Karmella Haynes, Ph.D.*
Biological and Health Systems Engineering
Arizona State University
Synthetic Chromatin-based Regulators as Biologics for Cancer Therapy
Nuclear protein complexes, collectively known as chromatin, spatially organize genomic DNA and support stability and plasticity of gene expression in eukaryotic cells. Decades of basic research has paved the way for scientists and engineers to manipulate the epigenetic information encoded in chromatin to regulate transcription and to control accessibility of DNA to gene-editing nucleases. Customizable fusion transcription factors (TF’s) have been an indispensable tool to artificially regulate nearly any target gene of interest. Typically, the mode of target site recognition is an interaction of the TF peptide (e.g., Gal4, TAL, ZF, etc.) or an RNA adapter (i.e., CRISPR gRNA) with DNA sequences at promoters or enhancers. Our work represents a unique innovation for fusion TF targeting: proteins that bind posttranslational marks on histones instead of DNA sequences. We developed the “Polycomb-based transcription factor” (PcTF), a fusion protein that reads histone modifications via affinity of an evolutionarily conserved N-terminal Polycomb chromodomain (PCD from CBX8) for trimethylated lysine 27 of histone H3 (H3K27me3). The C-terminal VP64 domain of PcTF recruits endogenous activators to silenced targets. We observed that PcTF-mediated activation of target genes was accompanied by the loss of H3K27me3 and the accumulation of the activation-associated H3K4me3 mark over time. Expression of PcTF in a panel of breast cancer cell lines revealed broad regulation of tumor suppressor, developmental, and innate immunity genes. Activation of these genes has also been observed for FDA-approved epigenetic anti-cancer drugs. To explore the tunability of PcTF function, we have implemented a cell-free to in-cell workflow to quickly identify more robust configurations of the protein. Enzyme-linked immunosorbent assay (ELISA), microspot array experiments, and expression in cells (HEK293) showed that tandem PCD domains conferred increased, specific interaction with H3K27me3 in vitro and enhanced activity at a silenced target gene. PcTF provides unique opportunities for cancer research and medicine. In contrast to epigenetic drugs, PcTF can be mapped at target sites to track the steps involved in broad gene co-regulation, or altered to stimulate distinct classes of promoters. This presentation will provide an overview of our development of PcTF, as well as our progress in DNA-free delivery of cell-penetrating PcTF proteins to cultured cells and manipulation of repressive Polycomb chromatin to enhance CRISPR-mediated editing at target genes.
Host: Manu Platt, Ph.D.