Scalable platforms for understudied histone modifications and modifiers

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.