Chemical modifications on DNA, RNA and histones are recognized by an array of ‘reader’ modules to regulate transcriptional programming and cell fate. However, identification of reader-specific interacting partners in a dynamic cellular environment remains a significant challenge. Herein, we report a chemoproteomic approach termed ‘interaction-based protein profiling’ (IBPP) to characterize novel interacting partners of potentially any reader protein. IBPP harnesses a photosensitive amino acid introduced into the hydrophobic pocket of a reader module to crosslink and enrich transient interacting partners that are inaccessible to traditional methods. Using bromodomain-containing protein 4 (BRD4) as a paradigm, we engineer an ‘aromatic cage’ of the bromodomain to introduce 4-azido-L-phenylalanine (pAzF) without compromising its ability to recognize acetylated lysine residues in histone proteins. We establish the binding efficiency, substrate specificity and crosslinking ability of the engineered ‘reader’ module in biochemical assays. Applying IBPP, we uncovered novel acetylated interacting partners of BRD4, such as transcription factors, expanding on its previously unappreciated role in diverse biological processes. By setting up an azide-acetyllysine photoreaction deep inside the bromodomain aromatic cage as a means to detect protein acetylation, our approach provides a potentially general platform for rapid and unbiased profiling of interacting partners of diverse epigenetic readers whose functions in eukaryotic gene regulation remain convoluted.

•Active demethylation of 5mC by TET enzymes is one of the key mechanisms of epigenetic regulation in vertebrates.•MALDI-based assay is developed to characterize catalytic activity of TET enzymes.•The method is employed to determine kinetic parameters of TETs and IC50 of TET inhibitors.•The assay is characterized by its minimum sample preparation, rapid and straightforward analysis.Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET2 and 3, Michaleis-Menten parameters (KM and kcat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by β-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.Download high-res image (113KB)Download full-size image

Oxidative C–H hydroxylation of methyl groups, followed by their removal from DNA, RNA, or histones, is an epigenetic process critical to transcriptional reprogramming and cell fate determination. This reaction is catalyzed by Fe(II)-dependent dioxygenases using the essential metabolite 2-ketoglutarate (2KG) as a cofactor. Given that the human genome encodes for more than 60 2KG-dependent dioxygenases, assigning their individual functions remains a significant challenge. Here we describe a protein–ligand interface engineering approach to break the biochemical degeneracy of these enzymes. Using histone lysine demethylase 4 as a proof-of-concept, we show that the enzyme active site can be expanded to employ bulky 2KG analogues that do not sensitize wild-type demethylases. We establish the orthogonality, substrate specificity, and catalytic competency of the engineered demethylation apparatus in biochemical assays. We further demonstrate demethylation of cognate substrates in physiologically relevant settings. Our results provide a paradigm for rapid and conditional manipulation of histone demethylases to uncloak their isoform-specific functions.

The relationship between DNA and protein sequences is well understood, yet because the members of a protein family/subfamily often carry out the same biochemical reaction, elucidating their individual role in cellular processes presents a challenge. Forward and reverse genetics have traditionally been employed to understand protein functions with considerable success. A fundamentally different approach that has gained widespread application is the use of small organic molecules, known as chemical genetics. However, the slow time-scale of genetics and inherent lack of specificity of small molecules used in chemical genetics have limited the applicability of these methods in deconvoluting the role of individual proteins involved in fast, dynamic biological events. Combining the advantages of both the techniques, the specificity achieved with genetics along with the reversibility and tunability of chemical genetics, has led to the development of a powerful approach to uncover protein functions in complex biological processes. This technique is known as allele-specific chemical genetics and is rapidly becoming an essential toolkit to shed light on proteins and their mechanism of action. The current review attempts to provide a comprehensive description of this approach by discussing the underlying principles, strategies, and successful case studies. Potential future implications of this technology in expanding the frontiers of modern biology are discussed.