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

Abstract

Chromatin is a system of nuclear proteins and nucleic acids that plays a pivotal role in gene expression and cell behavior and is therefore the subject of intense study for cell development and cancer research. Biochemistry, crystallography, and reverse genetics have elucidated the macromolecular interactions that drive chromatin regulation. One of the central mechanisms is the recognition of post-translational modifications (PTMs) on histone proteins by a family of nuclear proteins known as "readers". This knowledge has launched a wave of activity around the rational design of proteins that interact with histone PTMs. Useful molecular tools have emerged from this work, enabling researchers to probe and manipulate chromatin states in live cells. Chromatin-based proteins represent a vast design space that remains underexplored. Therefore, we have developed a rapid prototyping platform to identify engineered fusion proteins that bind histone PTMs in vitro and regulate genes near the same histone PTMs in living cells. We have used our system to build gene activators with strong avidity for the gene silencing-associated histone PTM H3K27me3. Here, we describe procedures and data for cell-free production of fluorescently tagged fusion proteins, enzyme-linked immunosorbent assay-based measurement of histone PTM binding, and a live cell assay to demonstrate that the fusion proteins modulate transcriptional activation at a site that carries the target histone PTM. This pipeline will be useful for synthetic biologists who are interested in designing novel histone PTM-binding actuators and probes.

Mapping chromatin modifications at the single cell level

“…2011). Although binding affinities for their targets are often lower than those of antibodies (Kungulovski et al, 2014), reader domains have known sequences and thus can be evolved to improve affinity and specificity (Tekel et al, 2018). Similarly, fluorescent modification-specific intracellular antibodies (mintbodies) are small, GFP-tagged, single-chain variable fragments that can be expressed in vivo to allow live imaging of histone modification dynamics spatiotemporally (Sato et al, 2013) (Fig.…”

Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes

“…We constructed mammalian expression vector 14 (MV14) for the overexpression of Gal4-mCherry-AAP fusion proteins in-frame with a nuclear localization sequence and 6X-histidine tag. First, plasmid MV13 was built by inserting a Gal4-mCherry fragment into MV10 [100] directly downstream of the CMV promoter. Next, MV14 was built by inserting a SpeI/PstlI (FastDigest enzymes, ThermoFisher Scientific) -digested gBlock Gene Fragment (Integrated DNA Technologies), which encoded a XbaI/NotI multiple cloning site, into MV13 downstream of mCherry.…”

“…Luciferase assays were performed as previously described in Tekel et al [100]. In brief, a single well of cells from a 12 well tissue culture plate was collected per independent transfection in 1.5mL 1× PBS.…”

Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones

“…[20][21][22][23][24][25][26] Reader protein families have evolved to be site-specific binders of different histone PTMs, and the mechanisms by which reader domains recognize these marks are generally well characterized. 27 The recognition capability of reader scaffolds has been exploited to enrich chromatin samples for mass spectrometry analysis 26,28 , to generate artificial histone PTM-dependent transcriptional activators 29,30 , to visualize multiple PTMs simultaneously in stem cells 31 , and to employ histone interacting domains in chromatin immunoprecipitation assays. 21,22,32,33 While the utilization of reader domains as tools has promising potential given their small size and evolved specificity, a major limitation is the generally weak dissociation constants for their respective PTM ligands, often in the midmicromolar range.…”