Refined read-out: The hUHRF1 Tandem-Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3.

UHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem-Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1-K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read-out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. Individual TTD mutants showed up to a 10,000-fold preference for the double-modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1-K9me2 regions in human chromatin demonstrated in our TTD chromatin pull-down and ChIP-western blot data suggests that it has specific biological roles. Chromatin pull-down of TTD from HepG2 cells and full-length murine UHRF1 ChIP-seq data correlate with H3K4me1 profiles indicating that the H3K4me1-K9me2/3 interaction of TTD influences chromatin binding of full-length UHRF1. We demonstrate the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of cell-type-specific genes at the flanks of cell-type-specific transcription factor binding sites, and provided evidence supporting an H3K4me1-K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1-TTD binding to H3K4me1-K9me2/3 double marks in a cellular context.

Application of modified histone peptide arrays in chromatin research.

Various post-translational modifications (PTMs) have been identified on histone proteins, which occur at hundreds of different sites. Histone PTMs influence the chromatin structure and serve as binding sites for reading domains, which further mediate downstream effects. Histone PTM antibodies or recombinant proteins derived from reading domains are unique research reagents essentially required to study histone modifications. To validate their specificity, histone PTM peptide arrays are used, because they allow to investigate the binding of proteins to a large number of different peptides in one experiment. Furthermore, histone PTM peptide arrays can be used to characterize reading domains and study the specificity of histone modifying enzymes. Here, we provide an overview of histone PTM peptide arrays, highlight some of their applications and compare different commercial histone PTM peptide arrays, viz. MODified Histone Peptide Array, AbSurance Pro Histone Peptide Microarrays, EpiTriton Histone Peptide Array and Histone Code Microarrays. These arrays contain histone peptides with several post-translational modifications in many different combinations, but they differ in peptide synthesis and immobilization methods, peptide and PTM coverage, and PTM combinatorial potential. In addition, some special applications of histone PTM peptide arrays like custom arrays or double peptide arrays are described.

Application of mixed peptide arrays to study combinatorial readout of chromatin modifications.

The N-terminal tails of histone proteins are massively decorated with post-translational modifications (PTMs), which play important roles in the regulation of gene expression. Several highly conserved chromatin interacting proteins can bind to histone modifications in a sequence and modification specific manner employing specific reading domains. These proteins often contain several reading domains, which can cooperate in the readout of different PTMs. To gain a better insight into the combinatorial readout of PTMs, we developed a method to study the binding of double reading domains to mixed peptide arrays containing two different peptides in each spot. For that, differently modified and unmodified peptides were prepared by SPOT synthesis and solubilized. Then, two peptides were mixed and spotted onto a glass slide creating peptide spots presenting two modifications on two different peptides. Different combinations of mixed spots containing modified and unmodified peptides were generated and incubated with recombinant double reading domains to study their synergistic binding. For validation of the method, we used the well-studied BPTF subunit of the NURF chromatin-remodeling complex. BPTF contains a plant homeodomain finger (PHD) and a Bromodomain recognizing H3K4me3 and H4K16ac, respectively. We first confirmed with peptide arrays and Fluorescence Anisotropy (FA) measurements that the BPTF PHD-Bromo (PB) domain interacts specifically with the expected modifications. Using our novel tool, we observed a strong and synergistic binding only to peptide spots containing both modifications, which was lost if one of the domains was inactivated by a mutation. These data indicate that BPTF-PB simultaneously interacts with both target modifications using its PHD and Bromodomain. In agreement with the synergistic peptide interaction on mixed peptide arrays, we also show that chromatin pulldown by BPTF-PB depends on the activity of both reading domains. We conclude that mixed peptide spot arrays are a powerful, cheap and novel method for screening the combinatorial interaction space of multidomain reading proteins. Using this approach hundreds of mixed peptide spots can be prepared and tested for binding in principle allowing for an unbiased medium throughput investigation.

Nucleosome stability measured in situ by automated quantitative imaging.

Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

Application of dual reading domains as novel reagents in chromatin biology reveals a new H3K9me3 and H3K36me2/3 bivalent chromatin state.

BACKGROUND: Histone post-translational modifications (PTMs) play central roles in chromatin-templated processes. Combinations of two or more histone PTMs form unique interfaces for readout and recruitment of chromatin interacting complexes, but the genome-wide mapping of coexisting histone PTMs remains an experimentally difficult task. RESULTS: We introduce here a novel type of affinity reagents consisting of two fused recombinant histone modification interacting domains (HiMIDs) for direct detection of doubly modified chromatin. To develop the method, we fused the MPP8 chromodomain and DNMT3A PWWP domain which have a binding specificity for H3K9me3 and H3K36me2/3, respectively. We validate the novel reagent biochemically and in ChIP applications and show its specific interaction with H3K9me3-H3K36me2/3 doubly modified chromatin. Modification specificity was confirmed using mutant double-HiMIDs with inactivated methyllysine binding pockets. Using this novel tool, we mapped coexisting H3K9me3-H3K36me2/3 marks in human cells by chromatin interacting domain precipitation (CIDOP). CIDOP-seq data were validated by qPCR, sequential CIDOP/ChIP and by comparison with CIDOP- and ChIP-seq data obtained with single modification readers and antibodies. The genome-wide distribution of H3K9me3-H3K36me2/3 indicates that it represents a novel bivalent chromatin state, which is enriched in weakly transcribed chromatin segments and at ZNF274 and SetDB1 binding sites. CONCLUSIONS: The application of double-HiMIDs allows the single-step study of co-occurrence and distribution of combinatorial chromatin marks. Our discovery of a novel H3K9me3-H3K36me2/3 bivalent chromatin state illustrates the power of this approach, and it will stimulate numerous follow-up studies on its biological functions.

Application of recombinant TAF3 PHD domain instead of anti-H3K4me3 antibody.

BACKGROUND: Histone posttranslational modifications (PTMs) represent a focal point of chromatin regulation. The genome-wide and locus-specific distribution and the presence of distinct histone PTMs is most commonly examined with the application of histone PTM-specific antibodies. In spite of their central role in chromatin research, polyclonal antibodies suffer from disadvantages like batch-to-batch variability and insufficient documentation of their quality and specificity. RESULTS: To mitigate some of the pitfalls of using polyclonal antibodies against H3K4me3, we successfully validated the application of a recombinant TAF3 PHD domain as anti-H3K4me3 affinity reagent in peptide array, western blot and ChIP-like experiments coupled with qPCR and deep sequencing. CONCLUSIONS: The successful addition of the TAF3 PHD domain to the growing catalog of recombinant affinity reagents for histone PTMs could help to improve the reproducibility, interpretation and cross-laboratory validation of chromatin data.

Affinity reagents for studying histone modifications & guidelines for their quality control.

Histone post-translational modifications (PTMs) have pivotal functions in many chromatin processes, which makes their detection and characterization an imperative in chromatin biology. The established approaches for histone PTM characterization are generally based on affinity reagents specific for modified histone tails such as antibodies and, most recently, recombinant reading domains. Hence, the proper performance of these reagents is a critical precondition for the validity of the generated experimental data. In this review, we evaluate and update the quality criteria for assessment of the binding specificity of histone PTM affinity reagents. In addition, we discuss in detail the advantages and pitfalls of using antibodies and recombinant reading domains in chromatin biology research. Reading domains provide key advantages, such as consistent quality and recombinant production, but the future will tell if this emerging technology keeps its promises.

Specificity Analysis of Histone Modification-Specific Antibodies or Reading Domains on Histone Peptide Arrays.

Histone posttranslational modifications (PTMs) have a crucial role in chromatin regulation and dynamics. They are specifically bound by so-called reading domains, which mediate the biological effects of histone PTMs. On a similar note, antibodies are invaluable reagents in chromatin biology for the detection, characterization, and mapping of histone PTMs. Despite these central roles in chromatin research and biology, the specificity of many antibodies and reading domains has been insufficiently characterized and documented. Here we describe in detail the application of the MODified™ Histone Peptide Array for the investigation of the binding specificity of histone binding antibodies or domains. The array contains 384 histone tail peptides carrying 59 posttranslational modifications in different combinations which can be used to study the primary binding specificity, but at the same time also allow to determine the combinatorial effect of secondary marks on antibody or reading domain binding.