Using Newly-Identified Epigenetic Reader-Effector BAHCC1 to Engineer a H3K27me3-Binding Transcriptional Activator

Epigenetic therapy has emerged as an approach to activate non-mutated tumor suppressor genes (TSGs) in cancer cells, which is an alternative to traditional chemotherapies that inhibit DNA replication. Currently, the design of epigenetic treatments for cancer is restricted to small molecules that bind the enzymes that generate biochemical marks (histone post-translational modifications and DNA methylation) within chromatin. Reader-effectors play a central role in translating these marks into gene expression states, but are less amenable to chemical inhibition. Our overall goal is to engineer reader-effectors that directly bind to modified histones in human cell nuclei for cancer epigenetics research and treatment. Our immediate goals are to identify binding modules with strong affinity and specificity for H3K27me3, and to use these modules to build reader-activators that can access TSGs in repressive chromatin and stimulate their expression. Previously we developed a synthetic reader-activator (SRA) protein with a binding module derived from the CBX8 protein. Doubling the number of CBX8 binding modules increases SRA activity at a H3K27me3-enriched reporter target. To further enhance SRA activity we set out to identify new functional variants of the CBX8-derived module. Here, we present an evolutionary analysis of metazoan CBX orthologs that identified 22 unique modifications that could be incorporated into our current SRA design to potentially increase binding affinity. We also considered a recently characterized reader-repressor, BAHCC1 (Fan et al. 2020, Nat. Genet.), which shows stronger H3K27me3-specific binding (Kd = 72.4 uM) than a CBX ortholog (Kd = 99.6 uM). We have isolated the H3K27me3-binding domain from BAHCC1 and incorporated it into an SRA. Functional characterization of SRAs that carry the new H3K27me3 modules is currently underway.