Abstract
Systematic identification of noncoding regulatory elements has, to date, mainly relied on large-scale reporter assays that do not reproduce endogenous conditions. We present two distinct CRISPR-Cas9 genetic screens to identify and characterize functional enhancers in their native context. Our strategy is to target Cas9 to transcription factor binding sites in enhancer regions. We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERα (ESR1) gene regulation. Moreover, we show that a genomic CRISPR-Cas9 tiling screen can precisely map functional domains within enhancer elements. Our approach expands the utility of CRISPR-Cas9 to elucidate the functions of the noncoding genome
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Acknowledgements
We would like to acknowledge K. Rooijers for help in bioinformatic analyses, insightful ideas and critical discussions. We thank W. de Laat, P. Krijger, B. Evers, M. Amendola and R. Maia for reagents and technical assistance. We also thank all members of the Agami group for helpful discussions. And we thank F. Zhang (MIT) for the gift of plasmid vector lentiCRISPRv2 and D. Root (MIT) for pLX304-GFP. R.L. is a fellow of the Fundação para a Ciência e Tecnologia de Portugal (SFRH/BD/74476/2010; POPH/FSE). This work was supported by funds of The Human Frontier Science Program (LT000640/2013) to A.P.U., and enhReg ERC-AdV and NWO (NGI 93512001/2012) to R.A.
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G.K., R.L. and R.A. conceived the project. G.K. and R.L. performed most of the experiments. A.P.U. did all luciferase assays. W.Z. and E.N. carried out and analyzed the ChIP-Seq data, respectively. R.H. helped during the library preparation. K.M. did cloning and validation of candidates from the screens. R.E. conducted all the bioinformatics analyses. G.K., R.L. and R.A. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Light microscopy images of cell populations transduced with the indicated sgRNA vectors.
(a, b and c) Images were taken after 15 days of HRASG12V induction.
Supplementary Figure 2 p53enh3508 region exhibits a p53-dependent enhancer activity that is required for OIS activation.
(a) Senescence induction was quantified using senescence-associated β-gal assay. (b) MCF-7 cells were transfected with the indicated vectors, treated with Nutlin-3a 5-10 hours later, and harvested 25-30hr after treatment. The relative luciferase activities (Firefly/Renilla) were normalized to the Ctrl reaction. (All p-values for luciferase assay were calculated by Student’s t-test. * indicates the significance relative to empty vector; # indicates the significance relative to untreated matching sample). (c) The same assay as in panel b, only that cells were co-transfected with control, or p53-targeting siRNAs. A reporter vector containing the enhancer region p53-BER420 was used as a positive control for p53-dependency. The efficiency of p53 knockdown was determined by immunoblot analysis. (All p-values for luciferase assay were calculated by Student’s t-test. * indicates the significance relative to empty vector).
Supplementary Figure 3 deCDKN1A contains key enhancer elements that regulate OIS in a p53-dependet fashion.
(a) The same cell extracts as in Figure 2g were blotted with an antibody against HRAS. (b) qRT-PCR analysis of CDKN1A mRNA levels performed with the indicated BJ-indRASG12V cell populations following induction of RASG12V. (n=3; *P<0.05, two-tailed Student’s t-test). (c) MCF-7 cells were transfected with the indicated reporter vectors, treated with Nutlin-3a 5-10 hours later, and harvested 25-30 hours later. The relative luciferase activities (Firefly/Renilla) were normalized to the Ctrl reaction. (d and e) The same cell populations as in b panel were subjected to BrdU labeling and β-gal assays to assess proliferation and OIS, respectively. N=2; for each condition at least 150 cells were count. *P<0.05, two-tailed Student’s t-test. (f) Western blot analysis of BJ-indRASG12V CDKN1A KO and control cells. P53 and HSP90 were used as controls.
Supplementary Figure 4 ERαenh588 regulates CCND1 activity in an estrogen-dependent manner.
(a) Screenshot of ChIA-PET analysis in MCF-7 cells showing a strong chromatin interaction between enh588 and CCND1 promoter. (b) MCF-7 cells were seeded with charcoal-treated medium and transfected with the indicated reporter vectors. 16 hours later the medium was refreshed and either supplemented or not with 17β-estradiol. 24 hours later cells were harvested and luciferase activity was measured. The relative luciferase activities (Firefly/Renilla) were normalized to the Ctrl reaction. (c) Complete blot shown in Figure 3h.
Supplementary Figure 5 Results of PROMO analyses are shown for regions surrounding the p53 BS and Hit38.
The p53 BS and the sgRNA-Hit38 targeting region are indicated, and the sequences representing potential CEBPB binding are underlined.
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Supplementary Figures 1–5 and Supplementary Tables 1–7 (PDF 3764 kb)
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Korkmaz, G., Lopes, R., Ugalde, A. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol 34, 192–198 (2016). https://doi.org/10.1038/nbt.3450
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DOI: https://doi.org/10.1038/nbt.3450
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