Scalable platforms for understudied histone modifications and modifiers

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PROJECT SUMMARY/ABSTRACT Eukaryotic genomic DNA is extensively associated with proteins and RNAs to form chromatin. Through its control of gene expression, changes in chromatin biochemistry and structure underlie nearly all cellular processes. Post-translational modifications of histone proteins that bind genomic DNA play an especially critical role in regulating chromatin structure and function. Modifications of certain histone residues influence the binding of histone proteins to DNA as well as the interactions of other proteins that specifically recognize these modifications. The specific pattern of histone modifications acts as a “histone code” to determine the set of proteins that interact with histones and histone-bound DNA, and consequently participate in diverse cellular processes. Therefore, identification of specific histone modifications, quantitative assessment of the interactions they mediate, and characterization of enzymes that modify histones is essential for understanding chromatin regulation of complex cellular behavior. However, unraveling the histone code is a daunting challenge due to its complexity – over eighty different amino acid residues on five histone proteins undergo over twenty distinct known post-translational modifications. Despite significant advances in research on some histone modifications such as acetylation and methylation, the vast majority of histone modifications remains understudied. Further, a vast majority of enzymes that write even extensively researched modifications like acetylation remain understudied. We propose to address the critical gap in essential biochemical tools and accessible experimental platforms that has hindered research on understudied histone modifications and modifiers. Specifically, we will harness next generation yeast surface display systems as scalable platforms for high throughput studies on understudied histone modifications and modifiers, as well as platforms for engineering biochemical reagents for chromatin research. In addition to understudied histone acetyltransferases, we will focus on three classes of histone modifications: (i) citrullination of arginine (ii) acyl modification of lysine by non-acetyl groups (propionylation, butyrylation, crotonylation), and (iii) monoamine modification of glutamine by serotonin and dopamine. In Aim 1, we will develop a platform for efficient generation of affinity reagents with high specificity that can serve as genetically encoded biosensors for live cell imaging, as well as in conventional analyses like ChIP-seq and CUT&Tag. In Aim 2, we will develop a platform for high throughput identification and quantification of binding interactions mediated by histone modifications. In Aim 3, we will develop a platform for high throughput interrogation of residue preferences of writers. Our work will develop “open source” platforms that are scalable, cost-efficient, and easily adopted by other investigators in chromatin biology. Such platforms will serve as strong complements to traditional biochemical assays by enabling research on both common and understudied histone modifications, and unlocking new high throughput measurements and research questions in chromatin biology.