Pioneer factors: nature or nurture?

Chromatin is densely packed with nucleosomes, which limits the accessibility of many chromatin-associated proteins. Pioneer factors (PFs) are usually viewed as a special group of sequence-specific transcription factors (TFs) that can recognize nucleosome-embedded motifs, invade compact chromatin, and generate open chromatin regions. Through this process, PFs initiate a cascade of events that play key roles in gene regulation and cell differentiation. A current debate in the field is if PFs belong to a unique subset of TFs with intrinsic "pioneering activity", or if all TFs have the potential to function as PFs within certain cellular contexts. There are also different views regarding the key feature(s) that define pioneering activity. In this review, we present evidence from the literature related to these alternative views and discuss how to potentially reconcile them. It is possible that both intrinsic properties, like tight nucleosome binding and structural compatibility, and cellular conditions, like concentration and co-factor availability, are important for PF function.

Genome-wide DNase I-hypersensitive site assay reveals distinct genomic distributions and functional features of open chromatin in autopolyploid sugarcane.

The characterization of cis-regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome-wide identification of DNase I-hypersensitive sites (DHSs) in leaf and stem tissues of the auto-octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf- and stem-dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue-specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene-responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue-specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding.

Right time, right place: the temporal regulation of developmental gene expression.

Many studies have focused on defining the critical transcription factors that specify tissue morphogenesis and differentiation. Our understanding of how these spatial regulators are deployed in the proper temporal order, however, has remained less clear. In this issue of Genes & Development, Uyehara and colleagues (pp. 862-875) provide new insights into the mechanisms by which temporal and spatial regulators are coordinated to control Drosophila wing development during metamorphosis.

Hormone-dependent control of developmental timing through regulation of chromatin accessibility.

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility.

CharID: a two-step model for universal prediction of interactions between chromatin accessible regions.

Open chromatin regions (OCRs) allow direct interaction between cis-regulatory elements and trans-acting factors. Therefore, predicting all potential OCR-mediated loops is essential for deciphering the regulation mechanism of gene expression. However, existing loop prediction tools are restricted to specific anchor types. Here, we present CharID (Chromatin Accessible Region Interaction Detector), a two-step model that combines neural network and ensemble learning to predict OCR-mediated loops. In the first step, CharID-Anchor, an attention-based hybrid CNN-BiGRU network is constructed to discriminate between the anchor and nonanchor OCRs. In the second step, CharID-Loop uses gradient boosting decision tree with chromosome-split strategy to predict the interactions between anchor OCRs. The performance was assessed in three human cell lines, and CharID showed superior prediction performance compared with other algorithms. In contrast to the methods designed to predict a particular type of loops, CharID can detect varieties of chromatin loops not limited to enhancer-promoter loops or architectural protein-mediated loops. We constructed the OCR-mediated interaction network using the predicted loops and identified hub anchors, which are highlighted by their proximity to housekeeping genes. By analyzing loops containing SNPs associated with cardiovascular disease, we identified an SNP-gene loop indicating the regulation mechanism of the GFOD1. Taken together, CharID universally predicts diverse chromatin loops beyond other state-of-the-art methods, which are limited by anchor types, and experimental techniques, which are limited by sensitivities drastically decaying with the genomic distance of anchors. Finally, we hosted Peaksniffer, a user-friendly web server that provides online prediction, query and visualization of OCRs and associated loops.

Functional Characterization of Accessible Chromatin in Common Wheat.

Eukaryotic gene transcription is fine-tuned by precise spatiotemporal interactions between cis-regulatory elements (CREs) and trans-acting factors. However, how CREs individually or coordinated with epigenetic marks function in regulating homoeolog bias expression is still largely unknown in wheat. In this study, through comprehensively characterizing open chromatin coupled with DNA methylation in the seedling and spikelet of common wheat, we observed that differential chromatin openness occurred between the seedling and spikelet, which plays important roles in tissue development through regulating the expression of related genes or through the transcription factor (TF)-centered regulatory network. Moreover, we found that CHH methylation may act as a key determinant affecting the differential binding of TFs, thereby resulting in differential expression of target genes. In addition, we found that sequence variations in MNase hypersensitive sites (MHSs) result in the differential expression of key genes responsible for important agronomic traits. Thus, our study provides new insights into the roles of CREs in regulating tissue or homoeolog bias expression, and controlling important agronomic traits in common wheat. It also provides potential CREs for genetic and epigenetic manipulation toward improving desirable traits for wheat molecule breeding.

Dynamics of Chromatin Opening across Larval Development in the Urochordate Ascidian Ciona savignyi.

Ascidian larvae undergo tail elongation and notochord lumenogenesis, making them an ideal model for investigating tissue morphogenesis in embryogenesis. The cellular and mechanical mechanisms of these processes have been studied; however, the underlying molecular regulatory mechanism remains to be elucidated. In this study, assays for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were applied to investigate potential regulators of the development of ascidian Ciona savignyi larvae. Our results revealed 351 and 138 differentially accessible region genes through comparisons of ATAC-seq data between stages 21 and 24 and between stages 24 and 25, respectively. A joint analysis of RNA-seq and ATAC-seq data revealed a correlation between chromatin accessibility and gene transcription. We further verified the tissue expression patterns of 12 different genes. Among them, Cs-matrix metalloproteinase 24 (MMP24) and Cs-krüppel-like factor 5 (KLF5) were highly expressed in notochord cells. Functional assay results demonstrated that both genes are necessary for notochord lumen formation and expansion. Finally, we performed motif enrichment analysis of the differentially accessible regions in different tailbud stages and summarized the potential roles of these motif-bearing transcription factors in larval development. Overall, our study found a correlation between gene expression and chromatin accessibility and provided a vital resource for understanding the mechanisms of the development of ascidian embryos.

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei.

BACKGROUND: The genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification. RESULTS: In this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels. CONCLUSIONS: We have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

Transcriptional and post-transcriptional regulation of young genes in plants.

BACKGROUND: New genes continuously emerge from non-coding DNA or by diverging from existing genes, but most of them are rapidly lost and only a few become fixed within the population. We hypothesized that young genes are subject to transcriptional and post-transcriptional regulation to limit their expression and minimize their exposure to purifying selection. RESULTS: We performed a protein-based homology search across the tree of life to determine the evolutionary age of protein-coding genes present in the rice genome. We found that young genes in rice have relatively low expression levels, which can be attributed to distal enhancers, and closed chromatin conformation at their transcription start sites (TSS). The chromatin in TSS regions can be re-modeled in response to abiotic stress, indicating conditional expression of young genes. Furthermore, transcripts of young genes in Arabidopsis tend to be targeted by nonsense-mediated RNA decay, presenting another layer of regulation limiting their expression. CONCLUSIONS: These data suggest that transcriptional and post-transcriptional mechanisms contribute to the conditional expression of young genes, which may alleviate purging selection while providing an opportunity for phenotypic exposure and functionalization.

Gene expression and chromatin conformation differs between worker bees performing different tasks.

BACKGROUND: Revealing the effect of transcriptomic regulation on behavioral differences is a fundamental goal in biology, but the relationship between gene regulatory networks and individual behavior differences remains largely unknown. Honey bees are considered as good models for studying the mechanisms underlying gene expression changes and behavioral differences since they exhibit strong and obvious differences in tasks between individuals. The cis-regulatory regions usually contain the binding sites of diverse transcription factor (TFs) influencing bee behavior. Thus, the identification of cis-regulatory elements in the brains across different behavioral states is important for understanding how genomic and transcriptomic variations affect different tasks in honeybees. METHODS: In this study, we employed transcriptome and genome-wide chromatin accessibility assays to analyze brain tissues of honey bees in different behavioral states for examining the relationship between individual behavior differences and brain gene expression changes. We also used the obtained open chromatin regions to identify cis-motifs associating differentially expressed TFs and genes in order to reveal the transcriptional regulatory mechanism related to the different tasks. RESULTS: We identified genetic regulatory modules regulating different tasks that contained key TFs (CTCF, Trl and schlank) associated with open chromatin regions enriched for DNA sequence motifs belonging to the family of the corresponding TFs. The most prominent transcriptomic changes, which correlated with chromatin accessibility modifications within their proximal promoter regions, occurred in nervous system development, and were associated with behavior switch. CONCLUSIONS: Our results revealed the regulatory landscape among three behavioral stages in honeybees and identified interactive molecular networks regulating different tasks. These results provide a comprehensive insight into behavioral differences of honeybees, which offers reference for future study.

ATAC-Seq Reveals the Landscape of Open Chromatin and cis-Regulatory Elements in the Phytophthora sojae Genome.

Nucleosome-free open chromatin often harbors transcription factor (TF)-binding sites that are associated with active cis-regulatory elements. However, analysis of open chromatin regions has rarely been applied to oomycete or fungal plant pathogens. In this study, we performed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to identify open chromatin and cis-regulatory elements in Phytophthora sojae at the mycelial stage. We identified 10,389 peaks representing nucleosome-free regions (NFRs). The peaks were enriched in gene-promoter regions and associated with 40% of P. sojae genes; transcription levels were higher for genes with multiple peaks than genes with a single peak and were higher for genes with a single peak than genes without peak. Chromatin accessibility was positively correlated with gene transcription level. Through motif discovery based on NFR peaks in core promoter regions, 25 candidate cis-regulatory motifs with evidence of TF-binding footprints were identified. These motifs exhibited various preferences for location in the promoter region and associations with the transcription level of their target genes, which included some putative pathogenicity-related genes. As the first study revealing the landscape of open chromatin and the correlation between chromatin accessibility and gene transcription level in oomycetes, the results provide a technical reference and data resources for future studies on the regulatory mechanisms of gene transcription.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin.

BACKGROUND: Evolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation of many enhancers, distal regulatory elements that control spatial and temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high. RESULTS: We present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that can predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Next, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss of regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity have a stronger tendency than the general population to have predicted lineage-specific open chromatin. CONCLUSION: The framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements.

A clinician's handbook for using ctDNA throughout the patient journey.

BACKGROUND: The promise of precision cancer medicine presently centers around the genomic sequence of a patient's tumor being translated into timely, actionable information to inform clinical care. The analysis of cell-free DNA from liquid biopsy, which contains circulating tumor DNA (ctDNA) in patients with cancer, has proven to be amenable to various settings in oncology. However, open questions surrounding the clinical validity and utility of plasma-based analyses have hindered widespread clinical adoption. MAIN BODY: Owing to the rapid evolution of the field, studies supporting the use of ctDNA as a biomarker throughout a patient's journey with cancer have accumulated in the last few years, warranting a review of the latest status for clinicians who may employ ctDNA in their precision oncology programs. In this work, we take a step back from the intricate coverage of detection approaches described extensively elsewhere and cover basic concepts around the practical implementation of next generation sequencing (NGS)-guided liquid biopsy. We compare relevant targeted and untargeted approaches to plasma DNA analysis, describe the latest evidence for clinical validity and utility, and highlight the value of genome-wide ctDNA analysis, particularly as it relates to early detection strategies and discovery applications harnessing the non-coding genome. CONCLUSIONS: The maturation of liquid biopsy for clinical application will require interdisciplinary efforts to address current challenges. However, patients and clinicians alike may greatly benefit in the future from its incorporation into routine oncology care.

Selection on Accessible Chromatin Regions in Capsella grandiflora.

Accurate estimates of genome-wide rates and fitness effects of new mutations are essential for an improved understanding of molecular evolutionary processes. Although eukaryotic genomes generally contain a large noncoding fraction, functional noncoding regions and fitness effects of mutations in such regions are still incompletely characterized. A promising approach to characterize functional noncoding regions relies on identifying accessible chromatin regions (ACRs) tightly associated with regulatory DNA. Here, we applied this approach to identify and estimate selection on ACRs in Capsella grandiflora, a crucifer species ideal for population genomic quantification of selection due to its favorable population demography. We describe a population-wide ACR distribution based on ATAC-seq data for leaf samples of 16 individuals from a natural population. We use population genomic methods to estimate fitness effects and proportions of positively selected fixations (α) in ACRs and find that intergenic ACRs harbor a considerable fraction of weakly deleterious new mutations, as well as a significantly higher proportion of strongly deleterious mutations than comparable inaccessible intergenic regions. ACRs are enriched for expression quantitative trait loci (eQTL) and depleted of transposable element insertions, as expected if intergenic ACRs are under selection because they harbor regulatory regions. By integrating empirical identification of intergenic ACRs with analyses of eQTL and population genomic analyses of selection, we demonstrate that intergenic regulatory regions are an important source of nearly neutral mutations. These results improve our understanding of selection on noncoding regions and the role of nearly neutral mutations for evolutionary processes in outcrossing Brassicaceae species.

Single-Cell Chromatin Accessibility Data Combined with GWAS Improves Detection of Relevant Cell Types in 59 Complex Phenotypes.

Several disease risk variants reside on non-coding regions of DNA, particularly in open chromatin regions of specific cell types. Identifying the cell types relevant to complex traits through the integration of chromatin accessibility data and genome-wide association studies (GWAS) data can help to elucidate the mechanisms of these traits. In this study, we created a collection of associations between the combinations of chromatin accessibility data (bulk and single-cell) with an array of 201 complex phenotypes. We integrated the GWAS data of these 201 phenotypes with bulk chromatin accessibility data from 137 cell types measured by DNase-I hypersensitive sequencing and found significant results (FDR adjusted p-value ≤ 0.05) for at least one cell type in 21 complex phenotypes, such as atopic dermatitis, Graves' disease, and body mass index. With the integration of single-cell chromatin accessibility data measured by an assay for transposase-accessible chromatin with high-throughput sequencing (scATAC-seq), taken from 111 adult and 111 fetal cell types, the resolution of association was magnified, enabling the identification of further cell types. This resulted in the identification of significant correlations (FDR adjusted p-value ≤ 0.05) between 15 categories of single-cell subtypes and 59 phenotypes ranging from autoimmune diseases like Graves' disease to cardiovascular traits like diastolic/systolic blood pressure.

CHIPIN: ChIP-seq inter-sample normalization based on signal invariance across transcriptionally constant genes.

BACKGROUND: Multiple studies rely on ChIP-seq experiments to assess the effect of gene modulation and drug treatments on protein binding and chromatin structure. However, most methods commonly used for the normalization of ChIP-seq binding intensity signals across conditions, e.g., the normalization to the same number of reads, either assume a constant signal-to-noise ratio across conditions or base the estimates of correction factors on genomic regions with intrinsically different signals between conditions. Inaccurate normalization of ChIP-seq signal may, in turn, lead to erroneous biological conclusions. RESULTS: We developed a new R package, CHIPIN, that allows normalizing ChIP-seq signals across different conditions/samples when spike-in information is not available, but gene expression data are at hand. Our normalization technique is based on the assumption that, on average, no differences in ChIP-seq signals should be observed in the regulatory regions of genes whose expression levels are constant across samples/conditions. In addition to normalizing ChIP-seq signals, CHIPIN provides as output a number of graphs and calculates statistics allowing the user to assess the efficiency of the normalization and qualify the specificity of the antibody used. In addition to ChIP-seq, CHIPIN can be used without restriction on open chromatin ATAC-seq or DNase hypersensitivity data. We validated the CHIPIN method on several ChIP-seq data sets and documented its superior performance in comparison to several commonly used normalization techniques. CONCLUSIONS: The CHIPIN method provides a new way for ChIP-seq signal normalization across conditions when spike-in experiments are not available. The method is implemented in a user-friendly R package available on GitHub: https://github.com/BoevaLab/CHIPIN.

ATAC-Seq identifies regions of open chromatin in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus.

BACKGROUND: Bovine Respiratory Syncytial Virus (BRSV) is a cause of Bovine Respiratory Disease (BRD). DNA-based biomarkers contributing to BRD resistance are potentially present in non-protein-coding regulatory regions of the genome, which can be determined using ATAC-Seq. The objectives of this study were to: (i) identify regions of open chromatin in DNA extracted from bronchial lymph nodes (BLN) of healthy dairy calves experimentally challenged with BRSV and compare them with those from non-challenged healthy control calves, (ii) elucidate the chromatin regions that were differentially or uniquely open in the BRSV challenged relative to control calves, and (iii) compare the genes found in regions proximal to the differentially open regions to the genes previously found to be differentially expressed in the BLN in response to BRSV and to previously identified BRD susceptibility loci. This was achieved by challenging clinically healthy Holstein-Friesian calves (mean age 143 ± 14 days) with either BRSV inoculum (n = 12) or with sterile phosphate buffered saline (PBS) (n = 6) and preparing and sequencing ATAC-Seq libraries from fresh BLN tissues. RESULTS: Using Diffbind, 9,144 and 5,096 differentially accessible regions (P < 0.05, FDR < 0.05) were identified between BRSV challenged and control calves employing DeSeq2 and EdgeR, respectively. Additionally, 8,791 chromatin regions were found to be uniquely open in BRSV challenged calves. Seventy-six and 150 of the genes that were previously found to be differentially expressed using RNA-Seq, were located within 2 kb downstream of the differentially accessible regions, and of the regions uniquely open in BRSV challenged calves, respectively. Pathway analyses within ClusterProfiler indicated that these genes were involved in immune responses to infection and participated in the Th1 and Th2 pathways, pathogen recognition and the anti-viral response. There were 237 differentially accessible regions positioned within 40 previously identified BRD susceptibility loci. CONCLUSIONS: The identified open chromatin regions are likely to be involved in the regulatory response of gene transcription induced by infection with BRSV. Consequently, they may contain variants which impact resistance to BRD that could be used in breeding programmes to select healthier, more robust cattle.

OCRDetector: Accurately Detecting Open Chromatin Regions via Plasma Cell-Free DNA Sequencing Data.

Open chromatin regions (OCRs) are special regions of the human genome that can be accessed by DNA regulatory elements. Several studies have reported that a series of OCRs are associated with mechanisms involved in human diseases, such as cancers. Identifying OCRs using ATAC-seq or DNase-seq is often expensive. It has become popular to detect OCRs from plasma cell-free DNA (cfDNA) sequencing data, because both the fragmentation modes of cfDNA and the sequencing coverage in OCRs are significantly different from those in other regions. However, it is a challenging computational problem to accurately detect OCRs from plasma cfDNA-seq data, as multiple factors-e.g., sequencing and mapping bias, insufficient read depth, etc.-often mislead the computational model. In this paper, we propose a novel bioinformatics pipeline, OCRDetector, for detecting OCRs from whole-genome cfDNA sequencing data. The pipeline calculates the window protection score (WPS) waveform and the cfDNA sequencing coverage. To validate the proposed pipeline, we compared the percentage overlap of our OCRs with those obtained by other methods. The experimental results show that 81% of the TSS regions of housekeeping genes are detected, and our results have obvious tissue specificity. In addition, the overlap percentage between our OCRs and the high-confidence OCRs obtained by ATAC-seq or DNase-seq is greater than 70%.

Chromatin Profiling Techniques: Exploring the Chromatin Environment and Its Contributions to Complex Traits.

The genetic architecture of complex traits is multifactorial. Genome-wide association studies (GWASs) have identified risk loci for complex traits and diseases that are disproportionately located at the non-coding regions of the genome. On the other hand, we have just begun to understand the regulatory roles of the non-coding genome, making it challenging to precisely interpret the functions of non-coding variants associated with complex diseases. Additionally, the epigenome plays an active role in mediating cellular responses to fluctuations of sensory or environmental stimuli. However, it remains unclear how exactly non-coding elements associate with epigenetic modifications to regulate gene expression changes and mediate phenotypic outcomes. Therefore, finer interrogations of the human epigenomic landscape in associating with non-coding variants are warranted. Recently, chromatin-profiling techniques have vastly improved our understanding of the numerous functions mediated by the epigenome and DNA structure. Here, we review various chromatin-profiling techniques, such as assays of chromatin accessibility, nucleosome distribution, histone modifications, and chromatin topology, and discuss their applications in unraveling the brain epigenome and etiology of complex traits at tissue homogenate and single-cell resolution. These techniques have elucidated compositional and structural organizing principles of the chromatin environment. Taken together, we believe that high-resolution epigenomic and DNA structure profiling will be one of the best ways to elucidate how non-coding genetic variations impact complex diseases, ultimately allowing us to pinpoint cell-type targets with therapeutic potential.

Genomic Marks Associated with Chromatin Compartments in the CTCF, RNAPII Loop and Genomic Windows.

The nature of genome organization into two basic structural compartments is as yet undiscovered. However, it has been indicated to be a mechanism of gene expression regulation. Using the classification approach, we ranked genomic marks that hint at compartmentalization. We considered a broad range of marks, including GC content, histone modifications, DNA binding proteins, open chromatin, transcription and genome regulatory segmentation in GM12878 cells. Genomic marks were defined over CTCF or RNAPII loops, which are basic elements of genome 3D structure, and over 100 kb genomic windows. Experiments were carried out to empirically assess the whole set of features, as well as the individual features in classification of loops/windows, into compartment A or B. Using Monte Carlo Feature Selection and Analysis of Variance, we constructed a ranking of feature importance for classification. The best simple indicator of compartmentalization is DNase-seq open chromatin measurement for CTCF loops, H3K4me1 for RNAPII loops and H3K79me2 for genomic windows. Among DNA binding proteins, this is RUNX3 transcription factor for loops and RNAPII for genomic windows. Chromatin state prediction methods that indicate active elements like promoters, enhancers or heterochromatin enhance the prediction of loop segregation into compartments. However, H3K9me3, H4K20me1, H3K27me3 histone modifications and GC content poorly indicate compartments.