Programmable control over histone acetylation at human regulatory elements using precision epigenome editing

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PROJECT SUMMARY/ABSTRACT Dysregulated gene expression contributes to nearly every human disease. The level at which we understand the inherent complexity of gene regulation ultimately dictates our ability to link and/or manipulate biomolecular changes and pathology. Beyond their function as a structural substrate, histone proteins play key roles in gene expression by selectively gating access to DNA and thereby guiding when and how transcriptional machinery engages the human genome. Histone-based gene regulatory control largely stems from post-translational modifications (PTMs) to histone proteins themselves. One type of PTM, histone lysine acetylation, is particularly critical for gene regulation and human health, because overall acetylation levels tightly correlate with genomic activity and gene expression, and inappropriate histone acetylation patterns are linked to diverse human diseases. In fact, small molecules that globally change histone acetylation across the human genome have emerged as important therapeutics. However, in many patients these drugs can be ineffective and/or can result in toxic side effects. Furthermore, small molecules that globally alter histone acetylation patterns cannot be used to dissect how changes at specific locations in the human genome drive disease pathology. Robust tools that enable precise and targeted control over endogenous histone acetylation are urgently needed, because these technologies could illuminate the function(s) that this complex epigenomic signature plays within the human genome and open the door to new sophisticated epigenetic therapies. In this project, we will fulfill this urgent need by building synthetic and precisely targetable biomolecules that mimic the spectrum of activities displayed by natural human histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Aim 1). Specifically, we will combine the programmability of the nuclease null CRISPR/Cas9 (dCas9) scaffold with different classes of human HATs/HDACs and we will use these technologies to probe the selectivity of different HATs/HDACs in vitro and within native human chromatin. We will integrate our results with epigenomic profiling data to define how epigenetic marks, nucleosome occupancy, and cis regulatory element proximity influence the effects of histone acetylation at human enhancers and promoters. We will also use mass spectrometry, dCas9-based transcriptional activators and HATs, and genome-scale knockout screening to define the proteins/protein complexes that support histone acetylation-based gene activation (Aim 2). Finally, we will establish the impact of precisely targeted histone acetylation/deacetylation on enhancer activity and enhancer-promoter interactions (Aim 3). Experiments will be conducted at testbed human loci that have broad significance to human health and mechanistic epigenetics, and that will serve as proof-of-principle for interrogating virtually any regulatory element or locus in the human genome in future efforts. To accomplish the aims of this project, we have assembled an experienced team with synergistic expertise in CRISPR/Cas- based epigenome editing, histone PTM analysis, and functional dissection of enhancer activity.