter the effect of TALER.

(a–d) Stage 5 embryos stained for Eve protein in either a wild-type background (a) or carrying a single ubiquitous nos::GAL4 driver (b,c) or two nos::GAL4 drivers (d) and the indicated UAS::stripe 3+7 TALE. (e–h) Profiles of average expression levels for the indicated genotype (n = 10 for each genotype). Bounding areas around experimental data indicate 1 s.d. AU, arbitrary units of fluorescence intensity.

Supplementary Figure 2 The logistic eve 4+6 model accurately predicts expression in different genetic backgrounds.

(a–c) Virtual Embryo predictions from the eve 4+6 model, for eve 4+6 expression in the indicated genetic background. The knirps (kni) heterozygote prediction has knirps at 75% of normal levels, which implies compensatory regulation; in the homozygote, knirps is not expressed. (d–f) Stage 5 embryos stained for Even-skipped protein (Eve) in either a wild-type background (d) or the indicated genetic background (e,f) with the third Eve stripe labeled in each panel. (g–i) Larval cuticle preparations for the indicated genomic backgrounds, with the A1 segment labeled in each panel. Red brackets highlight the modified larval segments.

Supplementary Figure 3 The logistic eve 4+6 model accurately predicts expression patterns resulting from input misexpression.

(a) Ventral view of the even-skipped (eve) expression pattern in a wild-type stage 5 embryo. (b) Ventral view of the Virtual Embryo eve 4+6 model prediction. (c) Expression pattern of an embryo carrying ectopic knirps (kni) expression driven from a snail (sna) enhancer. (d) Virtual embryo with ectopic knirps expression. (e,g) Ventral views showing stripe-specific changes in expression resulting from ectopic knirps expression, in embryos carrying one (e) or two (g) copies of the sna::kni transgene. (f,h) Ventral view of the eve 4+6 model predictions with ectopic expression of knirps in the snail domain (see also e and g). In situ images are reproduced from Figure 1 of Clyde et al.32.

Supplementary Figure 4 Hairy and Krüppel repressive domains display quantitatively different repressive strength.

(a–d) Stage 5 embryos stained for Eve protein in either a wild-type background (a) or carrying a ubiquitous nos::GAL4 driver and the indicated UAS::stripe 3+7–TALE (b–d). (e–h) Profiles of average expression levels for the indicated genotype (n = 10 for each genotype). In all plots, the solid blue line denotes model predictions; black, green, red and orange lines denote wild-type, enhancer-GFP, enhancer-TALER-hairy and enhancer-TALER-Krüppel constructs, respectively. Bounding areas around experimental data indicate 1 s.d. AU, arbitrary units of fluorescence intensity.

Supplementary Figure 5 The logistic eve 4+6 model accurately predicts expression in tailless (tll)-mutant embryos.

(a,b) Predicted expression levels for the eve 3+7 (pink) and eve 4+6 (blue) enhancers in wild-type (a) or tailless-mutant (b) embryos, based on previously published primary expression data37. Dashed lines indicate actual Even-skipped protein (Eve) expression levels for each background. (c,d) Stage 5 embryos stained for Eve protein in either a wild-type background (c) or the tailless-mutant background (d), with the third Eve stripe labeled in each panel. (e,f) Larval cuticle preparations for the indicated genomic backgrounds, with the A1 segment labeled in each panel.

Supplementary Figure 6 Cooperative interactions between Dorsal (Dl) and Twist (Twi) are combined with snail (sna) in a linear manner.

(a) A logistic model using linear combinations of previously published expression data for the known regulators of rhomboid (rho)39, indicating the model output (dashed black line), the regulatory inputs (blue, green and orange) and native rhomboid enhancer expression (pink). The cyan dashed line indicates the model prediction for three TALEAs. (b) Cooperativity between Dorsal and Twist is modeled by the addition of a nonlinear term to the model. Note the differences in the model predictions in a and b in the dorsal and ventral regions with the addition of TALEAs (blue dashed lines).

Supplementary Figure 7 The rhomboid (rho) enhancer model including Dorsal-Twist cooperativity accurately predicts expression patterns resulting from binding site modifications.

(a–d) rhomboid model predictions in wild-type (a), snail (sna)-deficient (b), twist-deficient (c) and dorsal-deficient backgrounds (d). (e–h) Lateral views of rhomboid enhancer expression with the indicated binding site modifications; see also Figure 4 of Ip et al.40.

Supplementary Figure 8 A balance of transcriptional activators and repressors at the rhomboid enhancer.

(a) Schematic of the rhomboid enhancer region, with known binding sites indicated40. (b–d) Stage 5 embryos from either wild-type rho-lacZ reporter (b), a reporter carrying a mutation in the Twist site of rho-lacZ (c) or the Twist-mutant rho-lacZ reporter together with UAS::rho-TALEA-VP64 (d) stained for lacZ (β-gal) RNA.

Supplementary Figure 9 TALEAs can drive increased activation of native enhancers in multiple developmental contexts.

(a,b) In the neuroectoderm, stage 5 embryos display increased expression of vndNEE-lacZ when targeted with vnd-enhancer-TALEA-VP64 by nos::GAL4 (b) as compared with a wild-type background (a). (c–e) In the wing imaginal disc, a TALEA increases enhancer expression. (c) A schematic of Decapentaplegic (dpp) regulation of optomotor blind (omb) in the larval imaginal wing disc. (d,e) Larval imaginal wing discs show stronger expression of omb-lacZ transgenes when targeted with omb-enhancer-TALEA-VP64 by Act5c::GAL4 (e) as compared with wild-type background (d).

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Crocker, J., Ilsley, G. & Stern, D. Quantitatively predictable control of Drosophila transcriptional enhancers in vivo with engineered transcription factors. Nat Genet 48, 292–298 (2016). https://doi.org/10.1038/ng.3509

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