Getting a handle on chemical probes of chomatin readers

Abstract

The dynamic nature of histone post-translational modifications such as methylation or acetylation makes possible the alteration of disease associated epigenetic states through the manipulation of the associated epigenetic machinery. One approach is through small molecule perturbation. Chemical probes of epigenetic reader domains have been critical in improving our understanding of the biological consequences of modulating their targets, while also enabling the development of novel probe-based reagents. By appending a functional handle to a reader domain probe, a chemical toolbox of reagents can be created to facilitate chemiprecipitation of epigenetic complexes, evaluate probe selectivity, develop in vitro screening assays, visualize cellular target localization, enable target degradation and recruit epigenetic machinery to a site within the genome in a highly controlled fashion.

Keywords: biotin; bivalent ligand; chemical probe; chemical tool; chromatin reader; epigenetics.

Figure 1.. Functionalization of chemical probes enhances their utility.

The addition of a variety of functional handles to previously characterized chemical probes allows for their use in a wide array of biological and biochemical platforms.

Figure 2.. Applications of biotinylated reader domain chemical probes.

(A) The general structure of a chemical probe (orange star) tagged with biotin (green circle), with the two moieties connected via a PEG-linker (yellow oval). (B) Biotin tagged probes targeting reader domains are capable of pulling down intact protein complexes as determined by western blot or proteomics analysis. (C) The genomic localization of the probe and its target reader protein can be assessed via Chem-seq which couples chemical affinity capture via a biotinylated ligand with massively parallel DNA sequencing. (D) Biotinylated chemical tools can serve as tracer ligands in in vitro TR-FRET assays to screen for new reader domain ligands and evaluate ligand potency to establish SAR. (E) Protein microarrays can be used to assess chemical probe selectivity within the reader target class by incubation with a biotinylated chemical tool and visualization with fluorescent streptavidin. FL: Fluorophore; PEG: Polyethylene glycol; SA: Streptavidin; SAR: Structure–activity relationships; TR-FRET: Time-resolved fluorescence energy transfer.

Figure 3.. Fluorescent chemical probes demonstrate cellular target engagement.

UNC1215 was tagged with the cell penetrant, long wavelength merocyanine dye mero76 and mero76-UNC1215 was shown to colocalize with GFP-L3MBTL3, indicating that UNC1215 engages its target in cells. Localization of mero76-UNC1215 was perturbed with the addition of untagged UNC1215.

Figure 4.. Bivalent degrader approach affords selective degradation of BRD4.

(A) Structure of the bivalent degrader, MZ1, which contains a bromo- and extra-terminal domain targeting ligand (JQ1) and a ligand for VHL. (B) MZ1 achieves selective degradation of BRD4 even though JQ1 is about equipotent for BRD2, BRD3 and BRD4. VHL: von Hippel-Lindau.

Figure 5.. Bivalent degrader targeting EED leads to polycomb repressive complex 2 degradation.

(A) Structure of UNC6852 which is composed of the EED ligand, EED226, tethered via an alkyl linker to a VHL ligand. (B) In the catalytic cycle of UNC6852, a ternary complex is first formed between PRC2, UNC6852 and VHL. Subsequent PRC2 ubiquitylation marks the complex for degradation and it is recruited to the proteasome, resulting in degradation of the core PRC2 components (EZH2, EED and SUZ12). VHL: von Hippel-Lindau.

Figure 6.. Recruitment of reader proteins to specific genomic loci to control gene expression.

(A) General strategy of using bivalent molecules containing a BET bromodomain ligand and a genomic targeting moiety to site specifically recruit BRD4 to perturb gene expression. (B) Syn-TEF1 recruits BRD4 to the FXN gene by localizing to GAA repeats via its polyamide DNA-binding moiety, in turn activating FXN expression. (C) Mixing JQ1 fused to IntC with a guide RNA and dCas-IntN in cell media results in an assembled dCas-JQ1: guide RNA complex which can then be delivered to cells via cationic lipid-mediated delivery and localize to its target DNA sequence. (D) A dCas9-FKBP fusion protein targets CEM-87 to a gene of interest, which induces transcription at that loci through the recruitment of BRD4. CEM: Chemical epigenetic modifier