The epigenome is an additional layer of chemical information that acts on top of the genome and informs gene expression. These combinatorial posttranslational modifications (PTMs) could give rise to a histone ‘code’ or ‘language’, which is interpreted by enzyme complexes to mediate transcriptional responses. New information suggests that ‘reader’ domains, which are protein:protein interaction modules, act within enzyme complexes to function as molecular interpreters of this combinatorial PTM code. Mounting genetic evidence has linked mutations in chromatin ‘reader’ domains to human disease, and there are recent reports that ‘reader’ proteins can be targeted for drug development.
Recent work from our lab suggests that tandem reader domains can engage two histone tails, resulting in increased binding affinity and specificity. We propose that such reader domain organization is a critical feature of chromatin proteins that permits facile recognition of specific PTM states within nucleosomes. Here, we will address the question: What are the basic biochemical principles that govern epigenetic information written onto histones? We will employ a number of innovative biochemical approaches to investigate the existence of a functional histone code and how this epigenetic language is read to control gene expression.
Current research highlights:
1.) To elucidate the binding mechanisms of tandem-domain chromatin readers.
Previously, we developed combinatorial histone peptide libraries (N-terminal of H3 and H4) using on-bead SPOT technology for studying reader domain binding specificities. Such bindings were found to be multi-site, meaning that PTM state on multiple residues within the same stretch of histone sequence rather than single residue governs reader recognition. More recently, we developed spatially-addressed combinatorial peptide arrays to determine the specificity and binding mechanisms of monovalent and multivalent reader proteins. These histone arrays contain sequences (>800 peptides) from all human histones, their genetic variants, and ~600 peptides with PTMs either as single modifications or in combinatorial fashion. These histone-on-a-slide arrays will be used to reveal the binding specificity and mechanisms of recognition for multivalent readers.
2.) To define the PTM state of nucleosomes recognized by multivalent chromatin reader proteins.
Histone antibodies are important affinity reagents in chromatin biology research, however, more and more evidence suggests batch-to-batch variations and specificity issues with antibodies. We recently developed a novel antibody-free affinity capture technique (Matrix-Assisted Reader Chromatin Capture or MARCC) by utilizing PTM-specific reader domains and HaloTag technology. Co-occuring mononucleosomal PTM patterns and DNA fragments can be enriched and quantified by this reader-based platform. By combining chromatin reader proteins, quantitative proteomics and next-generation sequencing, this approach will allow us to define the combinatorial PTM code associated with unique genomic loci. Further development to expand this platform is ongoing.
3.) To determine how chromatin enzymes utilize multivalent protein modules to read the PTM code written in nucleosomes.
A lot of chromatin-modifying enzymes have one or more reader domains within the enzyme protein or enzyme complex. Earlier work in the lab focuses on characterization of enzyme kinetics of several histone lysine acetyltransfarases (HATs). Using several histone lysine methyltransferases, demethylases and acetyltransferases as our model system and combining cell biology with biochemical approaches, we can now test the hypothesis of chromatin-modifying enzymes (acetyltransferases, methyltransferases, demethylases, helicases, etc.) using their tandem reader domains to target specific PTM states of chromatin. This information with high-throughput screening platforms we developed will also be used to find specific inhibitors to disrupt reader binding and downstream disease-related enzyme function.
4.) To discover cancer-related histone PTM markers.
Utilizing the histone peptide microarray technology, we are also interested in identifying biologically significant histone PTMs in a hope to develop it into a diagnostic biomarker as well as a prognostic tool for cancer state.
Review papers to get started:
- Dynamic interplay between histone H3 modifications and protein interpreters emerging evidence for a histone language. – Oliver, Denu – 2011
- Reading the combinatorial histone language. – Su, Denu -2016
Research papers to get started:
- Garske AL, Oliver SS, Wagner EK, Musselman CA, LeRoy G, Garcia BA, Kutateladze TG, Denu JM: Combinatorial profiling of chromatin binding modules reveals multisite discrimination. Nature chemical biology 2010, 6(4):283-290.
In this paper, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail.
- Oliver SS, Musselman CA, Srinivasan R, Svaren JP, Kutateladze TG, Denu JM: Multivalent Recognition of Histone Tails by the PHD Fingers of CHD5. Biochemistry 2012, 51(33):6534-6544.
In this study, we demonstrated the ability of the CHD5 PHD fingers to specifically recognize the unmodified N-terminus of histone H3. We use two distinct modified peptide-library platforms (beads and glass slides) to determine the detailed histone binding preferences of PHD1 and PHD2 alone and the tandem PHD1−2 construct. Using NMR, surface plasmon resonance, and two novel biochemical assays, we demonstrate that CHD5−PHD1−2 simultaneously engages two H3 N-termini and results in a 4−11-fold increase in affinity compared with either PHD finger alone. These studies provide biochemical evidence for the utility of tandem PHD fingers to recruit protein complexes at targeted genomic loci and provide the framework for understanding how multiple chromatin- binding modules function to interpret the combinatorial PTM capacity written in chromatin.
- Wagner EK, Nath N, Flemming R, Feltenberger JB, Denu JM: Identification and characterization of small molecule inhibitors of a plant homeodomain finger. Biochemistry 2012, 51(41):8293-8306.
In this study, we developed a novel small molecule screening strategy that utilizes HaloTag technology to identify several small molecules that disrupt binding of the JARID1A PHD finger to histone peptides. Small molecule inhibitors were validated biochemically through affinity pull downs, fluorescence polarization, and histone reader specificity studies. One compound was modified through medicinal chemistry to improve its potency while retaining histone reader selectivity.
- Su Z, Boersma MD, Lee J, Oliver SS, Liu S, Garcia BA, Denu JM: ChIP-less analysis of chromatin states. Epigenetics & Chromatin 2014, 7:7.
Here we demonstrated 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 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.