Histone Lysine and Genomic Targets of Histone Acetyltransferases in Mammals

Technologies that facilitate the 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 have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.

A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover because they are scattered among the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here we present the results of chromatin immunoprecipitation with the enhancer-associated protein p300 followed by massively parallel sequencing, and map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases demonstrated reproducible enhancer activity in the tissues that were predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities, and suggest that such data sets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.

Histone acetyltransferases (HATs) and deacetylases (HDACs) function antagonistically to control histone acetylation. As acetylation is a histone mark for active transcription, HATs have been associated with active and HDACs with inactive genes. We describe here genome-wide mapping of HATs and HDACs binding on chromatin and find that both are found at active genes with acetylated histones. Our data provide evidence that HATs and HDACs are both targeted to transcribed regions of active genes by phosphorylated RNA Pol II. Furthermore, the majority of HDACs in the human genome function to reset chromatin by removing acetylation at active genes. Inactive genes that are primed by MLL-mediated histone H3K4 methylation are subject to a dynamic cycle of acetylation and deacetylation by transient HAT/HDAC binding, preventing Pol II from binding to these genes but poising them for future activation. Silent genes without any H3K4 methylation signal show no evidence of being bound by HDACs.