Histone Tails Research Articles

Non-canonical bivalent H3K4me3K9me3 recognition by Spindlin1/C11orf84 complex.

Bivalent chromatin with active H3K4me3 and repressive H3K27me3 was initially identified in embryonic stem cells (ESCs) to poise expression of developmental genes upon lineage commitment. Since then, many more different bivalent modifications have been demonstrated in both ESCs and fully differentiated cells. Bivalency not only spatiotemporally controls gene transcription but also acts to fine-tune the level of transcription during development. Although increasing number of studies demonstrated the functional significance of bivalent chromatin, the molecular connection of bivalent chromatin and transcriptional regulation remains largely elusive. Recently, we showed Spindlin1/C11orf84 complex prefers to recognize the non-canonical histone H3K4me3K9me3 bivalent mark, which is required for timely ribosomal RNA transcription. Here, we hypothesize the recognition of K4me3 and K9me3 at the same histone tail by Spindlin1/C11orf84 complex may serve as a general mechanism of conversion from a repressed to an active chromatin structure for transcriptional activation.

Structural basis for the recognition of methylated histone H3 by the Arabidopsis LHP1 chromodomain

Arabidopsis LHP1 (LIKE HETEROCHROMATIN PROTEIN 1), a unique homolog of HP1 in Drosophila, plays important roles in plant development, growth, and architecture. In contrast to specific binding of the HP1 chromodomain to methylated H3K9 histone tails, the chromodomain of LHP1 has been shown to bind to both methylated H3K9 and H3K27 histone tails, and LHP1 carries out its function mainly via its interaction with these two epigenetic marks. However, the molecular mechanism for the recognition of methylated histone H3K9/27 by the LHP1 chromodomain is still unknown. In this study, we characterized the binding ability of LHP1 to histone H3K9 and H3K27 peptides and found that the chromodomain of LHP1 binds to histone H3K9me2/3 and H3K27me2/3 peptides with comparable affinities, although it exhibited no binding or weak binding to unmodified or monomethylated H3K9/K27 peptides. Our crystal structures of the LHP1 chromodomain in peptide-free and peptide-bound forms coupled with mutagenesis studies reveal that the chromodomain of LHP1 bears a slightly different chromodomain architecture and recognizes methylated H3K9 and H3K27 peptides via a hydrophobic clasp, similar to the chromodomains of human Polycomb proteins, which could not be explained only based on primary structure analysis. Our binding and structural studies of the LHP1 chromodomain illuminate a conserved ligand interaction mode between chromodomains of both animals and plants, and shed light on further functional study of the LHP1 protein.

Design, Synthesis, and Characterization of I-BET567, a Pan-Bromodomain and Extra Terminal (BET) Bromodomain Oral Candidate

Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.

Functional characterization of the tomato HAIRPLUS gene reveals the implication of the epigenome in the control of glandular trichome formation.

Trichomes are specialised epidermal cells developed in the aerial surface of almost every terrestrial plant. These structures form physical barriers, which combined with their capability of synthesis of complex molecules, prevent plagues from spreading and confer trichomes a key role in the defence against herbivores. In this work, the tomato gene HAIRPLUS (HAP) that controls glandular trichome density in tomato plants was characterised. HAP belongs to a group of proteins involved in histone tail modifications although some also bind methylated DNA. HAP loss of function promotes epigenomic modifications in the tomato genome reflected in numerous differentially methylated cytosines and causes transcriptomic changes in hap mutant plants. Taken together, these findings demonstrate that HAP links epigenome remodelling with multicellular glandular trichome development and reveal that HAP is a valuable genomic tool for pest resistance in tomato breeding.

Targeting the epigenome to treat neurodegenerative diseases or delay their onset: a perspective

Targeting the epigenome to treat neurodegenerative diseases or delay their onset: a perspective

Эпигенетические основы программирования хронического воспаления: роль питания и микробиома

The origins of major non-communicable diseases are programmed early in development, in the first 1,000 days, from conception to the end of the 2nd year of life. The mechanisms of this programming are associated with epigenetic modifications of genes that determine human metabolism and phenotype, disease predisposition, resistance to stress and adverse environmental factors. Such mechanisms include DNA methylation, non-coding RNA activity, and modifications of histone tails. All three mechanisms affect genome organization, gene expression, and nuclear functions. This review reveals the main known mechanisms of epigenetic programming. Eating disorders in pregnant women (under- or overnutrition), the presence of chronic diseases (obesity, diabetes, etc.) are risk factors for disease development in offspring. The perinatal period includes the formation of the gastrointestinal tract, intestinal integrity, microbiome, and intestinal immune system. Epigenetic processes play an important role in the development of chronic inflammatory bowel diseases, which implement the effect of environmental factors, influence gene expression, and change the inflammatory phenotype in the presence of genetic predisposition. Key words: epigenetics, mechanisms, nutrition, microbiome, inflammation, inflammatory bowel diseases

New "Omics" Approaches as Tools to Explore Mechanistic Nanotoxicology.

In the last years, "omics" approaches have been applied to study the toxicity of nanomaterials (NM) with the aim of obtaining insightful information on their biological effects. One of the most developed "omics" field, transcriptomics, expects to find unique profiles of differentially-expressed genes after exposure to NM that, besides providing evidence of their mechanistic mode of action, may also be used as biomarkers for biomonitoring purposes. Moreover, several NM have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by differential DNA methylation, histone tail modification and microRNA expression. Epigenomics research focusing on DNA methylation is increasingly common and the role of microRNAs is being better understood, either promoting or suppressing biological pathways. Moreover, the proteome is a highly dynamic system that changes constantly in response to a stimulus. Therefore, proteomics can identify changes in protein abundance and/or variability that lead to a better understanding of the underlying mechanisms of action of NM while discovering biomarkers. As to genomics, it is still not well developed in nanotoxicology. Nevertheless, the individual susceptibility to NM mediated by constitutive or acquired genomic variants represents an important component in understanding the variations in the biological response to NM exposure and, consequently, a key factor to evaluate possible adverse effects in exposed individuals. By elucidating the molecular changes that are involved NM toxicity, the new "omics" studies are expected to contribute to exclude or reduce the handling of hazardous NM in the workplace and support the implementation of regulation to protect human health.

Multiple interfaces to recognize nucleosomal targets.

In eukaryotic cells, DNA is tightly compacted as chromatin. Chromatin states must be dynamically changed to increase the accessibility of transcription factors (TFs) to chromatin or to stably silence genes by higher-order chromatin structures known as heterochromatin. The regulation of chromatin needs cooperative action performed by a variety of proteins. Specific binding of TFs to target DNA is the initial step of chromatin regulation and promotes changes in the post-translational modifications of histone tails, which themselves are recognized by a set of histone reader proteins. Recent biochemical studies have revealed that some TFs that recognize specific DNA sequences can also interact with histones. Furthermore, histone reader proteins that recognize specific histone tail modifications have been shown to have the ability to directly bind to DNA. In this commentary, we introduce recent advances in the elucidation of how chromatin regulating factors recognize nucleosomal targets.

Epigenetic modifications of tumor necrosis factor-alpha in joint cartilage tissue from osteoarthritis patients - CONSORT.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

The Cross Marks the Spot: The Emerging Role of JmjC Domain-Containing Proteins in Myeloid Malignancies.

Histone methylation tightly regulates chromatin accessibility, transcription, proliferation, and cell differentiation, and its perturbation contributes to oncogenic reprogramming of cells. In particular, many myeloid malignancies show evidence of epigenetic dysregulation. Jumonji C (JmjC) domain-containing proteins comprise a large and diverse group of histone demethylases (KDMs), which remove methyl groups from lysines in histone tails and other proteins. Cumulating evidence suggests an emerging role for these demethylases in myeloid malignancies, rendering them attractive targets for drug interventions. In this review, we summarize the known functions of Jumonji C (JmjC) domain-containing proteins in myeloid malignancies. We highlight challenges in understanding the context-dependent mechanisms of these proteins and explore potential future pharmacological targeting.

Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation.

SUMMARYRegulatory T (Treg) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how Treg cells are mechanistically induced in vitro (iTreg) and stabilized via transcriptional regulation of Treg lineage-specifying factor Foxp3. We find that acetylation of histone tails at the Foxp3 promoter is required for inducing Foxp3 transcription. Upon induction, histone acetylation signals via bromodomain-containing proteins, particularly targets of inhibitor JQ1, and sustains Foxp3 transcription via a global or trans effect. Subsequently, Tet-mediated DNA demethylation of Foxp3 cis-regulatory elements, mainly enhancer CNS2, increases chromatin accessibility and protein binding, stabilizing Foxp3 transcription and obviating the need for the histone acetylation signal. These processes transform stochastic iTreg induction into a stable cell fate, with the former sensitive and the latter resistant to genetic and environmental perturbations. Thus, sequential histone acetylation and DNA demethylation in Foxp3 induction and maintenance reflect stepwise mechanical switches governing iTreg cell lineage specification.

Effect of histone H4 tail on nucleosome stability and internucleosomal interactions

Chromatin structure is dictated by nucleosome assembly and internucleosomal interactions. The tight wrapping of nucleosomes inhibits gene expression, but modifications to histone tails modulate chromatin structure, allowing for proper genetic function. The histone H4 tail is thought to play a large role in regulating chromatin structure. Here we investigated the structure of nucleosomes assembled with a tail-truncated H4 histone using Atomic Force Microscopy. We assembled tail-truncated H4 nucleosomes on DNA templates allowing for the assembly of mononucleosomes or dinucleosomes. Mononucleosomes assembled on nonspecific DNA led to decreased DNA wrapping efficiency. This effect is less pronounced for nucleosomes assembled on positioning motifs. Dinucleosome studies resulted in the discovery of two effects- truncation of the H4 tail does not diminish the preferential positioning observed in full-length nucleosomes, and internucleosomal interaction eliminates the DNA unwrapping effect. These findings provide insight on the role of histone H4 in chromatin structure and stability.

TAMI-43. HYPOXIC REGULATION OF GLIOBLASTOMA TUMOR GROWTH THROUGH L(3)MBTL1

Abstract Glioblastoma (GBM) is the deadliest and most common of all primary brain tumors. Drosophila brain tumor models have uncovered signaling pathways regulating tumor growth that are highly conserved in GBM. Our search for a novel tumor suppressor using Drosophila led to Lethal (3) malignant brain tumor [l(3)mbt], temperature-sensitive mutants of which cause neuroepithelial tumor-like overproliferation in optic lobes. dL(3)MBT and its human orthologs L3MBTL1-L3MBTL4 all harbor Malignant Brain Tumor (MBT) domains that recognize methylated lysines on histone tails. Like dL(3)MBT, hL(3)MBTL1 acts as a chromatin compaction factor that represses transcription and inhibits cytokinesis in GBM cell lines. The highly hypoxic tumor microenvironment (TME) in GBM drives its progression, recurrence, and therapeutic resistance. However, it remains unclear if L(3)MBTL1 is regulated by TME cues to promote GBM growth. Based on this knowledge gap and our preliminary data, we hypothesize that hypoxia directly regulates L(3)MBTL1 in favor of GBM growth. Analysis of TCGA data for IDH-wildtype gliomas revealed that L3MBTL1 gene expression is downregulated in GBM, which are necrotic and severely hypoxic, compared to histologic grade 2/3 gliomas, which do not contain necrosis, indicating that hypoxia could potentially suppress L3MBTL1 to enhance glioma progression. TCGA data also revealed a number of HIF pathway and hypoxia-inducible genes strongly correlating with L3MBTL1 expression, including HIF1a and VHL. Using patient-derived GBM neurosphere cultures, we exposed glioma cells to hypoxia (1% O2 for 24hrs) and found that L3MBTL1 protein levels were suppressed compared to normoxia (21%). Under these same conditions, we found more rapid cell proliferation under hypoxia. Exploration of hypoxic TME regulation of the novel tumor suppressor L3MBTL1 in glioma progression has the potential to uncover novel mechanisms involving epigenetic modulation and potentially new therapeutic strategies.

Conserved Structure and Evolution of DPF Domain of PHF10-The Specific Subunit of PBAF Chromatin Remodeling Complex.

Transcription activation factors and multisubunit coactivator complexes get recruited at specific chromatin sites via protein domains that recognize histone modifications. Single PHDs (plant homeodomains) interact with differentially modified H3 histone tails. Double PHD finger (DPF) domains possess a unique structure different from PHD and are found in six proteins: histone acetyltransferases MOZ and MORF; chromatin remodeling complex BAF (DPF1–3); and chromatin remodeling complex PBAF (PHF10). Among them, PHF10 stands out due to the DPF sequence, structure, and functions. PHF10 is ubiquitously expressed in developing and adult organisms as four isoforms differing in structure (the presence or absence of DPF) and transcription regulation functions. Despite the importance of the DPF domain of PHF10 for transcription activation, its structure remains undetermined. We performed homology modeling of the human PHF10 DPF domain and determined common and distinct features in structure and histone modifications recognition capabilities, which can affect PBAF complex chromatin recruitment. We also traced the evolution of DPF1–3 and PHF10 genes from unicellular to vertebrate organisms. The data reviewed suggest that the DPF domain of PHF10 plays an important role in SWI/SNF-dependent chromatin remodeling during transcription activation.

Epigenetic modification for horticultural plant improvement comes of age

Our era has witnessed tremendous technique advances and mechanically epigenetic improvement in plant epigenetics, mainly including histone post-translational modifications (PTMs) and DNA methylation, which have been characterized as playing vital roles in development processes and plant response to environmental factors. Recently, chemical modifications on RNAs like 5-methylcytosine (m5C) and N6-methyladenosine (m6A) have been revealed as a new layer of epigenetic marks to regulate gene translation efficiency in model plant Arabidopsis thaliana and with later discovery of horticultural species like tomato (Solanum lycopersicum) and poplar (Populus trichocarpa). In model plants, these epigenetic modifications on DNA, RNA, and histone tails largely trigger innumerable studies on how epigenetic mechanisms are involved in gene regulation and biological functions. As an emerging research field in horticultural plants, epigenetic modifications have bloomed in fruit development and ripening, grafting, and bud dormancy. In this Review, we have demonstrated recent advances of high-throughput sequencing methods, summarized epigenetic enzymatic systems to install, remove and recognize epigenetic marks, discussed essential roles of epigenetic regulation, and proposed how innovative computation techniques like machine learning and deep learning are set to understanding epigenetic regulation mechanisms in horticultural plants. We also raise future perspectives on how epigenetic modifications act as new additions for understanding their roles in gene expression that is required for development and environmental adaptation in horticultural plants.

Heterodimerization of H3K9 histone methyltransferases G9a and GLP activates methyl reading and writing capabilities

Unique among metazoan repressive histone methyltransferases, G9a and GLP, which chiefly target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) in vivo. Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form in vivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl binding (“reading”) and deposition (“writing”) activity of G9a and GLP and why heterodimerization is essential in vivo remains opaque. Here, we examine the H3K9me “reading” and “writing” activities of defined, recombinantly produced homo- and heterodimers of G9a and GLP. We find that both reading and writing are significantly enhanced in the heterodimer. Compared with the homodimers, the heterodimer has higher recognition of H3K9me2, and a striking ∼10-fold increased turnover rate for nucleosomal substrates under multiple turnover conditions, which is not evident on histone tail peptide substrates. Cross-linking Mass Spectrometry suggests that differences between the homodimers and the unique activity of the heterodimer may be encoded in altered ground state conformations, as each dimer displays different domain contacts. Our results indicate that heterodimerization may be required to relieve autoinhibition of H3K9me reading and chromatin methylation evident in G9a and GLP homodimers. Relieving this inhibition may be particularly important in early differentiation when large tracts of H3K9me2 are typically deposited by G9a-GLP, which may require a more active form of the enzyme.

Design, synthesis, and biological evaluation of indole-based hydroxamic acid derivatives as histone deacetylase inhibitors

The equilibrium between histone acetylation and deacetylation plays an important role in cancer initiation and progression. The histone deacetylases (HDACs) are a class of key regulators of gene expression that enzymatically remove an acetyl moiety from acetylated lysine ε-amino groups on histone tails. Therefore, HDAC inhibitors have recently emerged as a promising strategy for cancer therapy and several pan-HDAC inhibitors have globally been approved for clinical use. In the present study, we designed and synthesized a series of substituted indole-based hydroxamic acid derivatives that exhibited potent anti-proliferative activities in various tumor cell lines. Among the compounds tested, compound 4o, was found to be among the most potent in the inhibition of HDAC1 (half maximal inhibitory concentration, IC50 = 1.16 nM) and HDAC6 (IC50 = 2.30 nM). It also exhibited excellent in vitro anti-tumor proliferation activity. Additionally, compound 4o effectively increased the acetylation of histone H3 in a dose-dependent manner and inhibited cell proliferation by inducing cell cycle arrest and apoptosis. Moreover, compound 4o remarkably blocked colony formation in HCT116 cancer cells. Based on its favorable in vitro profile, compound 4o was further evaluated in an HCT116 xenograft mouse model, in which it demonstrated better in vivo efficacy than the clinically used HDAC inhibitor, suberanilohydroxamic acid. Interestingly, compound 4k was found to have a preference for the inhibition of HDAC6, with IC50 values of 115.20 and 5.29 nM against HDAC1 and HDAC6, respectively.

Dynamic changes of histone methylation in mammalian oocytes and early embryos.

Histone methylation is a key epigenetic mechanism and plays a major role in regulating gene expression during oocyte maturation and early embryogenesis. This mechanism can be briefly defined as the process by which methyl groups are transferred to lysine and arginine residues of histone tails extending from nucleosomes. While methylation of the lysine residues is catalyzed by histone lysine methyltransferases (KMTs), protein arginine methyltransferases (PRMTs) add methyl groups to the arginine residues. When necessary, the added methyl groups can be reversibly removed by histone demethylases (HDMs) by a process called histone demethylation. The spatiotemporal regulation of methylation and demethylation in histones contributes to modulating the expression of genes required for proper oocyte maturation and early embryonic development. In this review, we comprehensively evaluate and discuss the functional importance of dynamic histone methylation in mammalian oocytes and early embryos, regulated by KMTs, PRMTs, and HDMs.

Probing multiple enzymatic methylation events in real time with NMR spectroscopy

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

Targeting the Interplay between HDACs and DNA Damage Repair for Myeloma Therapy.

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells, and accounts for 10% of all hematologic malignancies and 1% of all cancers. MM is characterized by genomic instability which results from DNA damage with certain genomic rearrangements being prognostic factors for the disease and patients’ clinical response. Following genotoxic stress, the evolutionary conserved DNA damage response (DDR) is activated and, in turn, coordinates DNA repair with cell-cycle events. However, the process of carcinogenesis cannot be attributed only to the genetic alterations, but also involves epigenetic processes. Regulation of expression and activity of key players in DNA repair and checkpoint proteins are essential and mediated partly by posttranslational modifications (PTM), such as acetylation. Crosstalk between different PTMs is important for regulation of DNA repair pathways. Acetylation, which is mediated by acetyltransferases (HAT) and histone deacetylases (HDAC), not only affects gene expression through its modulation of histone tails but also has recently been implicated in regulating non-histone proteins. Currently, several HDAC inhibitors (HDACi) have been developed both in pre-clinical and clinical studies, with some of them exhibiting significant anti-MM activities. Due to reversibility of epigenetic changes during the evolutionary process of myeloma genesis, the potency of epigenetic therapies seems to be of great importance. The aim of the present paper is the summary of all data on the role of HDACi in DDR, the interference with each DNA repair mechanism and the therapeutic implications of HDACi in MM.