Tandem histone-binding domains enhance the activity of a synthetic chromatin effector

Abstract

Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the "Polycomb based transcription factor" (PcTF), a fusion protein that recognizes histone modifications through a protein protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl histone enriched loci in cancer derived cell lines. However, PcTF induces modest activation of a methyl histone associated reporter compared to a DNA binding activator. Therefore, we modified PcTF to enhance its target affinity. Here, we demonstrate the activity of a modified regulator called Pc 2 TF, which has two tandem copies of the H3K27me3 binding PCD at the N terminus. Pc 2 TF shows higher affinity for H3K27me3 in vitro and shows enhanced gene activation in HEK293 cells compared to PcTF.These results provide compelling evidence that the intrinsic histone binding activity of the PCD motif can be used to tune the activity of synthetic histone binding transcriptional regulators.

The synthetic histone-binding regulator protein PcTF activates interferon genes in breast cancer cells

“…In this report we present a synthetic approach to macrogenomic engineering, a fusion protein that physically bridges a chromatin feature at silenced genes (H3K27me3) with proteins that drive gene activation. Our previous studies have established that PcTF specifically interacts with H3K27me3 in vitro [47] , and drives the activation of hundreds of repressed loci including master regulators and tumor suppressors in bone, blood, and brain cancer derived model cell lines [48] . In our current report, we discovered a core set of interferon-pathway-related genes that responded to PcTF in three distinct breast cancer cell lines.…”

“…It will be eventually important to determine if PcTF proteins meet or exceed the efficacy of low molecular weight epigenetic drugs in tumor and patient-derived models. At present, PcTF an its variants [47] represent a new exploration space for rationally-designed epigenetic interventions.…”

“…So far, our results suggest that PcTF-mediated activation requires a moderate level of basal expression at the target gene. This idea may be counterintuitive since H3K27me3 mark, the target of PcTF [47] , is essential for transcriptional repression according to the model for Polycomb-mediated regulation, which is supported by a wealth of data [77] .…”

“…Our group developed a fusion protein called Polycomb-based Transcription Factor (PcTF), which specifically binds H3K27me3 [47] and recruits endogenous transcription factors to PRC-silenced genes ( Fig. 1).…”

Delivery of cell-penetrating chromatin sensor-actuators to human osteosarcoma cells

“…However, experiments with epigenome editors and sensor-actuators have relied heavily on the delivery and overexpression of fusion-encoding transgenes. Having recently demonstrated that purified recombinant PcTF specifically binds its target H3K27me3 in vitro [20] , we set out to determine if purified PcTF proteins maintain their function as potent gene activators after they are delivered directly to cells.…”