Methods for Genome-Wide Chromatin Interaction Analysis.

Recent analyses revealed the essential function of chromatin structure in maintaining and regulating genomic information. Advancements in microscopy, nuclear structure observation techniques, and the development of methods utilizing next-generation sequencers (NGSs) have significantly progressed these discoveries. Methods utilizing NGS enable genome-wide analysis, which is challenging with microscopy, and have elucidated concepts of important chromatin structures such as a loop structure, a domain structure called topologically associating domains (TADs), and compartments. In this chapter, I introduce chromatin interaction techniques using NGS and outline the principles and features of each method.

Micro-C Analysis Workflow Using Pairtools and Juicer.

Three-dimensional (3D) chromosome structures are closely related to various chromosomal functions, and deep analysis of the structures is crucial for the elucidation of the functions. In recent years, chromosome conformation capture (3C) techniques combined with next-generation sequencing analysis have been developed to comprehensively reveal 3D chromosome structures. Micro-C is one such method that can detect the structures at nucleosome resolution. In this chapter, I provide a basic method for Micro-C analysis. I present and discuss a series of data analyses ranging from mapping to basic downstream analyses, including loop detection.

Single-Cell Hi-C Analysis Workflow with Pairtools.

Single-cell Hi-C (scHi-C) is a collection of protocols for studying genomic interactions within individual cells. Although data analysis for scHi-C resembles data analysis for bulk Hi-C, the unique challenges of scHi-C, such as high noise and protocol-specific biases, require specialized data processing strategies. In this tutorial chapter, we focus on using pairtools, a suite of tools optimized for scHi-C data, demonstrating its application on a Drosophila snHi-C dataset. While centered on pairtools for snHi-C data, the principles outlined are applicable across scHi-C variants with minor adjustments. This educational chapter aims to guide researchers in using open-source tools for scHi-C analysis, emphasizing critical steps of contact pair extraction, detection of ligation junctions, filtration, and deduplication.

Supervised Chromatin Loop Detection Using Peakachu Version 2.

Peakachu is a supervised-learning-based approach that identifies chromatin loops from chromatin contact data. Here, we present Peakachu version 2, an updated version that significantly improves extensibility, usability, and computational efficiency compared to its predecessor. It features pretrained models tailored for a wide range of experimental platforms, such as Hi-C, Micro-C, ChIA-PET, HiChIP, HiCAR, and TrAC-loop. This chapter offers a step-by-step tutorial guiding users through the process of training Peakachu models from scratch and utilizing pretrained models to predict chromatin loops across various platforms.

Integrative Modeling of 3D Genome Organization by Bayesian Molecular Dynamics Simulations with Hi-C Metainference.

Polymer modeling has been playing an increasingly important role in complementing 3D genome experiments, both to aid their interpretation and to reveal the underlying molecular mechanisms. This chapter illustrates an application of Hi-C metainference, a Bayesian approach to explore the 3D organization of a target genomic region by integrating experimental contact frequencies into a prior model of chromatin. The method reconstructs the conformational ensemble of the target locus by combining molecular dynamics simulation and Monte Carlo sampling from the posterior probability distribution given the data. Using prior chromatin models at both 1 kb and nucleosome resolution, we apply this approach to a 30 kb locus of mouse embryonic stem cells consisting of two well-defined domains linking several gene promoters together. Retaining the advantages of both physics-based and data-driven strategies, Hi-C metainference can provide an experimentally consistent representation of the system while at the same time retaining molecular details necessary to derive physical insights.

Prediction of Enhancer-Gene Interactions Using Chromatin-Conformation Capture and Epigenome Data Using STARE.

Disentangling the relationship of enhancers and genes is an ongoing challenge in epigenomics. We present STARE, our software to quantify the strength of enhancer-gene interactions based on enhancer activity and chromatin contact data. It implements the generalized Activity-by-Contact (gABC) score, which allows predicting putative target genes of candidate enhancers over any desired genomic distance. The only requirement for its application is a measurement of enhancer activity. In addition to regulatory interactions, STARE calculates transcription factor (TF) affinities on gene level. We illustrate its usage on a public single-cell data set of the human heart by predicting regulatory interactions on cell type level, by giving examples on how to integrate them with other data modalities, and by constructing TF affinity matrices.

Exploring Cohesin-Related Multiomics Information in the Web Browser.

Cohesin is a protein complex that plays a key role in regulating chromosome structure and gene expression. While next-generation sequencing technologies have provided extensive information on various aspects of cohesin, integrating and exploring the vast datasets associated with cohesin are not straightforward. CohesinDB ( https://cohesindb.iqb.u-tokyo.ac.jp ) offers a web-based interface for browsing, searching, analyzing, visualizing, and downloading comprehensive multiomics cohesin information in human cells. In this protocol, we introduce how to utilize CohesinDB to facilitate research on transcriptional regulation and chromatin organization.

Machine and Deep Learning Methods for Predicting 3D Genome Organization.

Three-dimensional (3D) chromatin interactions, such as enhancer-promoter interactions (EPIs), loops, topologically associating domains (TADs), and A/B compartments, play critical roles in a wide range of cellular processes by regulating gene expression. Recent development of chromatin conformation capture technologies has enabled genome-wide profiling of various 3D structures, even with single cells. However, current catalogs of 3D structures remain incomplete and unreliable due to differences in technology, tools, and low data resolution. Machine learning methods have emerged as an alternative to obtain missing 3D interactions and/or improve resolution. Such methods frequently use genome annotation data (ChIP-seq, DNAse-seq, etc.), DNA sequencing information (k-mers and transcription factor binding site (TFBS) motifs), and other genomic properties to learn the associations between genomic features and chromatin interactions. In this review, we discuss computational tools for predicting three types of 3D interactions (EPIs, chromatin interactions, and TAD boundaries) and analyze their pros and cons. We also point out obstacles to the computational prediction of 3D interactions and suggest future research directions.

Characterization of open chromatin sensitive to actin polymerization and identification of core-binding factor subunit beta as mechanosensitive nucleocytoplasmic shuttling protein.

Mechanotransduction leads to a variety of biological responses including gene expression, changes in cell shape, migration, tissue development, and immune responses. Dysregulation of mechanotransduction is implicated in the progression of various diseases such as cardiovascular diseases and cancer. The actin cytoskeleton plays a crucial role in transmitting mechanical stimuli. Actin filaments, essential for cell motility and shape changes, respond to mechanical cues by remodeling, influencing gene expression via the linker of nucleoskeleton and cytoskeleton complex and mechanosensitive transcription factors. This study employs the dithiobis(succinimidyl propionate) (DSP)-micrococcal nuclease (MNase) proteogenomics method to explore the relationship between cellular mechanosensing, chromatin architecture, and the identification of proteins involved in mechanosensitive nucleocytoplasmic shuttling, revealing how actin polymerization affects chromatin and gene expression. We found that depolymerization of actin filaments by latrunculin B (Lat B) for 30 min is sufficient to alter open chromatin and identified core-binding factor subunit beta as mechanosensitive nucleocytoplasmic shuttling protein.

Implicating type 2 diabetes effector genes in relevant metabolic cellular models using promoter-focused Capture-C.

AIMS/HYPOTHESIS: Genome-wide association studies (GWAS) have identified hundreds of type 2 diabetes loci, with the vast majority of signals located in non-coding regions; as a consequence, it remains largely unclear which 'effector' genes these variants influence. Determining these effector genes has been hampered by the relatively challenging cellular settings in which they are hypothesised to confer their effects. METHODS: To implicate such effector genes, we elected to generate and integrate high-resolution promoter-focused Capture-C, assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq datasets to characterise chromatin and expression profiles in multiple cell lines relevant to type 2 diabetes for subsequent functional follow-up analyses: EndoC-BH1 (pancreatic beta cell), HepG2 (hepatocyte) and Simpson-Golabi-Behmel syndrome (SGBS; adipocyte). RESULTS: The subsequent variant-to-gene analysis implicated 810 candidate effector genes at 370 type 2 diabetes risk loci. Using partitioned linkage disequilibrium score regression, we observed enrichment for type 2 diabetes and fasting glucose GWAS loci in promoter-connected putative cis-regulatory elements in EndoC-BH1 cells as well as fasting insulin GWAS loci in SGBS cells. Moreover, as a proof of principle, when we knocked down expression of the SMCO4 gene in EndoC-BH1 cells, we observed a statistically significant increase in insulin secretion. CONCLUSIONS/INTERPRETATION: These results provide a resource for comparing tissue-specific data in tractable cellular models as opposed to relatively challenging primary cell settings. DATA AVAILABILITY: Raw and processed next-generation sequencing data for EndoC-BH1, HepG2, SGBS_undiff and SGBS_diff cells are deposited in GEO under the Superseries accession GSE262484. Promoter-focused Capture-C data are deposited under accession GSE262496. Hi-C data are deposited under accession GSE262481. Bulk ATAC-seq data are deposited under accession GSE262479. Bulk RNA-seq data are deposited under accession GSE262480.

Unraveling the key role of chromatin structure in cancer development through epigenetic landscape characterization of oral cancer.

Epigenetic alterations, such as those in chromatin structure and DNA methylation, have been extensively studied in a number of tumor types. But oral cancer, particularly oral adenocarcinoma, has received far less attention. Here, we combined laser-capture microdissection and muti-omics mini-bulk sequencing to systematically characterize the epigenetic landscape of oral cancer, including chromatin architecture, DNA methylation, H3K27me3 modification, and gene expression. In carcinogenesis, tumor cells exhibit reorganized chromatin spatial structures, including compromised compartment structures and altered gene-gene interaction networks. Notably, some structural alterations are observed in phenotypically non-malignant paracancerous but not in normal cells. We developed transformer models to identify the cancer propensity of individual genome loci, thereby determining the carcinogenic status of each sample. Insights into cancer epigenetic landscapes provide evidence that chromatin reorganization is an important hallmark of oral cancer progression, which is also linked with genomic alterations and DNA methylation reprogramming. In particular, regions of frequent copy number alternations in cancer cells are associated with strong spatial insulation in both cancer and normal samples. Aberrant methylation reprogramming in oral squamous cell carcinomas is closely related to chromatin structure and H3K27me3 signals, which are further influenced by intrinsic sequence properties. Our findings indicate that structural changes are both significant and conserved in two distinct types of oral cancer, closely linked to transcriptomic alterations and cancer development. Notably, the structural changes remain markedly evident in oral adenocarcinoma despite the considerably lower incidence of genomic copy number alterations and lesser extent of methylation alterations compared to squamous cell carcinoma. We expect that the comprehensive analysis of epigenetic reprogramming of different types and subtypes of primary oral tumors can provide additional guidance to the design of novel detection and therapy for oral cancer.

Epigenetic Phenomenon of Paramutation in Plants and Animals.

The phenomenon of paramutation describes the interaction between two alleles, in which one allele initiates inherited epigenetic conversion of another allele without affecting the DNA sequence. Epigenetic transformations due to paramutation are accompanied by the change in DNA and/or histone methylation patterns, affecting gene expression. Studies of paramutation in plants and animals have identified small non-coding RNAs as the main effector molecules required for the initiation of epigenetic changes in gene loci. Due to the fact that small non-coding RNAs can be transmitted across generations, the paramutation effect can be inherited and maintained in a population. In this review, we will systematically analyze examples of paramutation in different living systems described so far, highlighting common and different molecular and genetic aspects of paramutation between organisms, and considering the role of this phenomenon in evolution.

RNA-protein interactions reveals the pivotal role of lncRNA1840 in tomato fruit maturation.

Long non-coding RNAs (lncRNAs) play crucial roles in various biological processes in plants. However, the functional mechanism of lncRNAs in fruit ripening, particularly the transition from unripe to ripe stages, remains elusive. One such lncRNA1840, reported by our group, was found to have important role in tomato fruit ripening. In the present study, we gain insight into its functional role in fruit ripening. CRISPR-Cas9 mediated lncRNA1840 mutants caused the delayed tomato fruit ripening. Notably, loss function of lncRNA1840 did not directly impact ethylene signaling but rather delay ethylene synthesis. Transcriptomic analysis revealed differences in the expression of ripening related genes in lncRNA1840 mutants, suggesting that it is involved in gene regulation of fruit ripening. We used Chromatin Isolation by RNA Purification (ChIRP)-Seq to identify lncRNA1840 binding sites on chromatin. ChIRP-seq suggested that lncRNA1840 had occupancy on 40 genes, but none of them is differentially expressed genes in transcriptomic analysis, which indicated lncRNA1840 might indirectly modulate the gene expression. ChIRP-mass spectrometry analysis identified potential protein interactors of lncRNA1840, Pre-mRNA processing splicing factor 8, highlighting its involvement in post-transcriptional regulatory pathways. In summary, lncRNA1840 is key player in tomato plant growth and fruit ripening, with multifaceted roles in gene expression and regulatory networks.

Stabilization of the hexasome intermediate during histone exchange by yeast SWR1 complex.

The yeast SWR1 complex catalyzes the exchange of histone H2A/H2B dimers in nucleosomes with Htz1/H2B dimers. We use cryoelectron microscopy to determine the structure of an enzyme-bound hexasome intermediate in the reaction pathway of histone exchange, in which an H2A/H2B dimer has been extracted from a nucleosome prior to the insertion of a dimer comprising Htz1/H2B. The structure reveals a key role for the Swc5 subunit in stabilizing the unwrapping of DNA from the histone core of the hexasome. By engineering a crosslink between an Htz1/H2B dimer and its chaperone protein Chz1, we show that this blocks histone exchange by SWR1 but allows the incoming chaperone-dimer complex to insert into the hexasome. We use this reagent to trap an SWR1/hexasome complex with an incoming Htz1/H2B dimer that shows how the reaction progresses to the next step. Taken together the structures reveal insights into the mechanism of histone exchange by SWR1 complex.

Monoubiquitinated H2B, a Main Chromatin Target of Formaldehyde, Is Important for S-Phase Checkpoint Signaling and Genome Stability.

Formaldehyde (FA) is a human carcinogen with ubiquitous environmental exposures and significant endogenous formation. Genotoxic activity of FA stems from its reactivity with DNA-NH2 groups. Histone lysines are another source of aldehyde-reactive amino groups in chromatin, however, chromatin/histone damage responses to FA and their biological significance are poorly understood. We examined histone posttranslational modifications in FA-treated human lung cells and found that the majority of the most prominent small lysine modifications associated with active or inactive chromatin were unchanged. FA moderately decreased H3K9 and H3K27 acetylation and H2A-K119 monoubiquitination but caused surprisingly severe losses of H2B-K120 monoubiquitination, especially in primary and stem-like cells. H2Aub1 decreases reflected its slower ubiquitination linked to a lower ubiquitin availability due to K48-polyubiquitination of FA-damaged proteins. Depletion of H2Bub1 resulted from its rapid deubiquitination in part by ATXN7L3-associated deubiquitinases and was independent on DNA damage signaling, indicating a direct chromatin damage response. Manipulations of H2Bub1 abundance showed that it was important for robust ATM and ATR signaling, efficient S-phase checkpoint, and suppression of mitotic transmission of unreplicated DNA and formation of micronuclei. Our findings identified H2B deubiquitination as a major FA-induced chromatin damage response that regulates S-phase checkpoint signaling and genome stability.

Enhlink infers distal and context-specific enhancer-promoter linkages.

Enhlink is a computational tool for scATAC-seq data analysis, facilitating precise interrogation of enhancer function at the single-cell level. It employs an ensemble approach incorporating technical and biological covariates to infer condition-specific regulatory DNA linkages. Enhlink can integrate multi-omic data for enhanced specificity, when available. Evaluation with simulated and real data, including multi-omic datasets from the mouse striatum and novel promoter capture Hi-C data, demonstrate that Enhlink outperfoms alternative methods. Coupled with eQTL analysis, it identified a putative super-enhancer in striatal neurons. Overall, Enhlink offers accuracy, power, and potential for revealing novel biological insights in gene regulation.

Transcriptional activation domains interact with ATPase subunits of yeast chromatin remodelling complexes SWI/SNF, RSC and INO80.

Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.

Temporally discordant chromatin accessibility and DNA demethylation define short and long-term enhancer regulation during cell fate specification.

Epigenetic mechanisms govern the transcriptional activity of lineage-specifying enhancers; but recent work challenges the dogma that joint chromatin accessibility and DNA demethylation are prerequisites for transcription. To understand this paradox, we established a highly-resolved timeline of DNA demethylation, chromatin accessibility, and transcription factor occupancy during neural progenitor cell differentiation. We show thousands of enhancers undergo rapid, transient accessibility changes associated with distinct periods of transcription factor expression. However, most DNA methylation changes are unidirectional and delayed relative to chromatin dynamics, creating transiently discordant epigenetic states. Genome-wide detection of 5-hydroxymethylcytosine further revealed active demethylation begins ahead of chromatin and transcription factor activity, while enhancer hypomethylation persists long after these activities have dissipated. We demonstrate that these timepoint specific methylation states predict past, present and future chromatin accessibility using machine learning models. Thus, chromatin and DNA methylation collaborate on different timescales to mediate short and long-term enhancer regulation during cell fate specification.

Comprehensive posttranslational modifications in the testis-specific histone variant H3t protein validated in tagged knock-in mice.

During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo.

Unveiling the role of epigenetics in leaf senescence: a comparative study to identify different epigenetic regulations of senescence types in barley leaves.

BACKGROUND: Developmental leaf senescence (DLS) is an irreversible process followed by cell death. Dark-induced leaf senescence (DILS) is a reversible process that allows adaptations to changing environmental conditions. As a result of exposure to adverse environmental changes, plants have developed mechanisms that enable them to survive. One of these is the redirection of metabolism into the senescence pathway. The plant seeks to optimise resource allocation. Our research aims to demonstrate how epigenetic machinery regulates leaf senescence, including its irreversibility. RESULTS: In silico analyses allowed the complex identification and characterisation of 117 genes involved in epigenetic processes in barley. These genes include those responsible for DNA methylation, post-translational histone modifications, and ATP-dependent chromatin remodelling complexes. We then performed RNAseq analysis after DILS and DLS to evaluate their expression in senescence-dependent leaf metabolism. Principal component analysis revealed that evaluated gene expression in developmental senescence was similar to controls, while induced senescence displayed a distinct profile. Western blot experiments revealed that senescence engages senescence-specific histone modification. During DILS and DLS, the methylation of histone proteins H3K4me3 and H3K9me2 increased. H3K9ac acetylation levels significantly decreased during DILS and remained unchanged during DLS. CONCLUSIONS: The study identified different epigenetic regulations of senescence types in barley leaves. These findings are valuable for exploring epigenetic regulation of senescence-related molecular mechanisms, particularly in response to premature, induced leaf senescence. Based on the results, we suggest the presence of an epigenetically regulated molecular switch between cell survival and cell death in DILS, highlighting an epigenetically driven cell survival metabolic response.