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

Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA - Nat. Biotechnol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA. [2] Center for Genomic &Computational Biology, Duke University, Durham, North Carolina, USA.

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- 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.

No MeSH data available.


Related in: MedlinePlus

The dCas9p300 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.).
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Figure 4: The dCas9p300 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.).

Mentions: Activity of regulatory elements correlates with covalent histone modifications such as acetylation and methylation1, 2. Of those histone modifications, acetylation of lysine 27 on histone H3 (H3K27ac) is one of the most widely documented indicators of enhancer activity29–31, 36, 37. Acetylation of H3K27 is catalyzed by p300 and is also correlated with endogenous p300 binding profiles36, 37. Therefore we used H3K27ac enrichment as a measurement of relative dCas9p300 Core-mediated acetylation at the genomic target site. To quantify targeted H3K27 acetylation by dCas9p300 Core we performed chromatin immuno-precipitation with an anti-H3K27ac antibody followed by quantitative PCR (ChIP-qPCR) in HEK293T cells co-transfected with four HS2 enhancer-targeted gRNAs and either dCas9, dCas9VP64, dCas9p300 Core, or dCas9p300 Core (D1399Y) (Fig. 4). We analyzed three amplicons at or around the target site in the HS2 enhancer or within the promoter regions of the HBE and HBG genes (Fig. 4a). Notably, H3K27ac is enriched in each of these regions in the human K562 erythroid cell line that has a high level of globin gene expression (Fig. 4a). We observed significant H3K27ac enrichment at the HS2 enhancer target locus compared to treatment with dCas9 in both the dCas9VP64 (P-value 0.0056 for ChIP Region 1and P-value 0.0029 for ChIP Region 3) and dCas9p300 Core (P-value 0.0013 for ChIP Region 1and P-value 0.0069 for ChIP Region 3) co-transfected samples (Fig. 4b). A similar trend of H3K27ac enrichment was also observed when targeting the IL1RN promoter with dCas9VP64 or dCas9p300 Core (Supplementary Fig. 4). In contrast to these increases in H3K27ac at the target sites by both dCas9VP64 or dCas9p300 Core, robust enrichment in H3K27ac at the HS2-regulated HBE and HBG promoters was observed only with dCas9p300 Core treatment (Fig. 4c–d). Together these results demonstrate that dCas9p300 Core uniquely catalyzes H3K27ac enrichment at gRNA-targeted loci and at enhancer-targeted distal promoters. Therefore the acetylation established by dCas9p300 Core at HS2 may catalyze enhancer activity in a manner distinct from direct recruitment of preinitiation complex components by dCas9VP64(refs 27, 28), as indicated by the distal activation of the HBE, HBG, and HBD genes from the HS2 enhancer by dCas9p300 Core but not by dCas9VP64 (Fig. 2c, Supplementary Fig. 3).


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

Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA - Nat. Biotechnol. (2015)

The dCas9p300 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.).
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Figure 4: The dCas9p300 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.).
Mentions: Activity of regulatory elements correlates with covalent histone modifications such as acetylation and methylation1, 2. Of those histone modifications, acetylation of lysine 27 on histone H3 (H3K27ac) is one of the most widely documented indicators of enhancer activity29–31, 36, 37. Acetylation of H3K27 is catalyzed by p300 and is also correlated with endogenous p300 binding profiles36, 37. Therefore we used H3K27ac enrichment as a measurement of relative dCas9p300 Core-mediated acetylation at the genomic target site. To quantify targeted H3K27 acetylation by dCas9p300 Core we performed chromatin immuno-precipitation with an anti-H3K27ac antibody followed by quantitative PCR (ChIP-qPCR) in HEK293T cells co-transfected with four HS2 enhancer-targeted gRNAs and either dCas9, dCas9VP64, dCas9p300 Core, or dCas9p300 Core (D1399Y) (Fig. 4). We analyzed three amplicons at or around the target site in the HS2 enhancer or within the promoter regions of the HBE and HBG genes (Fig. 4a). Notably, H3K27ac is enriched in each of these regions in the human K562 erythroid cell line that has a high level of globin gene expression (Fig. 4a). We observed significant H3K27ac enrichment at the HS2 enhancer target locus compared to treatment with dCas9 in both the dCas9VP64 (P-value 0.0056 for ChIP Region 1and P-value 0.0029 for ChIP Region 3) and dCas9p300 Core (P-value 0.0013 for ChIP Region 1and P-value 0.0069 for ChIP Region 3) co-transfected samples (Fig. 4b). A similar trend of H3K27ac enrichment was also observed when targeting the IL1RN promoter with dCas9VP64 or dCas9p300 Core (Supplementary Fig. 4). In contrast to these increases in H3K27ac at the target sites by both dCas9VP64 or dCas9p300 Core, robust enrichment in H3K27ac at the HS2-regulated HBE and HBG promoters was observed only with dCas9p300 Core treatment (Fig. 4c–d). Together these results demonstrate that dCas9p300 Core uniquely catalyzes H3K27ac enrichment at gRNA-targeted loci and at enhancer-targeted distal promoters. Therefore the acetylation established by dCas9p300 Core at HS2 may catalyze enhancer activity in a manner distinct from direct recruitment of preinitiation complex components by dCas9VP64(refs 27, 28), as indicated by the distal activation of the HBE, HBG, and HBD genes from the HS2 enhancer by dCas9p300 Core but not by dCas9VP64 (Fig. 2c, Supplementary Fig. 3).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA. [2] Center for Genomic &Computational Biology, Duke University, Durham, North Carolina, USA.

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- 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.

No MeSH data available.


Related in: MedlinePlus