Histone Post-translational Modifications Research Articles

Emerging Epigenetic-Based Nanotechnology for Cancer Therapy: Modulating the Tumor Microenvironment.

Dysregulated epigenetic modifications dynamically drive the abnormal transcription process to affect the tumor microenvironment; thus, promoting cancer progression, drug resistance, and metastasis. Nowadays, therapies targeting epigenetic dysregulation of tumor cells and immune cells in the tumor microenvironment appear to be promising adjuncts to other cancer therapies. However, the clinical results of combination therapies containing epigenetic agents are disappointing due to systemic toxicities and limited curative effects. Here, the role of epigenetic processes, including DNA methylation, post-translational modification of histones, and noncoding RNAs is discussed, followed by detailed descriptions of epigenetic regulation of the tumor microenvironment, as well as the application of epigenetic modulators in antitumor therapy, with an emphasis on the epigenetic-based advanced drug delivery system in targeting the tumor microenvironment.

Point and counterpoint: Polatuzumab vedotin in the front-line therapy for diffuse large B- cell lymphoma.

Hepatocellular carcinoma (HCC) constitutes most primary liver cancers and is one of the most lethal and life-threatening malignancies globally. Unfortunately, a substantial proportion of HCC patients are identified at an advanced stage that is unavailable for curative surgery. Thus, palliative therapies represented by multi-tyrosine kinase inhibitors (TKIs) sorafenib remained the front-line treatment over the past decades. Recently, the application of immune checkpoint inhibitors (ICIs), especially targeting the PD-1/PD-L1/ CTLA-4 axis, has achieved an inspiring clinical breakthrough for treating unresectable solid tumors. However, many HCC patients with poor responses lead to limited benefits in clinical applications, which has quickly drawn researchers’ attention to the regulatory mechanisms of immune checkpoints in HCC immune evasion. Evasion of immune surveillance by cancer is attributed to intricate reprogramming modulation in the tumor microenvironment. Currently, more and more studies have found that epigenetic modifications, such as chromatin structure remodeling, DNA methylation, histone post-translational modifications, and non-coding RNA levels, may contribute significantly to remodeling the tumor microenvironment to avoid immune clearance, affecting the efficacy of immunotherapy for HCC. This review summarizes the rapidly emerging progress of epigenetic-related changes during HCC resistance to ICIs and discusses the mechanisms of underlying epigenetic therapies available for surmounting immune resistance. Finally, we summarize the clinical advances in combining epigenetic therapies with immunotherapy, aiming to promote the formation of immune combination therapy strategies.

Epigenetic modification-related mechanisms of hepatocellular carcinoma resistance to immune checkpoint inhibition.

Hepatocellular carcinoma (HCC) constitutes most primary liver cancers and is one of the most lethal and life-threatening malignancies globally. Unfortunately, a substantial proportion of HCC patients are identified at an advanced stage that is unavailable for curative surgery. Thus, palliative therapies represented by multi-tyrosine kinase inhibitors (TKIs) sorafenib remained the front-line treatment over the past decades. Recently, the application of immune checkpoint inhibitors (ICIs), especially targeting the PD-1/PD-L1/CTLA-4 axis, has achieved an inspiring clinical breakthrough for treating unresectable solid tumors. However, many HCC patients with poor responses lead to limited benefits in clinical applications, which has quickly drawn researchers' attention to the regulatory mechanisms of immune checkpoints in HCC immune evasion. Evasion of immune surveillance by cancer is attributed to intricate reprogramming modulation in the tumor microenvironment. Currently, more and more studies have found that epigenetic modifications, such as chromatin structure remodeling, DNA methylation, histone post-translational modifications, and non-coding RNA levels, may contribute significantly to remodeling the tumor microenvironment to avoid immune clearance, affecting the efficacy of immunotherapy for HCC. This review summarizes the rapidly emerging progress of epigenetic-related changes during HCC resistance to ICIs and discusses the mechanisms of underlying epigenetic therapies available for surmounting immune resistance. Finally, we summarize the clinical advances in combining epigenetic therapies with immunotherapy, aiming to promote the formation of immune combination therapy strategies.

Effects of the omega-3 fatty acid DHA on histone and p53 acetylation in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) often develops resistance and/or relapses in response to immunochemotherapy. Epigenetic modifiers are frequently mutated in DLBCL, i.e., the lysine (histone) acetyltransferases CREBBP and EP300. Mutations in CBP/p300 can prevent the proper acetylation and activation of (i) enhancer sequences of genes required for essential functions (e.g., germinal center exit and differentiation) and (ii) the tumor suppressor p53. Based on evidence that omega-3 fatty acids (ω-3 FAs) affect histone acetylation in various cancers, we investigated whether ω-3 FA docosahexaenoic acid (DHA) could modify levels of histone and p53 acetylation in three DLBCL cell lines (at different CREBBP/EP300 mutational status) versus normal B cells. Exposure to DHA at clinically attainable doses was shown to significantly alter the genome-wide levels of histone posttranslational modifications in a cell-line-dependent and dose-dependent manner. Although histone acetylation did not increase uniformly, as initially expected, levels of p53 acetylation increased consistently. Quantitative reverse transcription polymerase chain reaction results revealed significant changes in expression of multiple genes, including increased expression of CREBBP and of PRDM1 (required for differentiation into plasma cells or memory B cells). Taken together, our results provide (to our knowledge) the first characterization of the epigenetic effects of ω-3 FAs in DLBCL.

A clustering of heterozygous missense variants in the crucial chromatin modifier WDR5 defines a new neurodevelopmental disorder.

WDR5 is a broadly studied, highly conserved key protein involved in a wide array of biological functions. Among these functions, WDR5 is a part of several protein complexes that affect gene regulation via post-translational modification of histones. We collected data from 11 unrelated individuals with six different rare de novo germline missense variants in WDR5; one identical variant was found in five individuals and another variant in two individuals. All individuals had neurodevelopmental disorders including speech/language delays (n= 11), intellectual disability (n= 9), epilepsy (n= 7), and autism spectrum disorder (n= 4). Additional phenotypic features included abnormal growth parameters (n= 7), heart anomalies (n= 2), and hearing loss (n= 2). Three-dimensional protein structures indicate that all the residues affected by these variants are located at the surface of one side of the WDR5 protein. It is predicted that five out of the six amino acid substitutions disrupt interactions of WDR5 with RbBP5 and/or KMT2A/C, as part of the COMPASS (complex proteins associated with Set1) family complexes. Our experimental approaches in Drosophila melanogaster and human cell lines show normal protein expression, localization, and protein-protein interactions for all tested variants. These results, together with the clustering of variants in a specific region of WDR5 and the absence of truncating variants so far, suggest that dominant-negative or gain-of-function mechanisms might be at play. All in all, we define a neurodevelopmental disorder associated with missense variants in WDR5 and a broad range of features. This finding highlights the important role of genes encoding COMPASS family proteins in neurodevelopmental disorders.

Mechanistic aspects of reversible methylation modifications of arginine and lysine of nuclear histones and their roles in human colon cancer.

Developmental proceedings and maintenance of cellular homeostasis are regulated by the precise orchestration of a series of epigenetic events that eventually control gene expression. DNA methylation and post-translational modifications (PTMs) of histones are well-characterized epigenetic events responsible for fine-tuning gene expression. PTMs of histones bear molecular logic of gene expression at chromosomal territory and have become a fascinating field of epigenetics. Nowadays, reversible methylation on histone arginine and lysine is gaining increasing attention as a significant PTM related to reorganizing local nucleosomal structure, chromatin dynamics, and transcriptional regulation. It is now well-accepted and reported that histone marks play crucial roles in colon cancer initiation and progression by encouraging abnormal epigenomic reprogramming. It is becoming increasingly clear that multiple PTM marks at the N-terminal tails of the core histones cross-talk with one another to intricately regulate DNA-templated biological processes such as replication, transcription, recombination, and damage repair in several malignancies, including colon cancer. These functional cross-talks provide an additional layer of message, which spatiotemporally fine-tunes the overall gene expression regulation. Nowadays, it is evident that several PTMs instigate colon cancer development. How colon cancer-specific PTM patterns or codes are generated and how they affect downstream molecular events are uncovered to some extent. Future studies would address more about epigenetic communication, and the relationship between histone modification marks to define cellular functions in depth. This chapter will comprehensively highlight the importance of histone arginine and lysine-based methylation modifications and their functional cross-talk with other histone marks from the perspective of colon cancer development.

Epigenetics Mechanisms of Honeybees: Secrets of Royal Jelly.

Early diets in honeybees have effects on epigenome with consequences on their phenotype. Depending on the early larval diet, either royal jelly (RJ) or royal worker, 2 different female castes are generated from identical genomes, a long-lived queen with fully developed ovaries and a short-lived functionally sterile worker. To generate these prominent physiological and morphological differences between queen and worker, honeybees utilize epigenetic mechanisms which are controlled by nutritional input. These mechanisms include DNA methylation and histone post-translational modifications, mainly histone acetylation. In honeybee larvae, DNA methylation and histone acetylation may be differentially altered by RJ. This diet has biologically active ingredients with inhibitory effects on the de novo methyltransferase DNMT3A or the histone deacetylase 3 HDAC3 to create and maintain the epigenetic state necessary for developing larvae to generate a queen. DNMT and HDAC enzymes work together to induce the formation of a compacted chromatin structure, repressing transcription. Such dialog could be coordinated by their association with other epigenetic factors including the ubiquitin-like containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1 (UHRF1). Through its multiple functional domains, UHRF1 acts as an epigenetic reader of both DNA methylation patterns and histone marks. The present review discusses the epigenetic regulation of honeybee's chromatin and how the early diets in honeybees can affect the DNA/histone modifying types of machinery that are necessary to stimulate the larvae to turn into either queen or worker. The review also looks at future directions in epigenetics mechanisms of honeybees, mainly the potential role of UHRF1 in these mechanisms.

CUT&Tag for Mapping In Vivo Protein-DNA Interactions in Plants.

Histone post-translational modifications and transcription factors (TFs) play vital roles in regulating gene expression. A comprehensive understanding of transcriptional regulation requires genome-wide mapping of chromatin features such as histone modifications and TF binding sites. Here, we describe a detailed nucleus CUT&Tag (Cleavage Under Targets and Tagmentation) protocol, which is an antibody-guided in situ protein-DNA interaction mapping method using protein A/G fused Tn5 transposase. Compared with regular ChIP-seq in plants, nucleus CUT&Tag (nCUT&Tag) omits many steps such as sonication and immunoprecipitation, thus saving much time and making it possible to efficiently profile chromatin features from low-input and even single cells with higher signal-to-noise ratio.

Quantitative Comparison of Multiple Chromatin Immunoprecipitation-Sequencing (ChIP-seq) Experiments with spikChIP.

The chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) is a powerful technique that enables to characterize the genomic distribution of chromatin-associated proteins, histone posttranslational modifications, and histone variants. However, in the absence of a reference control for monitoring experimental and biological variations, the standard ChIP-seq scheme is unable to accurately assess changes in the abundance of chromatin targets across different experimental samples. To overcome this limitation, the combination of external spike-in material with the experimental chromatin is offered as an effective solution for quantitative comparison of ChIP-seq data across different conditions. Here, we detail (i) the experimental protocol for preparing quality control spike-in chromatin from Drosophila melanogaster cells and (ii) the computational protocol to compare ChIP-seq samples with spike-in based on the use of the spikChIP software.

Chromatin regulators in DNA replication and genome stability maintenance during S-phase.

The duplication of genetic information is central to life. The replication of genetic information is strictly controlled to ensure that each piece of genomic DNA is copied only once during a cell cycle. Factors that slow or stop replication forks cause replication stress. Replication stress is a major source of genome instability in cancer cells. Multiple control mechanisms facilitate the unimpeded fork progression, prevent fork collapse and coordinate fork repair. Chromatin alterations, caused by histone post-translational modifications and chromatin remodeling, have critical roles in normal replication and in avoiding replication stress and its consequences. This text reviews the chromatin regulators that ensure DNA replication and the proper response to replication stress. We also briefly touch on exploiting replication stress in therapeutic strategies. As chromatin regulators are frequently mutated in cancer, manipulating their activity could provide many possibilities for personalized treatment.

Epigenetic and epitranscriptomic mechanisms of chromium carcinogenesis.

Hexavalent chromium [Cr(VI)], a Group I carcinogen classified by the International Agency for Research on Cancer (IARC), represents one of the most common occupational and environmental pollutants. The findings from human epidemiological and laboratory animal studies show that long-term exposure to Cr(VI) causes lung cancer and other cancer. Although Cr(VI) is a well-recognized carcinogen, the mechanism of Cr(VI) carcinogenesis has not been well understood. Due to the fact that Cr(VI) undergoes a series of metabolic reductions once entering cells to generate reactive Cr metabolites and reactive oxygen species (ROS) causing genotoxicity, Cr(VI) is generally considered as a genotoxic carcinogen. However, more and more studies have demonstrated that acute or chronic Cr(VI) exposure also causes epigenetic dysregulations including changing DNA methylation, histone posttranslational modifications and regulatory non-coding RNA (microRNA and long non-coding RNA) expressions. Moreover, emerging evidence shows that Cr(VI) exposure is also capable of altering cellular epitranscriptome. Given the increasingly recognized importance of epigenetic and epitranscriptomic dysregulations in cancer initiation and progression, it is believed that Cr(VI) exposure-caused epigenetic and epitranscriptomic changes could play important roles in Cr(VI) carcinogenesis. The goal of this chapter is to review the epigenetic and epitranscriptomic effects of Cr(VI) exposure and discuss their roles in Cr(VI) carcinogenesis. Better understanding the mechanism of Cr(VI) carcinogenesis may identify new molecular targets for more efficient prevention and treatment of cancer resulting from Cr(VI) exposure.

Mass Spectrometry-Based Analysis of Histone Posttranslational Modifications from Laser Microdissected Samples.

The analysis of histone posttranslational modifications (PTMs) in clinical samples has gained considerable interest due to the increasing knowledge about the implication of epigenetics in a multitude of physiological and pathological processes. Mass spectrometry (MS) has emerged as the most accurate and versatile tool to detect and quantify histone PTMs and has also been applied to clinical specimens, thanks to protocols developed during the past years. However, the requirement for relatively large amounts of material has so far impaired the application of these approaches to samples available in limited amounts. To address this issue, we have recently streamlined the protein extraction procedure from low-amount clinical samples and optimized the digestion step, obtaining a protocol suitable for the analysis of the most common histone PTMs from laser microdissected tissue areas containing down to 1000 cells, which we will describe in this chapter.

Targeting epigenetic regulation for cancer therapy using small molecule inhibitors.

Cancer cells display pervasive changes in DNA methylation, disrupted patterns of histone posttranslational modification, chromatin composition or organization and regulatory element activities that alter normal programs of gene expression. It is becoming increasingly clear that disturbances in the epigenome are hallmarks of cancer, which are targetable and represent attractive starting points for drug creation. Remarkable progress has been made in the past decades in discovering and developing epigenetic-based small molecule inhibitors. Recently, epigenetic-targeted agents in hematologic malignancies and solid tumors have been identified and these agents are either in current clinical trials or approved for treatment. However, epigenetic drug applications face many challenges, including low selectivity, poor bioavailability, instability and acquired drug resistance. New multidisciplinary approaches are being designed to overcome these limitations, e.g., applications of machine learning, drug repurposing, high throughput virtual screening technologies, to identify selective compounds with improved stability and better bioavailability. We provide an overview of the key proteins that mediate epigenetic regulation that encompass histone and DNA modifications and discuss effector proteins that affect the organization of chromatin structure and function as well as presently available inhibitors as potential drugs. Current anticancer small-molecule inhibitors targeting epigenetic modified enzymes that have been approved by therapeutic regulatory authorities across the world are highlighted. Many of these are in different stages of clinical evaluation. We also assess emerging strategies for combinatorial approaches of epigenetic drugs with immunotherapy, standard chemotherapy or other classes of agents and advances in the design of novel epigenetic therapies.

Biological functions and therapeutic potential of acylation by histone acetyltransferases

Histone lysine acylation is a major class of histone post-translational modifications involved in essential biological activities, such as transcriptional regulation, DNA-damage repair, and cell-cycle progression. Abnormal acylation is strongly associated with various diseases, such as cancer. Thus, histone acetyltransferases (HATs), the “writers” that catalyze histone acylation, are promising targets for cancer treatment. Rapid developments in structural biology and artificial intelligence have facilitated the development of drugs targeting HATs. To provide new ideas for exploring novel HAT modifiers with high efficiency and selectivity, this article reviews the relationships between acylation and diseases, illustrates HAT catalytic mechanisms through structural biology, and summarizes research progress in HAT modifiers.

Impaired RNA Binding Does Not Prevent Histone Modification Changes in a FUS ALS/FTD Yeast Model.

Mutations in the RNA-binding protein FUS are linked to amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). FUS mutants mislocalize and aggregate in dying neurons. We previously established that FUS proteinopathy is linked to changes in the histone modification landscape in a yeast ALS/FTD model. Here, we examine whether FUS' RNA binding is necessary for this connection. We find that overexpression of a FUS mutant unable to bind RNA is still associated with reduced levels of H3S10ph, H3K14ac and H3K56ac. Hence, FUS' ability to bind RNA is not required in the mechanism connecting FUS proteinopathy to altered histone post-translational modifications.

Evolution of Epigenetic Mechanisms and Signatures

DNA methylation, histone posttranslational modifications, higher-order chromatin organization and regulation by noncoding RNAs are considered as the basic mechanisms underlying the epigenetic memory [...].

Discovery of DS-9300: A Highly Potent, Selective, and Once-Daily Oral EP300/CBP Histone Acetyltransferase Inhibitor

Histone acetylation is a post-translational modification of histones that is catalyzed by histone acetyltransferases (HATs) and plays an essential role in cellular processes. The HAT domain of EP300/CBP has recently emerged as a potential drug target for cancer therapy. Here, we describe the identification of the novel, highly potent, and selective EP300/CBP HAT inhibitor DS-9300. Our optimization efforts using a structure-based drug design approach based on the cocrystal structures of the EP300 HAT domain in complex with compounds 2 and 3 led to the identification of compounds possessing low-nanomolar EP300 HAT inhibitory potency and the ability to inhibit cellular acetylation of histone H3K27. Optimization of the pharmacokinetic properties in this series resulted in compounds with excellent oral systemic exposure, and once-daily oral administration of 16 (DS-9300) demonstrated potent antitumor effects in a castrated VCaP xenograft mouse model without significant body weight loss.

Mass-Cytometry-Based Quantification of Global Histone Post-Translational Modifications at Single-Cell Resolution Across Peripheral Immune Cells in IBD.

Current understanding of histone post-translational modifications [histone modifications] across immune cell types in patients with inflammatory bowel disease [IBD] during remission and flare is limited. The present study aimed to quantify histone modifications at a single-cell resolution in IBD patients during remission and flare and how they differ compared to healthy controls. We performed a case-control study of 94 subjects [83 IBD patients and 11 healthy controls]. IBD patients had either ulcerative colitis [n = 38] or Crohn's disease [n = 45] in clinical remission or flare. We used epigenetic profiling by time-of-flight [EpiTOF] to investigate changes in histone modifications within peripheral blood mononuclear cells from IBD patients. We discovered substantial heterogeneity in histone modifications across multiple immune cell types in IBD patients. They had a higher proportion of less differentiated CD34+ haematopoietic progenitors, and a subset of CD56bright natural killer [NK] cells and γδ T cells characterized by distinct histone modifications associated with gene transcription. The subset of CD56bright NK cells had increases in several histone acetylations. An epigenetically defined subset of NK cells was associated with higher levels of C-reactive protein in peripheral blood. CD34+ monocytes from IBD patients had significantly decreased cleaved H3T22, suggesting they were epigenetically primed for macrophage differentiation. We describe the first systems-level quantification of histone modifications across immune cells from IBD patients at a single-cell resolution, revealing the increased epigenetic heterogeneity that is not possible with traditional ChIP-seq profiling. Our data open new directions in investigating the association between histone modifications and IBD pathology using other epigenomic tools.

The anaphase promoting complex/cyclosome ubiquitylates histone H2B on the promoter during UbcH10 transactivation.

Among various post-translational modifications of histones, ubiquitylation plays a crucial role in transcription regulation. Histone mono-ubiquitylation by RING finger motif-containing ubiquitin ligases is documented in this respect. The RING finger ligases primarily regulate the cell cycle, where the anaphase-promoting complex/cyclosome (APC/C) takes charge as mitotic ubiquitin machinery. Reportedly, APC/C participates in transcriptional activation of the ubiquitin carrier protein UbcH10. However, the ubiquitylation activity of APC/C on the UBCH10 promoter remains elusive. This study shows that APC/C, with its adapter protein Cdc20, catalyses mono-ubiquitylation of Lysine-120 in histone 2B on the UBCH10 promoter. This study also identified a cell-cycle-specific pattern of this modification. Finally, APC/C-driven crosstalk of acetylation and ubiquitylation was found operational on UBCH10 trans-regulation. Together, these findings suggest a role for APC/C catalysed promoter ubiquitylation in managing transcription of cell cycle regulatory genes.

Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis

Post-translational histone modifications modulate chromatin activity to affect gene expression. How chromatin states underlie lineage choice in single cells is relatively unexplored. We develop sort-assisted single-cell chromatin immunocleavage (sortChIC) and map active (H3K4me1 and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in the mouse bone marrow. During differentiation, hematopoietic stem and progenitor cells (HSPCs) acquire active chromatin states mediated by cell-type-specifying transcription factors, which are unique for each lineage. By contrast, most alterations in repressive marks during differentiation occur independent of the final cell type. Chromatin trajectory analysis shows that lineage choice at the chromatin level occurs at the progenitor stage. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin states. This implies a hierarchical regulation of chromatin during hematopoiesis: heterochromatin dynamics distinguish differentiation trajectories and lineages, while euchromatin dynamics reflect cell types within lineages.