Chromatin Research Research Articles

Step-by-Step Protocol to Generate Hi-C Contact Maps Using the rfy_hic2 Pipeline.

The Hi-C method has emerged as an indispensable tool for analyzing the 3D organization of the genome, becoming increasingly accessible and frequently utilized in chromatin research. To effectively leverage 3D genomics data obtained through advanced technologies, it is crucial to understand what processes are undertaken and what aspects require special attention within the bioinformatics pipeline. This protocol aims to demystify the Hi-C data analysis process for field newcomers. In a step-by-step manner, we describe how to process Hi-C data, from the initial sequencing of the Hi-C library to the final visualization of Hi-C contact data as heatmaps. Each step of the analysis is clearly explained to ensure an understanding of the procedures and their objectives. By the end of this chapter, readers will be equipped with the knowledge to transform raw Hi-C reads into informative visual representations, facilitating a deeper comprehension of the spatial genomic structures critical to cellular functions.

Nucleosome Dynamics Derived at the Single-Molecule Level Bridges Its Structures and Functions.

Nucleosome, the building block of chromatin, plays pivotal roles in all DNA-related processes. While cryogenic-electron microscopy (cryo-EM) has significantly advanced our understanding of nucleosome structures, the emerging field of single-molecule force spectroscopy is illuminating their dynamic properties. This technique is crucial for revealing how nucleosome behavior is influenced by chaperones, remodelers, histone variants, and post-translational modifications, particularly in their folding and unfolding mechanisms under tension. Such insights are vital for deciphering the complex interplay in nucleosome assembly and structural regulation, highlighting the nucleosome's versatility in response to DNA activities. In this Perspective, we aim to consolidate the latest advancements in nucleosome dynamics, with a special focus on the revelations brought forth by single-molecule manipulation. Our objective is to highlight the insights gained from studying nucleosome dynamics through this innovative approach, emphasizing the transformative impact of single-molecule manipulation techniques in the field of chromatin research.

Beyond the tail: the consequence of context in histone post-translational modification and chromatin research.

The role of histone post-translational modifications (PTMs) in chromatin structure and genome function has been the subject of intense debate for more than 60 years. Though complex, the discourse can be summarized in two distinct - and deceptively simple - questions: What is the function of histone PTMs? And how should they be studied? Decades of research show these queries are intricately linked and far from straightforward. Here we provide a historical perspective, highlighting how the arrival of new technologies shaped discovery and insight. Despite their limitations, the tools available at each period had a profound impact on chromatin research, and provided essential clues that advanced our understanding of histone PTM function. Finally, we discuss recent advances in the application of defined nucleosome substrates, the study of multivalent chromatin interactions, and new technologies driving the next era of histone PTM research.

Two pioneers of epigenetics: their different paths to chromatin research and DNA methylation, and general reflections on epigenetics

The concept of chromatin as a complex of DNA (nuclein at the time) and proteins in the nucleus of eukaryotic cells was generated in the late 19th century. Since the late 20th century, research on DNA methylation that originated in the 1970s and chromatin research have also been labelled epigenetics, a term that originated in developmental biology in the 1940s. Epigenetics now comprises many different research strands related to the regulation of gene activity, such as chemical modifications of histones and DNA, chromatin organization, genome architecture, different types of RNA molecules, and others. To show the various paths on which epigenetic research has developed, I present research and reflections of two pioneers of what later became called epigenetics, Gary Felsenfeld and Adrian Bird. They began their scientific career in very different scientific contexts with both of them crucially contributing to the development of modern chromatin research and the understanding of DNA methylation, respectively. The article is based on authorized transcripts of interviews that I conducted with these researchers, focusing on those parts that are related to chromatin research and epigenetics as well as general reflections on epigenetics and biology.

Epigenetic moonlighting: Catalytic-independent functions of histone modifiers in regulating transcription

The past three decades have yielded a wealth of information regarding the chromatin regulatory mechanisms that control transcription. The "histone code" hypothesis-which posits that distinct combinations of posttranslational histone modifications are "read" by downstream effector proteins to regulate gene expression-has guided chromatin research to uncover fundamental mechanisms relevant to many aspects of biology. However, recent molecular and genetic studies revealed that the function of many histone-modifying enzymes extends independently and beyond their catalytic activities. In this review, we highlight original and recent advances in the understanding of noncatalytic functions of histone modifiers. Many of the histone modifications deposited by these enzymes-previously considered to be required for transcriptional activation-have been demonstrated to be dispensable for gene expression in living organisms. This perspective aims to prompt further examination of these enigmatic chromatin modifications by inspiring studies to define the noncatalytic "epigenetic moonlighting" functions of chromatin-modifying enzymes.

Contribution of smFRET to Chromatin Research

Chromatins are structural components of chromosomes and consist of DNA and histone proteins. The structure, dynamics, and function of chromatins are important in regulating genetic processes. Several different experimental and theoretical tools have been employed to understand chromatins better. In this review, we will focus on the literatures engrossed in understanding of chromatins using single-molecule Förster resonance energy transfer (smFRET). smFRET is a single-molecule fluorescence microscopic technique that can furnish information regarding the distance between two points in space. This has been utilized to efficiently unveil the structural details of chromatins.

Chromatin research and epigenetics - historical perspectives, current research, open questions, and misconceptions

The concept of chromatin as a complex of nucleic acid and proteins in the cell nucleus was developed by cytologists and biochemists in the late 19th century. It was the starting point for biochemical research on DNA and nuclear proteins. Interest in chromatin declined rapidly at the beginning of the 20th century, but a few decades later a new focus on chromatin emerged, which was not only related to its structure, but also to its function in gene regulatory processes in the development of higher organisms. Since the late 20th century, research on chromatin modifications as well as DNA methylation that emerged in the 1970s have also been conducted under the label epigenetics, a term originally introduced for the complex processes between genotype and phenotype during development. These processes - in particular gene regulation - were subsequently scrutinized by molecular biologists. Research termed epigenetics remained marginal until the end of the 20th century but experienced a rapid rise when heritability was added to its definition. This was accompanied by an increasing diversity in researchers' understanding and definitions of epigenetics. Epigenetics now includes research on histone and DNA-modifying enzymes, nucleosome remodelers, histone chaperones, chromatin-binding proteins to facilitate transcription factor and polymerase action, and the role of long non-coding RNA and small interfering RNA in transcriptional regulation. This article highlights the major phases of chromatin and epigenetics research until the present time and illuminates how different scientific contexts changed the relevance and meaning of chromatin from the 19th century. The paper also points to misconceptions and media hype about epigenetics, for example unsupported claims about transgenerational inheritance in humans or questioning of the basic biological principles of gene regulation based on specific regulatory sequences of the genome.

43rd International Asilomar Chromatin, Chromosomes, and Epigenetics Conference

The 43rd Asilomar Chromatin, Chromosomes, and Epigenetics Conference was held in an entirely online format from 9 to 11 December 2021. The conference enabled presenters at various career stages to share promising new findings, and presentations covered modern chromatin research across an array of model systems. Topics ranged from the fundamental principles of nuclear organization and transcription regulation to key mechanisms underlying human disease. The meeting featured five keynote speakers from diverse backgrounds and was organized by Juan Ausió, University of Victoria (British Columbia, Canada), James Davie, University of Manitoba (Manitoba, Canada), Philippe T. Georgel, Marshall University (West Virginia, USA), Michael Goldman, San Francisco State University (California, USA), LeAnn Howe, The University of British Columbia (British Columbia, Canada), Jennifer A. Mitchell, University of Toronto (Ontario, Canada), and Sally G. Pasion, San Francisco State University (California, USA).

Unconventional metabolites in chromatin regulation.

Chromatin, the complex of DNA and histone proteins, serves as a main integrator of cellular signals. Increasing evidence links cellular functional to chromatin state. Indeed, different metabolites are emerging as modulators of chromatin function and structure. Alterations in chromatin state are decisive for regulating all aspects of genome function and ultimately have the potential to produce phenotypic changes. Several metabolites such as acetyl-CoA, S-adenosylmethionine (SAM) or adenosine triphosphate (ATP) have now been well characterized as main substrates or cofactors of chromatin-modifying enzymes. However, there are other metabolites that can directly interact with chromatin influencing its state or that modulate the properties of chromatin regulatory factors. Also, there is a growing list of atypical enzymatic and nonenzymatic chromatin modifications that originate from different cellular pathways that have not been in the limelight of chromatin research. Here, we summarize different properties and functions of uncommon regulatory molecules originating from intermediate metabolism of lipids, carbohydrates and amino acids. Based on the various modes of action on chromatin and the plethora of putative, so far not described chromatin-regulating metabolites, we propose that there are more links between cellular functional state and chromatin regulation to be discovered. We hypothesize that these connections could provide interesting starting points for interfering with cellular epigenetic states at a molecular level.

Histone chaperone-mediated co-expression assembly of tetrasomes and nucleosomes.

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone‐mediated assembly of both tetrasomes and nucleosomes on the well‐defined Widom 601 positioning sequence using a co‐expression/reconstitution wheat germ cell‐free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell‐free platform, our method is expected to be useful for downstream applications in the field of chromatin research.

De novo reconstitution of chromatin using wheat germ cell-free protein synthesis.

DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell‐free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP‐1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone‐modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research.

Cell type-specific chromatin accessibility analysis in the mouse and human brain

ABSTRACT The Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) is becoming popular in the neuroscience field where chromatin regulation is thought to be involved in neurodevelopment, activity-dependent gene regulation, hormonal and environmental responses, and pathophysiology of neuropsychiatric disorders. The advantages of using ATAC-seq include a small amount of material needed, fast protocol, and the ability to capture a range of gene regulatory elements with a single assay. With increasing interest in chromatin research, it is an imperative to have feasible, reliable assays that are compatible with a range of neuroscience study designs. Here we tested three protocols for neuronal chromatin accessibility analysis, including a varying brain tissue freezing method followed by fluorescence-activated nuclei sorting (FANS) and ATAC-seq. Our study shows that the cryopreservation method impacts the number of open chromatin regions identified from frozen brain tissue using ATAC-seq. However, we show that all protocols generate consistent and robust data and enable the identification of functional regulatory elements in neuronal cells. Our study implies that the broad biological interpretation of chromatin accessibility data is not significantly affected by the freezing condition. We also reveal additional challenges of doing chromatin analysis on post-mortem human brain tissue. Overall, ATAC-seq coupled with FANS is a powerful method to capture cell-type-specific chromatin accessibility information in mouse and human brain. Our study provides alternative brain preservation methods that generate high-quality ATAC-seq data while fitting in different study designs, and further encourages the use of this method to uncover the role of epigenetic (dys)regulation in the brain.

Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top-Down Proteomic Analysis.

Histones are the primary protein component of chromatin and are involved in virtually all DNA-templated processes. Histones are abundantly post-translationally modified by a variety of chromatin-modifying machinery. These post-translational modifications (PTMs) are recognized by a range of "reader" proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: that is, histone PTMs function in combination to exert precise DNA-templated regulation. Thus, there is a need to identify and understand proteoforms, or unambiguously defined single protein molecules with all combinations of modifications. Top-down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high-throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocols described here, which increase throughput, reduce sample requirements, and maintain robust quantitation. Although originally designed for high-throughput quantitative top-down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two basic protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high-performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top-down proteomic analysis of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in culture/tissues Basic Protocol 2: HPLC fractionation of histones and histone H4 HPLC-MS/MS Support Protocol: Preparation of intact H3 histone tails by Glu-C digestion.

Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging

CCCTC-binding factor (CTCF) and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution stimulated emission depletion (STED) microscopy, we demonstrate that in living cells, CTCF forms clusters typically containing 2-8 molecules. A fraction of CTCF clusters, enriched for those with ≥3 molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition (TI) result in increased CTCF clustering. Furthermore, the effect of TI on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.

Transposase-assisted tagmentation of RNA/DNA hybrid duplexes.

Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed 'TRACE-seq') are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6 hr) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.

The SAGA continues: The rise of cis- and trans-histone crosstalk pathways

Fueled by key technological innovations during the last several decades, chromatin-based research has greatly advanced our mechanistic understanding of how genes are regulated by epigenetic factors and their associated histone-modifying activities. Most notably, the landmark finding that linked histone acetylation by Gcn5 of the Spt–Ada–Gcn5–acetyltransferase (SAGA) complex to gene activation ushered in a new area of chromatin research and a realization that histone-modifying activities have integral genome functions. This review will discuss past and recent studies that have shaped our understanding of how the histone-modifying activities of SAGA are regulated by, and modulate the outcomes of, other histone modifications during gene transcription. Because much of our understanding of SAGA was established with budding yeast, we will focus on yeast as a model. We discuss the actions of cis- and trans-histone crosstalk pathways that involve the histone acetyltransferase, deubiquitylase, and reader domains of SAGA. We conclude by considering unanswered questions about SAGA and related complexes.

Looking At the Past and Heading to the Future: Meeting Summary of the 6th European Workshop on Plant Chromatin 2019 in Cologne, Germany.

In June 2019, more than a hundred plant researchers met in Cologne, Germany, for the 6th European Workshop on Plant Chromatin (EWPC). This conference brought together a highly dynamic community of researchers with the common aim to understand how chromatin organization controls gene expression, development, and plant responses to the environment. New evidence showing how epigenetic states are set, perpetuated, and inherited were presented, and novel data related to the three-dimensional organization of chromatin within the nucleus were discussed. At the level of the nucleosome, its composition by different histone variants and their specialized histone deposition complexes were addressed as well as the mechanisms involved in histone post-translational modifications and their role in gene expression. The keynote lecture on plant DNA methylation by Julie Law (SALK Institute) and the tribute session to Lars Hennig, honoring the memory of one of the founders of the EWPC who contributed to promote the plant chromatin and epigenetic field in Europe, added a very special note to this gathering. In this perspective article we summarize some of the most outstanding data and advances on plant chromatin research presented at this workshop.

Application of CE-MS for the analysis of histones and histone modifications.

The analysis, identification and quantification of histones and their post-translational modifications plays a central role in chromatin research and in studying epigenetic regulations during physiological processes. In the last decade analytical strategies based on mass spectrometry have been greatly improved for providing a global view of single modification abundances or to determine combinatorial patterns of modifications. Presented here is a newly developed strategy for histone protein analysis and a number of applications are illustrated with an emphasis on PTM characterization. Capillary electrophoresis is coupled to mass spectrometry (CE-MS) and has proven to be a very promising concept as it enables to study intact histones (top-down proteomics) as well as the analysis of enzymatically digested proteins (bottom-up proteomics). This technology combines highly efficient low-flow CE separations with ionization in a single device and offers an orthogonal separation principle to conventional LC-MS analysis, thus expanding the existing analytical repertoire in a perfect manner.

Locus-specific chromatin isolation.

Identifying factors bound to specific genomic loci in an unbiased manner is the holy grail of chromatin research, because it would provide an ultimate description of locus function. In this Comment, we explain why purifying a single locus represents a major technical challenge and provide recommendations for achieving this goal. Michiel Vermeulen and Jerome Dejardin recommend how best to characterize the proteins that bind any specific genomic locus.

New Aspects of Magnesium Function: A Key Regulator in Nucleosome Self-Assembly, Chromatin Folding and Phase Separation.

Metal cations are associated with many biological processes. The effects of these cations on nucleic acids and chromatin were extensively studied in the early stages of nucleic acid and chromatin research. The results revealed that some monovalent and divalent metal cations, including Mg2+, profoundly affect the conformations and stabilities of nucleic acids, the folding of chromatin fibers, and the extent of chromosome condensation. Apart from these effects, there have only been a few reports on the functions of these cations. In 2007 and 2013, however, Mg2+-implicated novel phenomena were found: Mg2+ facilitates or enables both self-assembly of identical double-stranded (ds) DNA molecules and self-assembly of identical nucleosomes in vitro. These phenomena may be deeply implicated in the heterochromatin domain formation and chromatin-based phase separation. Furthermore, a recent study showed that elevation of the intranuclear Mg2+ concentration causes unusual differentiation of mouse ES (embryonic stem) cells. All of these phenomena seem to be closely related to one another. Mg2+ seems to be a key regulator of chromatin dynamics and chromatin-based biological processes.