A modified epigenetics toolbox to study histone modifications on the nucleosome core

In the eukaryotic cell nucleus, the DNA is packaged in a structure called chromatin. The fundamental building block of chromatin is the nucleosome, which is composed of DNA wrapped around an octamer of four distinct histone proteins. Post-translational modifications (PTMs) of histone proteins can affect chromatin structure and function and thereby play critical roles in regulating gene expression. Most histone PTMs are found in unstructured histone tails that protrude from the nucleosome core. As a consequence, (synthetic) peptide truncations of these tails provide convenient substrates for the analysis of histone binding proteins and modifying enzymes. Modifications located on residues that reside in the nucleosome core are more difficult to study because short peptides do not recapitulate this defined structured state well. Methylation of histone H3 on Lys79 (H3K79), mediated by the Dot1 enzyme, is an example of such a core PTM. This modification, which is highly conserved, is linked to human leukemia, and pharmacological modulation of Dot1 activity could be a strategy to treat leukemia. Here we review the available and emerging genetic, biochemical, and chemical methods that together are starting to reveal the function and regulation of this and other histone modifications on the nucleosome core.     

Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys¹⁸ in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg¹⁷ was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys¹⁸ does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.     

Acetylation-Specific Interference by Anti-Histone H3K9ac Intrabody Results in Precise Modulation of Gene Expression

Among Histone post-translational modifications (PTMs), lysine acetylation plays a pivotal role in the epigenetic regulation of gene expression, mediated by chromatin modifying enzymes. Due to their activity in physiology and pathology, several chemical compounds have been developed to inhibit the function of these proteins. However, the pleiotropy of these classes of proteins represents a weakness of epigenetic drugs. Ideally, a new generation of epigenetic drugs should target with molecular precision individual acetylated lysines on the target protein. We exploit a PTM-directed interference, based on an intrabody (scFv-58F) that selectively binds acetylated lysine 9 of histone H3 (H3K9ac), to test the hypothesis that targeting H3K9ac yields more specific effects than inhibiting the corresponding HAT enzyme that installs that PTM. In yeast scFv-58F modulates, gene expression in a more specific way, compared to two well-established HAT inhibitors. This PTM-specific interference modulated expression of genes involved in ribosome biogenesis and function. In mammalian cells, the scFv-58F induces exclusive changes in the H3K9ac-dependent expression of specific genes. These results suggest the H3K9ac-specific intrabody as the founder of a new class of molecules to directly target histone PTMs, inverting the paradigm from inhibiting the writer enzyme to acting on the PTM.     

Slowly on,Slowly off: Bisubstrate‐Analogue Conjugates of 5‐Iodotubercidin and Histone H3 Peptide Targeting Protein Kinase Haspin

The atypical protein kinase haspin is a key player in mitosis by catalysing the phosphorylation of Thr3 in histone H3, and thus ensuring the normal function of the chromosomal passenger complex. Here, we report the development of bisubstrate‐analogue inhibitors targeting haspin. The compounds were constructed by linking 5‐iodotubercidin to the N terminus of histone H3 peptide. The new conjugates show high affinity (sub‐nanomolar K_D) towards haspin as well as slow kinetics of association and dissociation (residence time of several hours). This reflects a unique binding mode and translated into improved selectivity. The latter was confirmed in a biochemical binding/displacement assay with a panel of ten protein kinases, in a thermal shift assay with off‐targets of 5‐iodotubercidin (adenosine kinase and the Cdc2‐like kinase family) and in assay with spiked HeLa cell lysate.     

The SUV4-20H Histone Methyltransferases in Health and Disease

The post-translational modification of histone tails is a dynamic process that provides chromatin with high plasticity. Histone modifications occur through the recruitment of nonhistone proteins to chromatin and have the potential to influence fundamental biological processes. Many recent studies have been directed at understanding the role of methylated lysine 20 of histone H4 (H4K20) in physiological and pathological processes. In this review, we will focus on the function and regulation of the histone methyltransferases SUV4-20H1 and SUV4-20H2, which catalyze the di- and tri-methylation of H4K20 at H4K20me2 and H4K20me3, respectively. We will highlight recent studies that have elucidated the functions of these enzymes in various biological processes, including DNA repair, cell cycle regulation, and DNA replication. We will also provide an overview of the pathological conditions associated with H4K20me2/3 misregulation as a result of mutations or the aberrant expression of SUV4-20H1 or SUV4-20H2. Finally, we will critically analyze the data supporting these functions and outline questions for future research.     

Traceless Semisynthesis of a Set of Histone 3 Species Bearing Specific Lysine Methylation Marks

Considerable mechanistic insight into the function of histone post‐translational modifications and the enzymes that install and remove them derives from in vitro experiments with modified histones, often embedded in nucleosomes. We report the first semisyntheses of native‐like histone 3 (H3) bearing tri‐ and dimethyllysines at position 79 and trimethyllysine at position 36, as well as more facile and traceless semisyntheses of K9 and K27 trimethylated species. These semisyntheses are practical on a multi‐milligram scale and can also generate H3 with combinations of marks. Each of these modifications has distinct functional consequences, although the pathways by which H3K36me3 and H3K79me2/3 act have not been entirely mapped. To this end, we demonstrated that our semisynthetic histones, when reconstituted into nucleosomes, are valuable affinity reagents for unbiased binding partner discovery and compare them to their methyllysine analogue (MLA) counterparts at the nucleosome level.     

TET3- and OGT-Dependent Expression of Genes Involved in Epithelial-Mesenchymal Transition in Endometrial Cancer

TET3 is a member of the TET (ten-eleven translocation) proteins family that catalyzes the conversion of the 5-methylcytosine into 5-hydroxymethylcytosine. TET proteins can also affect chromatin modifications and gene expression independently of their enzymatic activity via interactions with other proteins. O-GlcNAc transferase (OGT), the enzyme responsible for modification of proteins via binding of N-acetylglucosamine residues, is one of the proteins whose action may be dependent on TET3. Here, we demonstrated that in endometrial cancer cells both TET3 and OGT affected the expression of genes involved in epithelial to mesenchymal transition (EMT), i.e., FOXC1, TWIST1, and ZEB1. OGT overexpression was caused by an increase in TWIST1 and ZEB1 levels in HEC-1A and Ishikawa cells, which was associated with increased O-GlcNAcylation of histone H2B and trimethylation of H3K4. The TET3 had the opposite effect on gene expressions and histone modifications. OGT and TET3 differently affected FOXC1 expression and the migratory potential of HEC-1A and Ishikawa cells. Analysis of gene expressions in cancer tissue samples from endometrial cancer patients confirmed the association between OGT or TET3 and EMT genes. Our results contribute to the knowledge of the role of the TET3/OGT relationship in the complex mechanism supporting endometrial cancer progression.     

Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.     

The Histone Chaperone HIRA Is a Positive Regulator of Seed Germination

Histone chaperones regulate the flow and dynamics of histone variants and ensure their assembly into nucleosomal structures, thereby contributing to the repertoire of histone variants in specialized cells or tissues. To date, not much is known on the distribution of histone variants and their modifications in the dry seed embryo. Here, we bring evidence that genes encoding the replacement histone variant H3.3 are expressed in Arabidopsis dry seeds and that embryo chromatin is characterized by a low H3.1/H3.3 ratio. Loss of HISTONE REGULATOR A (HIRA), a histone chaperone responsible for H3.3 deposition, reduces cellular H3 levels and increases chromatin accessibility in dry seeds. These molecular differences are accompanied by increased seed dormancy in hira-1 mutant seeds. The loss of HIRA negatively affects seed germination even in the absence of HISTONE MONOUBIQUITINATION 1 or TRANSCRIPTION ELONGATION FACTOR II S, known to be required for seed dormancy. Finally, hira-1 mutant seeds show lower germination efficiency when aged under controlled deterioration conditions or when facing unfavorable environmental conditions such as high salinity. Altogether, our results reveal a dependency of dry seed chromatin organization on the replication-independent histone deposition pathway and show that HIRA contributes to modulating seed dormancy and vigor.     

A Transfer-Learning-Based Deep Convolutional Neural Network for Predicting Leukemia-Related Phosphorylation Sites from Protein Primary Sequences

As one of the most important post-translational modifications (PTMs), phosphorylation refers to the binding of a phosphate group with amino acid residues like Ser (S), Thr (T) and Tyr (Y) thus resulting in diverse functions at the molecular level. Abnormal phosphorylation has been proved to be closely related with human diseases. To our knowledge, no research has been reported describing specific disease-associated phosphorylation sites prediction which is of great significance for comprehensive understanding of disease mechanism. In this work, focusing on three types of leukemia, we aim to develop a reliable leukemia-related phosphorylation site prediction models by combing deep convolutional neural network (CNN) with transfer-learning. CNN could automatically discover complex representations of phosphorylation patterns from the raw sequences, and hence it provides a powerful tool for improvement of leukemia-related phosphorylation site prediction. With the largest dataset of myelogenous leukemia, the optimal models for S/T/Y phosphorylation sites give the AUC values of 0.8784, 0.8328 and 0.7716 respectively. When transferred learning on the small size datasets, the models for T-cell and lymphoid leukemia also give the promising performance by common sharing the optimal parameters. Compared with other five machine-learning methods, our CNN models reveal the superior performance. Finally, the leukemia-related pathogenesis analysis and distribution analysis on phosphorylated proteins along with K-means clustering analysis and position-specific conversation profiles on the phosphorylation site all indicate the strong practical feasibility of our easy-to-use CNN models.