Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

Abstract

Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

Figure 1

The dCas9^{p300 Core} fusion protein activates transcription of endogenous genes from proximal promoter regions. (a) Schematic of dCas9 fusion proteins dCas9^VP64, dCas9^{FL p300}, and dCas9^{p300 Core}. Streptococcus pyogenes dCas9 contains nuclease inactivating mutations D10A and H840A. The D1399 catalytic residue in the p300 HAT domain is indicated. (b) Western blot showing expression levels of dCas9 fusion proteins and GAPDH in co-transfected cells (full blot shown in Supplementary Fig. 1c). (c) Relative mRNA expression of IL1RN, MYOD, and OCT4, determined by qRT-PCR, by the indicated dCas9 fusion protein co-transfected with four gRNAs targeted to each promoter region (Tukey-test, *P-value < 0.05, n = 3 independent experiments each, error bars: s.e.m.). Numbers above bars indicate mean expression. FLAG, epitope tag; NLS, nuclear localization signal; HA, hemagglutinin epitope tag; CH, cysteine-histidine-rich region; Bd, bromodomain; HAT, histone acetyltransferase domain.

Figure 2

The dCas9^{p300 Core} fusion protein activates transcription of endogenous genes from distal enhancer regions. (a) Relative MYOD mRNA production in cells co-transfected with a pool of gRNAs targeted to either the proximal or distal regulatory regions and dCas9^VP64 or dCas9^{p300 Core}; promoter data from Fig. 1c (Tukey-test, *P-value <0.05 compared to mock-transfected cells, Tukey test †P-value <0.05 between dCas9^{p300 Core} and dCas9^VP64, n = 3 independent experiments, error bars: s.e.m.). The human MYOD locus is schematically depicted with corresponding gRNA locations in red. CE, MyoD core enhancer; DRR, MyoD distal regulatory region. (b) Relative OCT4 mRNA production in cells co-transfected with a pool of gRNAs targeted to the proximal and distal regulatory regions and dCas9^VP64 or dCas9^{p300 Core}; promoter data from Fig. 1c (Tukey-test, *P-value <0.05 compared to mock-transfected cells, Tukey test †P-value <0.05 between dCas9^{p300 Core} and dCas9^VP64, n = 3 independent experiments, error bars: s.e.m.). The human OCT4 locus is schematically depicted with corresponding gRNA locations in red. DE, Oct4 distal enhancer; PE, Oct4 proximal enhancer. (c) The human β-globin locus is schematically depicted with approximate locations of the hypersensitive site 2 (HS2) enhancer region and downstream genes (HBE, HBG, HBD, and HBB). Corresponding HS2 gRNA locations are shown in red. Relative mRNA production from distal genes in cells co-transfected with four gRNAs targeted to the HS2 enhancer and the indicated dCas9 proteins. Note logarithmic y-axis and dashed red line indicating background expression (Tukey test among conditions for each β-globin gene, †P-value <0.05, n = 3 independent experiments, error bars: s.e.m.). n.s., not significant.

Figure 3

dCas9^{p300 Core} targeted transcriptional activation is specific and robust. (a–c) MA plots generated from DEseq2 analysis of genome-wide RNA-seq data from HEK293T cells transiently co-transfected with dCas9^VP64 (a) dCas9^{p300 Core} (b) or dCas9^{p300 Core (D1399Y)} (c) and four IL1RN promoter-targeting gRNAs compared to HEK293T cells transiently co-transfected with dCas9 and four IL1RN promoter-targeting gRNAs. mRNAs corresponding to IL1RN isoforms are shown in blue and circled in each panel. Red labeled points in panels b–c correspond to off-target transcripts significantly enriched after multiple hypothesis testing (KDR, (FDR = 1.4 × 10⁻³); FAM49A, (FDR = 0.04); p300, (FDR = 1.7 × 10⁻⁴) in panel b; and p300, (FDR = 4.4 × 10⁻¹⁰) in panel c.

Figure 4

The dCas9^{p300 Core} fusion protein acetylates chromatin at a targeted enhancer and corresponding downstream genes. (a) The region encompassing the human β-globin locus on chromosome 11 (5,304,000 – 5,268,000; GRCh37/hg19 assembly) is shown. HS2 gRNA target locations are indicated in red and ChIP-qPCR amplicon regions are depicted in black with corresponding green numbers. ENCODE/Broad Institute H3K27ac enrichment signal in K562 cells is shown for comparison. Magnified insets for the HS2 enhancer, HBE, and HBG1/2 promoter regions are displayed below. (b–d) H3K27ac ChIP-qPCR enrichment (relative to dCas9; red dotted line) at the HS2 enhancer, HBE promoter, and HBG1/2 promoters in cells co-transfected with four gRNAs targeted to the HS2 enhancer and the indicated dCas9 fusion protein. HBG ChIP amplicons 1 and 2 amplify redundant sequences at the HBG1 and HBG2 promoters (denoted by ‡). Tukey test among conditions for each ChIP-qPCR region, *P-value <0.05 (n = 3 independent experiments, error bars: s.e.m.).

Figure 5

The dCas9^{p300 Core} fusion protein activates transcription of endogenous genes from regulatory regions with a single gRNA. (a–c) Relative (a) IL1RN, (b) MYOD or (c) OCT4 mRNA produced from cells co-transfected with dCas9^{p300 Core} or dCas9^VP64 and gRNAs targeting respective promtoters (n = 3 independent experiments, error bars: s.e.m.). Relative MYOD (d) or OCT4 (e) mRNA produced from cells co-transfected with dCas9^{p300 Core} and indicated gRNAs targeting the indicated MYOD or OCT4 enhancers (n = 3 independent experiments, error bars: s.e.m.). DRR, MYOD distal regulatory region; CE, MYOD core enhancer; PE, OCT4 proximal enhancer; DE, OCT4 distal enhancer. (Tukey test between dCas9^{p300 Core} and single OCT4 DE gRNAs compared to mock-transfected cells, *P-value <0.05, Tukey test among dCas9^{p300 Core} and OCT4 DE gRNAs compared to All, †P-value <0.05,). Relative HBE (f) or HBG (g) mRNA production in cells co-transfected with dCas9^{p300 Core} and the indicated gRNAs targeted to the HS2 enhancer (Tukey test between dCas9^{p300 Core} and single HS2 gRNAs compared to mock-transfected cells, *P-value <0.05, Tukey test among dCas9^{p300 Core} and HS2 single gRNAs compared to All, †P<0.05, n = 3 independent experiments, error bars: s.e.m.). HS2, β-globin locus control region hypersensitive site 2; n.s., not significant using Tukey test.

Figure 6

The p300 Core can be targeted to genomic loci by diverse programmable DNA-binding proteins. (a) Schematic of the Neisseria meningitidis (Nm) dCas9 fusion proteins Nm-dCas9^VP64 and Nm-dCas9^{p300 Core}. Neisseria meningitidis dCas9 contains nuclease-inactivating mutations D16A, D587A, H588A, and N611A. (b–c) Relative (b) HBE or (c) HBG mRNA in cells co-transfected with five individual or pooled (A–E) Nm gRNAs targeted to the HBE or HBG promoter and Nm-dCas9^VP64 or Nm-dCas9^{p300 Core}. (d–e) Relative (d) HBE or (e) HBG mRNA in cells co-transfected with five individual or pooled (A–E) Nm gRNAs targeted to the HS2 enhancer and Nm-dCas9^VP64 or Nm-dCas9^{p300 Core}. (f) Schematic of TALEs with domains containing IL1RN-targeted repeat variable diresidues (Repeat Domain). (g) Relative IL1RN mRNA in cells transfected with individual or pooled (A–D) IL1RN TALE^VP64 or IL1RN TALE^{p300 Core} encoding plasmids. (h) Schematic of ZF fusion proteins with zinc finger helices 1–6 (F1–F6) targeting the ICAM1 promoter. (i) Relative ICAM1 mRNA in cells transfected with ICAM1 ZF^VP64 or ICAM1 ZF^{p300 Core}. Tukey-test, *P-value <0.05 compared to mock-transfected control, n = 3 independent experiments each, error bars: s.e.m. NLS, nuclear localization signal; HA, hemagglutinin tag; Bd, bromodomain; CH, cysteine-histidine-rich region; HAT, histone acetyltransferase domain.