Speaker: Karmella Haynes, Ph.D.
Affiliation: School of Biological and Health Systems Engineering at Arizona State University
A pipeline to engineer synthetic regulators that read chromatin modifications
Recombinant DNA technology has empowered scientists to control gene expression at will. Fusion transcription factors (TF’s) are customizable proteins that can activate and repress virtually 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) with DNA at promoters or enhancers near target genes. Our work represents a unique approach to TF targeting: the use of fusion proteins that bind epigenetic marks on histones instead of DNA sequences. This approach enables efficient, broad macrogenomic engineering in cancer cells where hundreds of genes are misregulated as a cohort. In previous work, we developed and characterized the “Polycomb-based transcription factor” (PcTF), a fusion protein that reads histone modifications through a protein-protein interaction between its N-terminal Polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3). The C-terminal VP64 domain of PcTF recruits endogenous activators to silenced targets. We observed that dose-dependent, PcTF-mediated activation of target genes was accompanied by the loss of H3K27me3 and the accumulation of the activation-associated H3K4me3 mark over time. Several PcTF target genes are tumor suppressors, therefore PcTF has significant implications for cancer treatment. Recently, we have implemented a cell-free to in-cell workflow to quickly identify more robust configurations of the modular PcTF fusion protein. Enzyme-linked immunosorbent assay (ELISA) and microspot array experiments showed that tandem PCD domains conferred enhanced and specific interaction with H3K27me3 in vitro. We observed little cross-reactivity with unmodified histones and other histone marks. The double PCD fusion also showed enhanced target gene activation in a model cell line (HEK293). In conclusion, we have demonstrated a screening pipeline to support the design of functional histone-binding TF’s. Dozens of other known histone-binding peptides could be used to build TFs that recognize other histone marks. We believe that peptides that specifically interact with epigenetic marks are on the verge of becoming a new generation of synthetic transcriptional macro-regulators.
1. Tandem histone-binding domains enhance the activity of a synthetic chromatin effector. Tekel SJ, Vargas DA, Song L, LaBaer J, Haynes KA. (2017) ACS Synthetic Biol. (just accepted manuscript) https://pubs.acs.org/doi/pdf/10.1021/acssynbio.7b00281
2. Regulation of cancer epigenomes with a histone-binding synthetic transcription factor. Nyer DB, Daer R, Vargas D, Hom C, Haynes KA. (2017) Nature Genomic Medicine. http://rdcu.be/oqv7
Karmella Haynes is an Assistant Professor of Biomedical Engineering at Arizona State University. She earned her Ph.D. studying epigenetics and chromatin in Drosophila at Washington University, St. Louis. Postdoctoral fellowships at Davidson College and Harvard Medical School introduced her to synthetic biology. Today, her research aims to identify how the intrinsic properties of chromatin, the DNA-protein structure that packages eukaryotic genes, can be used to control cell development in tissues. Her HHMI postdoctoral fellowship project on bacterial computers was recognized as “Publication of the Year” in 2008 by the Journal of Biological Engineering. As an assistant professor at ASU, Dr. Haynes received an NIH Young Faculty Award (K01) and a Arizona Biomedical Early Stage Investigator Award (AZ ESI). She is currently a Councilor of the Engineering Biology Research Consortium (EBRC), a SynBioLEAP alum, and Advisor and Judge Emeritus for the International Genetically Engineered Machines (iGEM) competition. Her discovery of the impact of chromatin on CRISPR activity in human cells was featured on PRI’s Science Friday
Date(s) - May 17, 2018
12:00 pm - 1:00 pm