New Bifunctional Molecules Capture Chromatin Regulatory Machines to Control Gene Expression

Gene expression programs are intricately controlled at the level of chromatin by dynamic and complex pathways that regulate the accessibility of gene promoters to transcriptional machinery. In eukaryotic cells, DNA is packaged in the nucleus and wrapped around histone proteins to form nucleosomes, the central unit of chromatin. Heterochromatin (closed) regions of chromatin, are transcriptionally repressed while euchromatic (open) regions of the genome are associated with active transcription. Post-translational modifications of histone tails are an important determinant of chromatin structure. For instance, lysine acetylation decreases the positive charge on the histone tails and prevents the close compaction of the negatively charged DNA. Consequently, acetylation is associated with euchromatin and is regulated by a series of histone modifying proteins that deposit, read, and remove this mark. The regulation of chromatin compaction is commonly disrupted in human disease, yet much remains unknown about the mechanisms by which histone modifying enzymes regulate chromatin structure. A modular, reversible, and gene specific tool would enable selective recruitment to study the direct effects of these enzymes. To investigate these complex interactions in live cells, we developed a novel system that will unveil the underlying mechanisms of the proteins responsible for modulating chromatin states using a genome-wide manipulation strategy. To explore how dynamic changes in acetylation levels impact chromatin structure, we generated an endogenous reporter system at the murine Pou5F1 locus with a Gal4 DNA binding array upstream of a GFP reporter allele. In our system, a FKBP protein fused to a Gal4 DNA-binding-domain serves as a protein anchor to the DNA binding array. Next, we synthesized new cell-permeable, bifunctional molecules that include FK506 (which binds to FKBP) linked to a chemical moiety that selectively binds to a chromatin modifying enzyme. We determined the effects of the bifunctional molecules by quantifying the expression of GFP using flow cytometry and high content fluorescence microscopy. We have shown that our lead Chemical Epigenetic Modifier (CEM), a small molecule composed of FK506 linked to an HDAC3-specific inhibitor, can repress GFP expression by 50%. To study the addition of histone acetylation we used HAT-binding CEMs, which significantly accelerated reactivation of GFP coming out of heterochromatin release. Based on our preliminary data, we hypothesize that our CEM system can be further optimized and characterized. We are currently comparing activating and repressing CEMs, and determining their effects on gene expression and chromatin environment. Our system becomes more modular by creating a deactivated Cas9 (dCas9)-FKBP fusion protein, which will allow us to recruit chromatin modifiers to any region of the genome to which we design a guide RNA. We have begun to detail the direct results of histone acetylation or deacetylation on chromatin domain structure and gene expression in real time, and we plan to further develop and revolutionize the system. The end goal of our emerging approach is to generate a platform where expression of any gene can be either amplified or repressed in a dose dependent manner by simple addition of activating or repressive CEMs.