Design and Construction of a Focused DNA-Encoded Library for Multivalent Chromatin Reader Proteins.
ABSTRACT: Chromatin structure and function, and consequently cellular phenotype, is regulated in part by a network of chromatin-modifying enzymes that place post-translational modifications (PTMs) on histone tails. These marks serve as recruitment sites for other chromatin regulatory complexes that 'read' these PTMs. High-quality chemical probes that can block reader functions of proteins involved in chromatin regulation are important tools to improve our understanding of pathways involved in chromatin dynamics. Insight into the intricate system of chromatin PTMs and their context within the epigenome is also therapeutically important as misregulation of this complex system is implicated in numerous human diseases. Using computational methods, along with structure-based knowledge, we have designed and constructed a focused DNA-Encoded Library (DEL) containing approximately 60,000 compounds targeting bi-valent methyl-lysine (Kme) reader domains. Additionally, we have constructed DNA-barcoded control compounds to allow optimization of selection conditions using a model Kme reader domain. We anticipate that this target-class focused approach will serve as a new method for rapid discovery of inhibitors for multivalent chromatin reader domains.
Project description:Chromatin regulatory complexes localize to specific sites via recognition of posttranslational modifications (PTMs) on N-terminal tails of histone proteins (e.g., methylation, acetylation, and phosphorylation). Molecular recognition of modified histones is mediated by "reader" protein subunits. The recruited complexes govern processes such as gene transcription, DNA replication, and chromatin remodeling. Dysregulation of histone modifications and consequent downstream effects have been associated with a variety of disease states, leading to an interest in developing small-molecule inhibitors of reader proteins. Herein, we describe a generalized time-resolved fluorescence resonance energy transfer (TR-FRET) assay for a panel of methyl-lysine (Kme) reader proteins. These assays are facile, robust, and reproducible. Importantly, this plug-and-play assay can be used for high-throughput screening (HTS) campaigns, generation of structure-activity relationships (SARs), and evaluation of inhibitor selectivity. Successful demonstration of this assay format for compound screening is highlighted with a pilot screen of a focused compound set with CBX2. This assay platform enables the discovery and characterization of chemical probes that can potently and selectively inhibit Kme reader proteins to ultimately accelerate studies of chromatin reader proteins in normal biology and disease states.
Project description:Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.
Project description:We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.
Project description:Site-specific lysine acetylation and methylation on histones are critical post-translational modifications (PTMs) that govern ordered gene transcription in chromatin. Mis-regulation of these histone PTM-mediated processes has been shown to be associated with human diseases. Since the 2010 landmark reports of small molecules (+)-JQ1 and I-BET762 that target the acetyl-lysine 'reader' Bromodomain and Extra Terminal domain (BET) proteins, there have been relentless efforts to develop epigenetic therapy with small molecules to modulate molecular interactions of epigenome reader domain proteins with PTMs. In addition to BET, the other emerging targets include non-BET acetyl-lysine and methyl-lysine reader domains. This review covers the key chemical modulators of the aforementioned epigenome reader proteins.
Project description:Lysine methylation facilitates protein-protein interactions through the activity of methyllysine (Kme) "reader" proteins. Functions of Kme readers have historically been studied in the context of histone interactions, where readers aid in chromatin-templated processes such as transcription, DNA replication and repair. However, there is growing evidence that Kme readers also function through interactions with non-histone proteins. To facilitate expanded study of Kme reader activities, we developed a high-throughput binding assay to reveal the sequence determinants of Kme-driven protein interactions. The assay queries a degenerate methylated lysine-oriented peptide library (Kme-OPL) to identify the key residues that modulate reader binding. The assay recapitulated methyl order and amino acid sequence preferences associated with histone Kme readers. The assay also revealed methylated sequences that bound Kme readers with higher affinity than histones. Proteome-wide scoring was applied to assay results to help prioritize future study of Kme reader interactions. The platform was also used to design sequences that directed specificity among closely related reader domains, an application which may have utility in the development of peptidomimetic inhibitors. Furthermore, we used the platform to identify binding determinants of site-specific histone Kme antibodies and surprisingly revealed that only a few amino acids drove epitope recognition. Collectively, these studies introduce and validate a rapid, unbiased, and high-throughput binding assay for Kme readers, and we envision its use as a resource for expanding the study of Kme-driven protein interactions.
Project description:Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.
Project description:Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insight into complex biological functions. Reader assemblies connected via flexible linkages facilitate avidity and increase affinity, however little is known of the contribution to the recognition process of multiple PTMs due to rigidity in the absence of conformational flexibility. We report here the high resolution crystal structure of the triple-reader module (PHD-Bromo-PWWP) of ZMYND8 which forms a stable unit capable of simultaneously recognizing multiple histone PTMs, while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish proof-of-principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states, thus identifying an important underappreciated role for rigid multivalent reader modules in nucleosome binding and chromatin function. Overall design: Investigation of the genome-wide occupancy of ZMYND8 using H3K27ac as a landmark of enhancer regions, as well as the putative histone mark H3K14ac recognized by ZMYND8
Project description:BACKGROUND:Plant homeodomain (PHD) fingers are central "readers" of histone post-translational modifications (PTMs) with >?100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete. RESULTS:We expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk. CONCLUSIONS:The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain-histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.
Project description:This review focuses on a structure-based analysis of histone posttranslational modification (PTM) readout, where the PTMs serve as docking sites for reader modules as part of larger complexes displaying chromatin modifier and remodeling activities, with the capacity to alter chromatin architecture and templated processes. Individual topics addressed include the diversity of reader-binding pocket architectures and common principles underlying readout of methyl-lysine and methyl-arginine marks, their unmodified counterparts, as well as acetyl-lysine and phosphoserine marks. The review also discusses the impact of multivalent readout of combinations of PTMs localized at specific genomic sites by linked binding modules on processes ranging from gene transcription to repair. Additional topics include cross talk between histone PTMs, histone mimics, epigenetic-based diseases, and drug-based therapeutic intervention. The review ends by highlighting new initiatives and advances, as well as future challenges, toward the promise of enhancing our structural and mechanistic understanding of the readout of histone PTMs at the nucleosomal level.
Project description:<h4>Background</h4>Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools.<h4>Results</h4>Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA.<h4>Conclusions</h4>Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms.