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 binding avidity. Here, we demonstrate the activity of a modified regulator called PcTF, which has two tandem copies of the H3K27me3-binding PCD at the N-terminus. PcTF has a smaller apparent dissociation constant value 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.

ChromID identifies the protein interactome at chromatin marks

“…Single domain eCRs were often insufficient to achieve binding to histone modifications under physiological conditions. This is in line with several well-known examples where binding of full-length proteins or complexes to chromatin rely on multivalent interactions 4 , including recent studies that introduced synthetic multivalent chromatin readers for immunofluorescence or activation of reporter genes 57 – 59 . We made use of the required multivalent interactions to generate synthetic readers that recognise two modifications on the same nucleosome.…”

Faculty Opinions recommendation of ChromID identifies the protein interactome at chromatin marks.

“…Single domain eCRs were often insufficient to achieve binding to histone modifications under physiological conditions. This is in line with several wellknown examples where binding of full-length proteins or complexes to chromatin rely on multivalent interactions 4 , including recent studies that introduced synthetic multivalent chromatin readers for immunofluorescence or activation of reporter genes [57][58][59] . We made use of the required multivalent interactions to generate synthetic readers that recognise two modifications on the same nucleosome.…”

Design, Construction, and Validation of Histone-Binding Effectors in Vitro and in Cells

“…“Histone peptide capture: relative binding” is the mean of the four normalized values. Nonspecific binding was assessed by using a fusion protein that contained no HBD (PcΔ 14 ). We used two methods to determine histone peptide capture as percent fusion protein input, where either the mean six-His capture A 450 value or the TXTL RFP end point signal (533−610, a.u.)…”

“…Current approaches to validate the function of histone PTM-binding domains (HBDs) include fluorescence polarization (FP), 11 isothermal calorimetry (ITC), 12 surface plasmon resonance (SPR), 13 and peptide arrays. 14 The primary limitation of these approaches is the need for high yields of purified protein, which limits the number and variety of proteins that can be assayed efficiently. Recently, we demonstrated the rapid prototyping of novel multivalent histone-binding transcriptional activators built from a human orthologue called CBX8.…”

“…Recently, we demonstrated the rapid prototyping of novel multivalent histone-binding transcriptional activators built from a human orthologue called CBX8. 14 The results showed that small volumes (<12 μ L) of cell-free transcription−translation lysates (TXTL) can be used to produce sufficient RFP-tagged histone-binding fusion proteins to generate significant binding signal over background in an enzyme-linked immunosorbent assay (ELISA). Here as a proof of concept, we screened a small library of additional TXTL-expressed variants for H3K27me3 avidity.…”