Histone Post-translational Modifications Research Articles

Epigenetics as a Therapeutic Target in Osteoarthritis.

Osteoarthritis (OA) is a heterogenous, complex disease affecting the integrity of diarthrodial joints that, despite its high prevalence worldwide, lacks effective treatment. In recent years it has been discovered that epigenetics may play an important role in OA. Our objective is to review the current knowledge of the three classical epigenetic mechanisms-DNA methylation, histone post-translational modifications (PTMs), and non-coding RNA (ncRNA) modifications, including microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs)-in relation to the pathogenesis of OA and focusing on articular cartilage. The search for updated literature was carried out in the PubMed database. Evidence shows that dysregulation of numerous essential cartilage molecules is caused by aberrant epigenetic regulatory mechanisms, and it contributes to the development and progression of OA. This offers the opportunity to consider new candidates as therapeutic targets with the potential to attenuate OA or to be used as novel biomarkers of the disease.

Epigenetic Regulation of Macrophage Polarization in Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the leading cause of hospitalization and death worldwide, especially in developing countries. The increased prevalence rate and mortality due to CVDs, despite the development of several approaches for prevention and treatment, are alarming trends in global health. Chronic inflammation and macrophage infiltration are key regulators of the initiation and progression of CVDs. Recent data suggest that epigenetic modifications, such as DNA methylation, posttranslational histone modifications, and RNA modifications, regulate cell development, DNA damage repair, apoptosis, immunity, calcium signaling, and aging in cardiomyocytes; and are involved in macrophage polarization and contribute significantly to cardiac disease development. Cardiac macrophages not only trigger damaging inflammatory responses during atherosclerotic plaque formation, myocardial injury, and heart failure but are also involved in tissue repair, remodeling, and regeneration. In this review, we summarize the key epigenetic modifications that influence macrophage polarization and contribute to the pathophysiology of CVDs, and highlight their potential for the development of advanced epigenetic therapies.

Gene Expression and Epigenetic Regulation in the Prefrontal Cortex of Schizophrenia.

Schizophrenia pathogenesis remains challenging to define; however, there is strong evidence that the interaction of genetic and environmental factors causes the disorder. This paper focuses on transcriptional abnormalities in the prefrontal cortex (PFC), a key anatomical structure that determines functional outcomes in schizophrenia. This review summarises genetic and epigenetic data from human studies to understand the etiological and clinical heterogeneity of schizophrenia. Gene expression studies using microarray and sequencing technologies reported the aberrant transcription of numerous genes in the PFC in patients with schizophrenia. Altered gene expression in schizophrenia is related to several biological pathways and networks (synaptic function, neurotransmission, signalling, myelination, immune/inflammatory mechanisms, energy production and response to oxidative stress). Studies investigating mechanisms driving these transcriptional abnormalities focused on alternations in transcription factors, gene promoter elements, DNA methylation, posttranslational histone modifications or posttranscriptional regulation of gene expression mediated by non-coding RNAs.

Global profiling of protein lysine lactylation and potential target modified protein analysis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, often metastasizes to the lungs. The implications of lysine lactylation (Kla), a recently identified histone post-translational modification (PTM), in the pathology of HCC remain unclear. Here, we report the first proteomic survey of this specific modification in HCC (with no metastasis during 3 years of follow-up), normal liver tissues, and lung metastasis samples of HCC. Of the 2045 modification sites detected on 960 proteins, 1438 sites on 772 proteins contained quantitative information. Subsequently, we analyzed the differentially modified proteins among the different groups. Differentially lactylated proteins were found to be involved in several biological processes, including-but not limited to-amino acid metabolism, ribosomal protein synthesis, and fatty acid metabolism. In addition, we identified numerous highly valuable lactate-modified proteins from the literature. Among them, we verified the lactate modification levels of the following two tumor-related proteins and obtained similar results: USP14 and ABCF1. These two modified proteins will be further investigated in our future studies. This paper is the first report on the lactylome of HCC and it provides a reliable foundation for further research on Kla in HCC.

Epigenetic changes induced by pathogenic Chlamydia spp.

Chlamydia trachomatis, C. pneumoniae, and C. psittaci, the three Chlamydia species known to cause human disease, have been collectively linked to several pathologies, including conjunctivitis, trachoma, respiratory disease, acute and chronic urogenital infections and their complications, and psittacosis. In vitro, animal, and human studies also established additional correlations, such as between C. pneumoniae and atherosclerosis and between C. trachomatis and ovarian cancer. As part of their survival and pathogenesis strategies as obligate intracellular bacteria, Chlamydia spp. modulate all three major types of epigenetic changes, which include deoxyribonucleic acid (DNA) methylation, histone post-translational modifications, and microRNA-mediated gene silencing. Some of these epigenetic changes may be implicated in key aspects of pathogenesis, such as the ability of the Chlamydia spp. to induce epithelial-to-mesenchymal transition, interfere with DNA damage repair, suppress cholesterol efflux from infected macrophages, act as a co-factor in human papillomavirus (HPV)-mediated cervical cancer, prevent apoptosis, and preserve the integrity of mitochondrial networks in infected host cells. A better understanding of the individual and collective contribution of epigenetic changes to pathogenesis will enhance our knowledge about the biology of Chlamydia spp. and facilitate the development of novel therapies and biomarkers. Pathogenic Chlamydia spp. contribute to epigenetically-mediated gene expression changes in host cells by multiple mechanisms.

Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair.

The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via the concerted interplay among different layers, including DNA, histone post-translational modifications (hPTMs) and chromatin-associated proteins. Here, we employ multi-layered proteomics to characterize chromatin-mediated functional interactions of repair proteins, signatures of hPTMs and the DNA-bound proteome during DNA double-strand break (DSB) repair at high temporal resolution. Our data illuminate the dynamics of known and novel DDR-associated factors both at chromatin and at DSBs. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining (NHEJ), homologous recombination (HR) and DSB repair pathway choice. We reveal histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and involved in poly-ADP-ribose polymerase-inhibitor sensitivity. Furthermore, we distinguish hPTMs that are globally induced by DNA damage from those specifically acquired at sites flanking DSBs (γH2AX foci-specific) and profiled their dynamics during the DDR. Integration of complementary chromatin layers implicates G9A-mediated monomethylation of H3K56 in DSBs repair via HR. Our data provide a dynamic chromatin-centered view of the DDR that can be further mined to identify novel mechanistic links and cell vulnerabilities in DSB repair.

Characterizing crosstalk in epigenetic signaling to understand disease physiology.

Epigenetics, the inheritance of genomic information independent of DNA sequence, controls the interpretation of extracellular and intracellular signals in cell homeostasis, proliferation and differentiation. On the chromatin level, signal transduction leads to changes in epigenetic marks, such as histone post-translational modifications (PTMs), DNA methylation and chromatin accessibility to regulate gene expression. Crosstalk between different epigenetic mechanisms, such as that between histone PTMs and DNA methylation, leads to an intricate network of chromatin-binding proteins where pre-existing epigenetic marks promote or inhibit the writing of new marks. The recent technical advances in mass spectrometry (MS) -based proteomic methods and in genome-wide DNA sequencing approaches have broadened our understanding of epigenetic networks greatly. However, further development and wider application of these methods is vital in developing treatments for disorders and pathologies that are driven by epigenetic dysregulation.

The MOZ-BRPF1 acetyltransferase complex in epigenetic crosstalk linked to gene regulation, development, and human diseases.

Acetylation of lysine residues on histone tails is an important post-translational modification (PTM) that regulates chromatin dynamics to allow gene transcription as well as DNA replication and repair. Histone acetyltransferases (HATs) are often found in large multi-subunit complexes and can also modify specific lysine residues in non-histone substrates. Interestingly, the presence of various histone PTM recognizing domains (reader domains) in these complexes ensures their specific localization, enabling the epigenetic crosstalk and context-specific activity. In this review, we will cover the biochemical and functional properties of the MOZ-BRPF1 acetyltransferase complex, underlining its role in normal biological processes as well as in disease progression. We will discuss how epigenetic reader domains within the MOZ-BRPF1 complex affect its chromatin localization and the histone acetyltransferase specificity of the complex. We will also summarize how MOZ-BRPF1 is linked to development via controlling cell stemness and how mutations or changes in expression levels of MOZ/BRPF1 can lead to developmental disorders or cancer. As a last touch, we will review the latest drug candidates for these two proteins and discuss the therapeutic possibilities.

Microspore embryogenesis induction by mannitol and TSA results in a complex regulation of epigenetic dynamics and gene expression in bread wheat.

Reprogramming of microspores development towards embryogenesis mediated by stress treatment constitutes the basis of doubled haploid production. Recently, compounds that alter histone post-translational modifications (PTMs) have been reported to enhance microspore embryogenesis (ME), by altering histones acetylation or methylation. However, epigenetic mechanisms underlying ME induction efficiency are poorly understood. In this study, the epigenetic dynamics and the expression of genes associated with histone PTMs and ME induction were studied in two bread wheat cultivars with different ME response. Microspores isolated at 0, 3 and 5 days, treated with 0.7M mannitol (MAN) and 0.7M mannitol plus 0.4µM trichostatin A (TSA), which induced ME more efficiently, were analyzed. An additional control of gametophytic development was included. Microspores epigenetic state at the onset of ME induction was distinctive between cultivars by the ratio of H3 variants and their acetylated forms, the localization and percentage of labeled microspores with H3K9ac, H4K5ac, H4K16ac, H3K9me2 and H3K27me3, and the expression of genes related to pollen development. These results indicated that microspores of the high responding cultivar could be at a less advanced stage in pollen development. MAN and TSA resulted in a hyperacetylation of H3.2, with a greater effect of TSA. Histone PTMs were differentially affected by both treatments, with acetylation being most concerned. The effect of TSA was observed in the H4K5ac localization pattern at 3dT in the mid-low responding cultivar. Three gene networks linked to ME response were identified. TaHDT1, TaHAG2, TaYAO, TaNFD6-A, TabZIPF1 and TaAGO802-B, associated with pollen development, were down-regulated. TaHDA15, TaHAG3, TaHAM, TaYUC11D, Ta-2B-LBD16 TaMS1 and TaDRM3 constituted a network implicated in morphological changes by auxin signaling and cell wall modification up-regulated at 3dT. The last network included TaHDA18, TaHAC1, TaHAC4, TaABI5, TaATG18fD, TaSDG1a-7A and was related to ABA and ethylene hormone signaling pathways, DNA methylation and autophagy processes, reaching the highest expression at 5dT. The results indicated that TSA mainly modified the regulation of genes related to pollen and auxin signaling. This study represents a breakthrough in identifying the epigenetic dynamics and the molecular mechanisms governing ME induction efficiency, with relevance to recalcitrant wheat genotypes and other crops.

The Pathogenetic Basis of the Action of Bempedoic Acid

The modern cardiology has a wide range of medications which affect various pathogenetic links of atherosclerosis, but even the best of them still obtain disadvantages causing intolerance and medicine discontinuation. The development of new hypolipidemic medications will allow not only to introduce alternative therapies into the cardiology practice, but also to completely execute the strategy of residual risk reduction by utilizing rational combinations of medications. One of such alternatives could be bempedoic acid, which can have a positive effect on a number of endpoints as the results of third phase trials have shown. These effects are also confirmed in Mendelian randomization studies. The mechanism of action of bempedoic acid is presumably associated with inhibition of the activity of ATP citrate lyase – the enzyme responsible for the breakdown of citrate into acetyl-CoA and oxaloacetate. Acetyl-CoA, in turn, is used by the cell to synthesize cholesterol and fatty acids. Thus, bempedoic acid affects in the same metabolic pathway as statins, but at an earlier stage. According to this, it is possible that medications of these classes will have similar side effects and pleiotropic effects associated with modulation of the mevalonic pathway, such as prenylation regulatory proteins (small GTPases) or reduction of coenzyme Q synthesis. However, there are also some specific features of the pharmacodynamics and pharmacokinetics of bempedoic acid to be considered. In particular, once entered the body, it must be activated via esterification by very long-chain acyl-CoA synthetase-1. The enzyme isoform required for this process is expressed in a tissue-specific manner and, for example, is absent in skeletal myocytes. In addition, citrate, oxaloacetate, and acetyl-CoA are important regulators of many intracellular processes: metabolism, growth and proliferation, mechanotransduction, posttranslational modifications of histones and other proteins. The levels of all three substances are altered by bempedoic acid, although no firm conclusions about the effects of these changes can be drawn at this time. The mentioned features probably have a significant impact on the clinical profile of bempedoic acid and underlie the differences from statins already observed in third phase trials, including, for example, a reduced risk of the onset or worsening of diabetes mellitus while taking bempedoic acid.

FUNCTIONAL MAPPING OF NOVEL HISTONE POST-TRANSLATIONAL ALTERATIONS IN COLON CANCER

Abstract Introduction Epigenetic alterations are related to the process of tumorigenesis and resistance to oncological therapies. Although more than 60 types of histone post-translational modifications (HPTMs) have been described, the alterations that manifest simultaneously in tumor tissue have not been systematically studied in colorectal cancer (CRC). This project aims to develop a new screening methodology to delineate epigenetic alterations present in CRC and to obtain a set of biomarkers related to malignancy and tumor response. Methods Using state-of-the-art tandem mass spectrometry (MS-MS) techniques, a direct injection protocol will be performed to simultaneously identify the presence of >200 HPTMs-related peptides in a cohort of primary tumors and paired healthy CRC tissue. Patterning of spectra and statistical analyses will be performed with the automated processing algorithm EpiProfile 2.0. Results A histone extraction protocol optimized for human tumor samples was developed. Likewise, we managed to make MS/MS sample processing more flexible by implementing a multiplatform workflow (Thermo / Sciex), which will increase the screening and data analysis capacity. Through this process we managed to detect >200 peptides related to HPTMs in control samples, and we observed significant differences in certain HPTMs with pharmacological treatments in primary cultures. Conclusions These results will allow the identification of epigenetic biomarkers related to grade, stage and recurrence in the context of CRC.

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.