dCas9 fusion proteins can target and alter epigenetic marks in enhancers and promoters
Protein fusions of deactivated CRISPR-associated endonuclease Cas9 (dCas9) with transcription activator or repressor domains have been used to study gene regulation. However, such fusion proteins do not modulate chromatin conformation directly, limiting their use when studying epigenetic marks. Two studies now describe the fusion of dCas9 with epigenetic writer or eraser enzymes; these dCas9 fusion proteins can target and alter epigenetic marks in enhancers and promoters, thereby modulating gene expression.
The acetylation of histone H3 on Lys 27 (H3K27ac), which is characteristic of active enhancers, is catalysed by the transcription activator histone acetyltransferase p300. Hilton et al. fused dCas9 with the catalytic core domain of p300 (dCas9–p300Core) and found that dCas9–p300Core, but not a fusion with an inactivated core domain (dCas9–p300Core(D1399Y)), strongly induced transcription from guide RNA-targeted promoters in HEK293T cells, demonstrating that the dCas9–p300Core acetyltransferase activity was necessary for transcription activation. Targeting dCas9–p300Core to several enhancers had similar effects; for example, when the HS2 enhancer in the β-globin locus control region was targeted with dCas9–p300Core (but not when targeted with dCas9–p300Core(D1399Y)), the expression of three of the four distal globin genes was induced. H3K27ac was significantly enriched at the HS2 enhancer and at the promoters of two of the globin genes following targeting with dCas9–p300Core. These results demonstrate that dCas9–p300Core can potently activate gene expression through the targeted deposition of H3K27ac marks at proximal and distal regulatory sequences.
Kearns et al. fused dCas9 with the histone demethylase LSD1 (lysine-specific histone demethylase 1), which was previously implicated in enhancer repression. They generated mouse embryonic stem cells (ES cells) expressing dCas9–LSD1 or controls, and delivered into them single guide RNAs targeting two enhancers of the transcription factor Oct4 (also known as Pou5f1), which is an essential pluripotency factor. Targeting dCas9–LSD1 but not the other fusion proteins resulted in pluripotency enhancer-specific loss of Oct4 expression and change in colony morphology accompanied by transcriptome changes. Using dCas9–LSD1, the authors also identified four new enhancers required specifically for the expression of pluripotency maintenance genes. They further validated one of these as the enhancer of the pluripotency factor T-box 3 (Tbx3), as targeting it in dCas9–LSD1 ES cells resulted in loss of H3K4me2 around the enhancer, Tbx3 downregulation, change in colony morphology and an increase of differentiation-associated markers. An LSD1-specific inhibitor suppressed Tbx3 downregulation, which indicates that the enzymatic activity of LSD1 is essential for its gene regulation activity at enhancers.
Both studies demonstrate that epigenetic alterations in regulatory elements are sufficient to cause strong changes in gene expression. Such tools should therefore enable the functional annotation of regulatory elements in various cell types and conditions.
Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nature Biotech. http://dx.doi.org/10.1038/nbt.3199 (2015)
Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nature Methods http://dx.doi.org/10.1038/nmeth.3325 (2015)
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Zlotorynski, E. Characterizing enhancers with dCas9. Nat Rev Mol Cell Biol 16, 267 (2015). https://doi.org/10.1038/nrm3983