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Functional annotation of native enhancers with a Cas9–histone demethylase fusion

Abstract

Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type–specific function. Here, we use a nuclease-deficient Cas9 (dCas9)–histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.

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Figure 1: dCas9-effector fusions regulate cis-regulatory elements in an effector-dependent manner.
Figure 2: Enhancer targeting by dCas9-LSD1 or dCas9-KRAB.

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Acknowledgements

We thank T. Fazzio, J. Dekker and S. Wolfe for helpful discussions and E. Sontheimer and M. Ziller for critical reading of the manuscript. We thank K. Eggan (Harvard University) and A. McMahon (University of Southern California) for providing the V6.5 ESCs and the Rosa26 targeting vector backbone, respectively. We are grateful to S. Hainer, H. Belaghzal, K. Morrison and the University of Massachusetts Medical School morphology core for technical help and discussions. R.M. is supported by The Leona M. and Harry B. Helmsley Charitable Trust (2015PG-T1D057 and 2015PG-T1D035), a Charles H. Hood Foundation Child Health Research Award, the Glass Family Charitable Foundation and the US National Institutes of Health (NIH; UC4 DK104218). M.G. is supported by a grant from the NIH (1R01HD080224-01A1).

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Authors and Affiliations

Authors

Contributions

N.A.K., H.P. and R.M. were responsible for the conception, design and interpretation of experiments. M.G. conceived the bioinformatic approach for novel enhancer identification. N.A.K., H.P. and R.M.G. conducted experiments. B.T. and N.J.S. performed bioinformatics analyses. All authors wrote the manuscript.

Corresponding author

Correspondence to René Maehr.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Nm dCas9 amino acid sequences and sgRNA vector cloning region.

(a) Amino acid sequences of Nm dCas9-3xFLAG-BAT, dCas9-3xFLAG-KRAB, and dCas9-3xFLAG-LSD1 with dCas9 sequence in yellow, SV40 NLS in cyan, 3xFLAG tag in green, Biotin affinity tag in red, and effectors in magenta. (b) Nucleotide sequence of Lenti-U6 Nm sgRNA acceptor cassette. Type IIS BsmBI recognition sites used for cloning of aligned oligonucleotides encoding sgRNAs (bold-red), the Nm sgRNA-specific scaffold (bold-cursive)8 and the transcriptional terminator (underlined) are indicated.

Supplementary Figure 2 Generation of Rosa26 Nm dCas9–effector mESC lines.

(a) Targeting scheme for the insertions at Rosa26. A CAGS promoter and loxP site-flanked stop cassette containing the Nm dCas9-effector targeting construct (1) is depicted, along with the Rosa26 locus (2). The targeted Rosa26 locus contains a loxP-flanked stop cassette (3) that can be deleted in a cre-dependent manner (4). Primers used for validation of targeting and stop removal are indicated. (b) PCR validation of Rosa26 locus targeting (upper panel) and stop cassette removal (lower panel). Controls for targeting include a previously targeted Rosa26 ESC line (positive control) and parental V6.5 mouse ESCs (negative control). Amplicon size was monitored by DNA ladder (L). (c) Hematoxylin and eosin stain of structures in Nm dCas9-effector teratomas. Endodermal gut-epithelium (G), ectodermal neural-like cells (N) and mesodermal cartilage (C) are indicated. Scale bar represents 50μm.

Supplementary Figure 3 Nm dCas9–effector fusions target Oct4 cis-regulatory elements in an effector-specific manner.

(a) Immunofluorescence analysis of dCas9-effector mESCs targeted with sgRNAs specific to an unrelated control genomic region (Ctrl), distal enhancer (ODE), proximal enhancer (OPE) or the promoter (OPP) of Oct4. Cells are detected by a nuclear Hoechst stain, and protein expression of SOX2 or OCT4 is detected. White arrows indicate regions containing cells without detectable OCT4 or SOX2 expression. Images are representative of three independent experiments. Scale bar represents 100μm. (b) Phase contrast evaluation of ESC colony morphology in the indicated dCas9-effector/sgRNA combinations. White arrows indicate non-ESC morphologies. Images are representative of three independent experiments. Scale bar represents 100μm. (c) Heat map of gene expression microarray data from ESC cultures expressing specific Nm dCas9-effectors and sgRNAs displayed relative to average expression levels of dCas9-effector Ctrl-sgRNA samples. Unsupervised hierarchical clustering of 170 differentially expressed genes (listed in Supplementary Table 1) is displayed on the y-axis. Hierarchical clustering of samples based on similarities in gene expression profiles is displayed on the x-axis.

Supplementary Figure 4 Genomic organization of candidate 1 enhancer regions.

The genomic organization of the Tbx3 locus near Enhancer 1, genome accessibility (as measured by ATACseq signal on the y-axis), publically available H3K27ac ChIPseq data24, the target sites of the locus-specific sgRNAs and the location of sites used for ChIP qPCR evaluation. sgRNAs that result in loss of the ESC state in Nm dCas9-LSD1 mESCs are indicated in red.

Supplementary Figure 5 Genomic organization of candidate enhancers 2–8.

The genomic organization of candidate enhancers 2-8, genome accessibility (as measured by ATACseq signal on the y-axis), publically available H3K27ac ChIPseq data24 and the target sites of the locus-specific sgRNAs. sgRNAs that result in loss of the ESC state in Nm dCas9-LSD1 mESCs are indicated in red.

Supplementary Figure 6 Genomic organization of control enhancer regions.

The genomic organization of control enhancers Oct6 and N-Myc, genome accessibility (as measured by ATACseq signal on the y-axis), publically available H3K27ac ChIPseq data24 and the target sites of the locus-specific sgRNAs.

Supplementary Figure 7 dCas9-LSD1 screening identifies novel, distal cis-regulatory elements required for maintenance of ESC state.

(a) Strategy to functionally evaluate ESC-specific enhancers with the Nm dCas9-LSD1 effector. Comparative analysis of enhancer-associated H3K27ac ChIPseq24 signal together with RNAseq signal allows identification of putative ESC enhancers close to genes encoding transcription factors. ESC-specific enhancers with low or no change in mRNA abundance (blue, H3K27ac signal change >2-fold, and RNAseq signal enrichment <4-fold), and with high mRNA enrichment (red, H3K27ac signal change >2-fold, and RNAseq signal enrichment >4-fold) are displayed. Putative enhancers that are not significantly changed (light grey) or are EpiLC-specific (dark grey, H3K27ac signal change >2-fold) are indicated. sgRNAs targeting candidate enhancer elements with the highest scores (red) are delivered to Nm dCas9-LSD1-expressing mESCs. Morphological changes and ESC state-associated alkaline phosphatase activity are measured in an end-point assay. (b) Putative enhancer candidates tested in a dCas9-LSD1 screen with their relative ESC-EpiLC enrichment score. The number of sgRNAs that caused a loss of AP activity out of the total number of sgRNAs tested for each enhancer are listed and enhancers thereby identified as critical for ESC state are indicated (*). (c) Alkaline phosphatase staining of Nm dCas9-LSD1 cells with the indicated enhancer-specific sgRNA. sgRNAs resulting in a loss of AP activity are marked with (*). Scale bar represents 2mm. (d) Phase contrast evaluation of ESC colony morphology in Nm dCas9-LSD1 cells with the indicated enhancer-specific sgRNA. White arrows indicate non-ESC morphologies. Scale bar represents 200μm.

Supplementary Figure 8 Inactivation of an enhancer distal to Tbx3 leads to upregulation of OCT6.

(a) Immunofluorescence analysis of TBX3 in dCas9-effector mESCs targeted with sgRNAs specific to an unrelated control genomic region (Ctrl), distal enhancer (TDE) or proximal promoter (TPP) of Tbx3. Cells are detected by a nuclear Hoechst stain. White arrows indicate regions containing cells without detectable TBX3 expression. Images are representative of three independent experiments. Scale bar represents 100μm. (b) Phase contrast evaluation of ESC colony morphology in the indicated Nm dCas9-effector/sgRNA combinations. White arrows indicate non-ESC morphologies. Images are representative of three independent experiments. Scale bar represents 100μm. (c) Quantitative PCR analysis for Oct6 expression in dCas9-effector mESCs treated with sgRNAs specific to Ctrl, TDE or TPP regions. n=3 biological replicates +/- s.d. (d) Immunofluorescence analysis of OCT6 in Nm dCas9-effector mESCs targeted with sgRNAs specific to Ctrl, TDE or TPP regions. Cells are detected by a nuclear Hoechst stain. White arrows indicate regions containing cells with detectable OCT6 expression. Images are representative of three independent experiments. Scale bar represents 100μm.

Supplementary Figure 9 Enhancer targeting by dCas9-LSD1 does not disrupt the local genomic architecture.

(a) Quantitative PCR analysis for Tbx3 expression in Nm dCas9-effector mESCs treated with sgRNAs specific to an unrelated control genomic region (Ctrl) or the putative Tbx3 distal enhancer (TDE) prior to the onset of morphological changes. n=3 technical replicates +/- s.d. (b) The genomic organization of the Tbx3 locus, location of the putative distal enhancer and the location of HindIII sites (top panel). 3C interaction frequencies between the Tbx3 distal region and downstream fragments in dCas9-BAT ESCs with sgRNA to Tbx3 DE or control region and dCas9-LSD1 targeted to the Tbx3 DE (lower panel). The position of the anchor point is indicated (*). Error bars represent the s.d. of three independent PCR reactions.

Supplementary Figure 10 Enhancer targeting is dCas9-LSD1 specific and differs from the dCas9-KRAB effector mechanism.

(a) ChIP qPCR analysis of H3K4me2, H3K27ac, H3K27me3 and H3K9me3 at the Tbx3 enhancer 410 bases downstream of the sgRNA target site (primer position indicated in Supplementary Fig. 4) for Nm dCas9-effector mESCs in the presence of control sgRNA (Ctrl) or a sgRNA specific to the putative Tbx3 DE (TDE). The data are expressed as percent input values. Adjacent black and grey bars represent two independent experiments with mean +/- s.d. of three technical replicates. (b) ChIP qPCR analysis of H3K4me2 and H3K27ac at the Actb promoter for Nm dCas9-effector mESCs in the presence of control sgRNA (Ctrl) or a sgRNA specific to the putative Tbx3 DE (TDE). The data are expressed as percent input values. Adjacent black and grey bars represent two independent experiments with mean +/- s.d. of three technical replicates. (c) ChIP qPCR analysis of H3K27me3 and H3K9me3 at the control HoxC10 promoter for dCas9-effector mESCs in the presence of control sgRNA (Ctrl) or a sgRNA specific to the putative Tbx3 DE (TDE). The data are expressed as percent input values. Adjacent black and grey bars represent two independent experiments with mean +/- s.d. of three technical replicates. (d) ChIP qPCR analysis of H3 at the control Actb promoter, Tbx3 enhancer and Tbx3 promoter for Nm dCas9-effector mESCs in the presence of control sgRNA (Ctrl) or an sgRNA specific to the putative Tbx3 DE (TDE). The data are expressed as percent input values. Adjacent black and grey bars represent two independent experiments with mean +/- s.d. of three technical replicates.

Supplementary Figure 11 dCas9-LSD1–mediated Tbx3 downregulation is dependent on the enzymatic activity of LSD1.

(a) Quantitative PCR analysis for Tbx3 expression in dCas9-effector mESCs maintained under 500nM TCP conditions or after TCP withdrawal treated with sgRNAs specific to the putative Tbx3 distal enhancer (TDE) n=3 technical replicates +/- s.d. (b) Immunofluorescence analysis of Nm dCas9-effector mESCs targeted with sgRNAs specific to TDE. Cells are detected by a nuclear Hoechst stain, and protein expression of TBX3 is detected. White arrows indicate regions containing cells without detectable TBX3 expression. Scale bar represents 100μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Table 2 and 4–8 and and Supplementary Note (PDF 1917 kb)

Supplementary Table 1

Log2 expression levels of differentially expressed genes between specific Nm dCas9-effectors and Oct4 sgRNA combinations. (XLSX 30 kb)

Supplementary Table 3

Log2 expression levels of differentially expressed genes between specific Nm dCas9-effectors and Tbx3 sgRNA combinations. (XLSX 77 kb)

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Kearns, N., Pham, H., Tabak, B. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat Methods 12, 401–403 (2015). https://doi.org/10.1038/nmeth.3325

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