Differentially Isotope-Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time-Resolved NMR Spectroscopy.

Post-translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core histones, the establishment of different PTMs on individual "sister" histones in the same nucleosomal context, that is, asymmetric histone PTMs, are difficult to analyze. Here, we generated differentially isotope-labeled nucleosomes to study asymmetric histone modification crosstalk by time-resolved NMR spectroscopy. Specifically, we present mechanistic insights into nucleosomal histone H3 modification reactions in cis and in trans, that is, within individual H3 copies or between them. We validated our approach by using the H3S10phK14ac crosstalk mechanism, which is mediated by the Gcn5 acetyltransferase. Moreover, phosphorylation assays on methylated substrates showed that, under certain conditions, Haspin kinase is able to produce nucleosomes decorated asymmetrically with two distinct types of PTMs.

Methylation of lysine 9 in histone H3 directs alternative modes of highly dynamic interaction of heterochromatin protein hHP1ß with the nucleosome.

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1ß to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1ß with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1ß-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.

Analysis of phosphorylation-dependent protein-protein interactions of histone h3.

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails.

Quantitative assessment of protein interaction with methyl-lysine analogues by hybrid computational and experimental approaches.

In cases where binding ligands of proteins are not easily available, structural analogues are often used. For example, in the analysis of proteins recognizing different methyl-lysine residues in histones, methyl-lysine analogues based on methyl-amino-alkylated cysteine residues have been introduced. Whether these are close enough to justify quantitative interpretation of binding experiments is however questionable. To systematically address this issue, we developed, applied, and assessed a hybrid computational/experimental approach that extracts the binding free energy difference between the native ligand (methyl-lysine) and the analogue (methyl-amino-alkylated cysteine) from a thermodynamic cycle. Our results indicate that measured and calculated binding differences are in very good agreement and therefore allow the correction of measured affinities of the analogues. We suggest that quantitative binding parameters for defined ligands in general can be derived by this method with remarkable accuracy.

Phosphorylation of histone H3 Ser10 establishes a hierarchy for subsequent intramolecular modification events.

Phosphorylation of Ser10 of histone H3 regulates chromosome condensation and transcriptional activity. Using time-resolved, high-resolution NMR spectroscopy, we demonstrate that histone H3 Ser10 phosphorylation inhibits checkpoint kinase 1 (Chk1)- and protein kinase C (PKC)-mediated modification of Thr11 and Thr6, the respective primary substrate sites of these kinases. On unmodified H3, both enzymes also target Ser10 and thereby establish autoinhibitory feedback states on individual H3 tails. Whereas phosphorylated Ser10 does not affect acetylation of Lys14 by Gcn5, phosphorylated Thr11 impedes acetylation. Our observations reveal mechanistic hierarchies of H3 phosphorylation and acetylation events and provide a framework for intramolecular modification cross-talk within the N terminus of histone H3.

Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells.

BACKGROUND: Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation. RESULTS: We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation. CONCLUSIONS: Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.