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

Abstract

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.

Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery

“…We have previously demonstrated the ability of chemical epigenetic modifiers (CEMs) to modify chromatin and subsequently repress gene expression at engineered reporter loci 14 . In this study, we report CEM activating (CEMa) molecules that recruit endogenous gene activating machinery.…”

Chemical and Light Inducible Epigenome Editing

“…To address these concerns, synthetic bifunctional ligands, termed chemical epigenetic modifiers (CEMs), were developed to target endogenous epigenome modifiers (Figure 2c). The first generation CEMs conjugate FK506 (a ligand binds FKBP12) to histone deacetylase (HDAC) inhibitors that recruits HDAC-containing corepressor complexes [92,93]. Using these CEMs in the CiA system with the GAL4DBD-FKBP12 fusion protein, endogenous HDAC3 can be targeted to the CiA:OCT4 locus leading to the reduction of H3K27ac and gene silencing in revisable and dose-dependent manners [92].…”

“…The first generation CEMs conjugate FK506 (a ligand binds FKBP12) to histone deacetylase (HDAC) inhibitors that recruits HDAC-containing corepressor complexes [92,93]. Using these CEMs in the CiA system with the GAL4DBD-FKBP12 fusion protein, endogenous HDAC3 can be targeted to the CiA:OCT4 locus leading to the reduction of H3K27ac and gene silencing in revisable and dose-dependent manners [92]. To expand the CEM strategy for broader applications, a second generation CEM platform was recently developed that incorporated the CRISPR/dCas9 system for DNA targeting [93].…”

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

“…This is accomplished by (1) redirecting the HDAC to the locus, releasing the enzyme, and increasing the density of un-inhibited HDACs in the area and (2) maintaining HDAC inhibition at the locus, but recruiting repressive complexes that bind to the inhibited HDACs, or (3) a combination of both. In a previous study, we showed that the CEMs were able to successfully repress the GFP reporter in a dose-and time-dependent manner, as well as in a manner that was rapidly reversible (i.e., within 24 hours) 12 . We characterized the ability of the CEM technology presented here to control gene expression using fluorescence microscopy, flow cytometry, and the ability to control the chromatin environment using ChIP-qPCR 6 .…”

“…The linker length between FK506 and the recruiter moiety will influence the permeability of the CEMs, as well as the position of the recruited protein. When comparing two CEMs that differed only in linker length, the CEM with the shorter linker was more effective 12 . While it has not been tested directly here, the higher effectiveness is a result of greater cell permeability.…”