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In Vitro Development of Synthetic Chromatin Proteins That Function in Live Cells
Author(s) -
Haynes Karmella A.,
Tekel Stefan,
Vargas Daniel A.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.922.8
Subject(s) - fusion protein , h3k4me3 , biology , chromatin , histone , microbiology and biotechnology , histone h3 , promoter , enhancer , transcription factor , gene , genetics , gene expression , recombinant dna
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 rather than DNA sequences. 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 P olycomb c hromo d omain (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 surface plasmon resonance (SPR) 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 TF's that recognize other histone marks. We believe that peptides that specifically interact with epigenetic marks are on the verge of becoming the next generation of synthetic transcriptional regulators. Support or Funding Information KAH is supported by NIH NCI (K01 CA188164). S. Tekel is supported by ASU SBHSE. D. Vargas is supported by NSF DHRD WEASO (1401190).Workflow to identify modular, chromatin‐derived peptides that bind histone post‐translational modifications.