Targeted Gene Repression Using Novel Bifunctional Molecules to Harness Endogenous Histone Deacetylation Activity

Epigenome editing is a powerful method for life science research and could give rise to new therapies for diseases initiated or maintained by epigenetic dysregulation, including several types of cancers and autoimmune disorders. In addition, much is still unknown about the mechanisms by which histone-modifying proteins work in concert to properly regulate gene expression. To investigate and manipulate complex epigenetic interactions in live cells, we have developed a small molecule platform for specifically inducing gene repression and histone deacetylation at a reporter gene. We synthesized bifunctional ligands, or chemical epigenetic modifiers (CEMs), that contain two functional groups: a FK506 derivative capable of binding to a FKBP-Gal4 fusion transcription factor, and a histone deacetylase (HDAC) inhibitor that recruits HDAC-containing corepressor complexes. In our reporter cell line, which contains a GFP reporter allele upstream of a Gal4 DNA binding array in the murine Oct4 locus, our lead CEM repressed GFP expression by 50%. We also show that CEM recruitment of deacetylation activity causes marked deacetylation along our target loci. This system allowed us to detail the direct results of deacetylation to chromatin and measure the resulting gene expression in a chemically dependent and reversible manner. The CEMs system provides new insights into epigenetic gene regulation and has the potential to control disease-relevant gene regulation. The CEMs are derived from FDA-approved epigenetic modulator drugs, and use their pharmacology in a gene-specific way that avoids the toxicities and off-target effects caused by whole-cell application of these drugs.