Recent exome-sequencing data of patients revealed that de novo mutations in protein-coding regions drive almost half of all severe developmental disorders. While we may estimate haploinsufficient or dominant-negative effects from these mutations, we mostly lack precise information on the molecular chain-of- events from mutation to pathogenesis. One example is the rare neurodevelopmental disorder Coffin-Siris Syndrome (CSS), a rare neurodevelopmental disorder characterized by intellectual disability, delayed speech development, and certain facial abnormalities. Sequenced CSS patients have dominant heterozygous de novo mutations in subunits of the BAF chromatin remodeling complex: a majority putative haploinsufficient protein- truncating variants of ARID1B, and a minority putative dominant-negative missense variants of SMARCE1. Traditional molecular biology perturbation techniques to investigate mechanism, including CRISPR/dCas9, RNAi, small-molecule inhibitors, and protein overexpression, are hampered by off-target effects, limited design, and lack of temporal control. A general method to target and regulate any mutant gene, from synaptic component to chromatin remodeler, could help determine the clinical significance of mutations in many developmental disorders towards developing precision therapies. Chemical induced proximity is a strategy to use a two-sided small-molecule to co-localize two proteins of interest together in a biologically-relevant setting. To expand such a strategy to gene regulation, we developed an initial system to recruit a repressive epigenome regulator, Hp1, rapidly and reversibly to any locus with 10- fold higher locus-specificity than existing systems and precise temporal control. This proposal seeks to expand upon our hypothesis that chemical induced epigenome editing can provide highly-locus-specific, kinetic control of gene regulation for mechanistic studies of developmental disorders, using CSS-derived iPSCs as a model system. In this proposal, we will first expand the use of chemically- inducible systems by using genomics to examine the specificity of two types of activators: a transactivator (VPR) and a histone methyltransferase (Ash2l). To further provide a general strategy for future development, we will build a biophysical model clarifying the on-target activity to off-target specificity of chemical induced recruitment of epigenome editors in a genome-wide manner. We will finally apply inducible epigenome editing to study the precision and dosage effects on the genome of activating ARID1B and repressing SMARCE1Y73C, two variants implicated in CSS as haploinsufficient (ARID1B) or dominant-negative (SMARCE1Y73C), in iPSCs. At the culmination of this work, we will have not only developed a platform of epigenome editing for highly-specific gene regulation, but also have validated its use in defining molecular mechanisms in patient-relevant genetic contexts. The proposal presented also reflects my Training Goals of becoming skilled as an interdisciplinary researcher at the intersection of human genetics and chemical engineering.
Severe developmental disorders affect almost 1 in 213 children worldwide. Recent genetic sequencing has implicated mutant proteins in many such disorders, but we lack the molecular-biological knowledge needed to develop therapies for most of these disorders. The research proposed here develops and applies new chemical-genetic technology to regulate mutant genes for uncovering pathophysiological mechanisms and identifying therapeutic targets for patients with developmental diseases.
