Showing papers on "Chromosome conformation capture published in 2023"

Cell-type-specific prediction of 3D chromatin organization enables high-throughput in silico genetic screening

Abstract: Investigating how chromatin organization determines cell-type-specific gene expression remains challenging. Experimental methods for measuring three-dimensional chromatin organization, such as Hi-C, are costly and have technical limitations, restricting their broad application particularly in high-throughput genetic perturbations. We present C.Origami, a multimodal deep neural network that performs de novo prediction of cell-type-specific chromatin organization using DNA sequence and two cell-type-specific genomic features-CTCF binding and chromatin accessibility. C.Origami enables in silico experiments to examine the impact of genetic changes on chromatin interactions. We further developed an in silico genetic screening approach to assess how individual DNA elements may contribute to chromatin organization and to identify putative cell-type-specific trans-acting regulators that collectively determine chromatin architecture. Applying this approach to leukemia cells and normal T cells, we demonstrate that cell-type-specific in silico genetic screening, enabled by C.Origami, can be used to systematically discover novel chromatin regulation circuits in both normal and disease-related biological systems.

Synthetic analysis of chromatin tracing and live-cell imaging indicates pervasive spatial coupling between genes

Abstract: The role of the spatial organization of chromosomes in directing transcription remains an outstanding question in gene regulation. Here, we analyze two recent single-cell imaging methodologies applied across hundreds of genes to systematically analyze the contribution of chromosome conformation to transcriptional regulation. Those methodologies are (1) single-cell chromatin tracing with super-resolution imaging in fixed cells; and (2) high-throughput labeling and imaging of nascent RNA in living cells. Specifically, we determine the contribution of physical distance to the coordination of transcriptional bursts. We find that individual genes adopt a constrained conformation and reposition toward the centroid of the surrounding chromatin upon activation. Leveraging the variability in distance inherent in single-cell imaging, we show that physical distance - but not genomic distance - between genes on individual chromosomes is the major factor driving co-bursting. By combining this analysis with live-cell imaging, we arrive at a corrected transcriptional correlation of [Formula: see text] for genes separated by < 400 nm. We propose that this surprisingly large correlation represents a physical property of human chromosomes and establishes a benchmark for future experimental studies.

Mapping the semi-nested community structure of 3D chromosome contact networks

Abstract: Mammalian DNA folds into 3D structures that facilitate and regulate genetic processes such as transcription, DNA repair, and epigenetics. Several insights derive from chromosome capture methods, such as Hi-C, which allow researchers to construct contact maps depicting 3D interactions among all DNA segment pairs. These maps show a complex cross-scale organization spanning megabase-pair compartments to short-ranged DNA loops. To better understand the organizing principles, several groups analyzed Hi-C data assuming a Russian-doll-like nested hierarchy where DNA regions of similar sizes merge into larger and larger structures. Apart from being a simple and appealing description, this model explains, e.g., the omnipresent chequerboard pattern seen in Hi-C maps, known as A/B compartments, and foreshadows the co-localization of some functionally similar DNA regions. However, while successful, this model is incompatible with the two competing mechanisms that seem to shape a significant part of the chromosomes’ 3D organization: loop extrusion and phase separation. This paper aims to map out the chromosome’s actual folding hierarchy from empirical data. To this end, we take advantage of Hi-C experiments and treat the measured DNA-DNA interactions as a weighted network. From such a network, we extract 3D communities using the generalized Louvain algorithm. This algorithm has a resolution parameter that allows us to scan seamlessly through the community size spectrum, from A/B compartments to topologically associated domains (TADs). By constructing a hierarchical tree connecting these communities, we find that chromosomes are more complex than a perfect hierarchy. Analyzing how communities nest relative to a simple folding model, we found that chromosomes exhibit a significant portion of nested and non-nested community pairs alongside considerable randomness. In addition, by examining nesting and chromatin types, we discovered that nested parts are often associated with active chromatin. These results highlight that crossscale relationships will be essential components in models aiming to reach a deep understanding of the causal mechanisms of chromosome folding.

Multiscale 3D genome reorganization during skeletal muscle stem cell lineage progression and aging

Abstract: Little is known about three-dimensional (3D) genome organization in skeletal muscle stem cells [also called satellite cells (SCs)]. Here, we comprehensively map the 3D genome topology reorganization during mouse SC lineage progression. Specifically, rewiring at the compartment level is most pronounced when SCs become activated. Marked loss in topologically associating domain (TAD) border insulation and chromatin looping also occurs during early activation process. Meanwhile, TADs can form TAD clusters and super-enhancer–containing TAD clusters orchestrate stage-specific gene expression. Furthermore, we uncover that transcription factor PAX7 is pivotal in enhancer-promoter (E-P) loop formation. We also identify cis-regulatory elements that are crucial for local chromatin organization at Pax7 locus and Pax7 expression. Lastly, we unveil that geriatric SC displays a prominent gain in long-range contacts and loss of TAD border insulation. Together, our results uncover that 3D chromatin extensively reorganizes at multiple architectural levels and underpins the transcriptome remodeling during SC lineage development and SC aging.

Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and physical proximity

Abstract: Enhancers are often located tens to hundreds of kilobases away from the genes they regulate. How they exert this regulation in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we have used the activation of estrogen receptor-dependent enhancers in the breast cancer cell line MCF-7 as a model system to study enhancer-promoter communication. This system allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine both enhancer-promoter proximity by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation by Capture-C. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate the roles of enhancer-bound protein complexes and transcription in enhancer-promoter contact frequencies and proximity. Our work further emphasizes that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one, and is modulated by the biochemical and biophysical context of the loci under study.

Easy Hi-C: A Low-Input Method for Capturing Genome Organization.

Abstract: Chromosome conformation capture technology and its derivatives have been widely used to study genome organization. Among them, Hi-C (chromosome conformation capture coupling with high-throughput sequencing) is popular in dissecting chromatin architecture on the genome-wide level. However, the intrinsic limitations prevent its application when it comes to rare samples. Here, we present easy Hi-C, a biotin-free technology that dramatically reduces DNA loss and is suitable for low-input samples.

Polymer simulations guide the detection and quantification of chromatin loop extrusion by imaging

Abstract: Abstract Genome-wide chromosome conformation capture (Hi-C) has revealed the organization of chromatin into topologically associating domains (TADs) and loops, which are thought to help regulate genome functions. TADs and loops are understood as the result of DNA extrusion mediated by the cohesin complex. However, despite recent efforts, direct visualization and quantification of this process in single cells remains an open challenge. Here, we use polymer simulations and dedicated analysis methods to explore if, and under which conditions, DNA loop extrusion can be detected and quantitatively characterized by imaging pairs of fluorescently labeled loci located near loop or TAD anchors in fixed or living cells. We find that under realistic conditions, extrusion can be detected and the frequency of loop formation can be quantified from fixed cell images alone, while the lifetime of loops and the speed of extrusion can be estimated from dynamic live-cell data. Our delineation of appropriate imaging conditions and the proposed analytical methods lay the groundwork for a systematic quantitative characterization of loop extrusion in fixed or living cells.

Linking genome structures to functions by simultaneous single-cell Hi-C and RNA-seq

Abstract: Much progress has been made recently in single-cell chromosome conformation capture technologies. However, a method that allows simultaneous profiling of chromatin architecture and gene expression has not been reported. Here, we developed an assay named “Hi-C and RNA-seq employed simultaneously” (HiRES) and performed it on thousands of single cells from developing mouse embryos. Single-cell three-dimensional genome structures, despite being heavily determined by the cell cycle and developmental stages, gradually diverged in a cell type–specific manner as development progressed. By comparing the pseudotemporal dynamics of chromatin interactions with gene expression, we found a widespread chromatin rewiring that occurred before transcription activation. Our results demonstrate that the establishment of specific chromatin interactions is tightly related to transcriptional control and cell functions during lineage specification. Description Editor’s summary Whether changes in the three-dimensional (3D) structures of chromosomes regulate gene expression during development has been hotly debated. A major challenge to studying these systems is how to relate a long-range chromatin interaction thousands of base pairs away to its target gene. Liu et al. developed a multi-omics sequencing approach to profile 3D genome structures and gene expression at the same time in single cells, which enables a direct linkage between chromatin conformation and gene expression. Leveraging this new tool, the authors identified widespread chromatin interactions, especially between loci enriched for active enhancers, and found that they were rewired before gene activation during mouse embryonic development. This observation suggests that dynamic chromatin organization plays a critical role in the control of gene expression. —Di Jiang Joint profiling of a single cell’s 3D genome and transcriptome revealed specific chromatin dynamics associated with transcription during development.

A graph neural network-based interpretable framework reveals a novel DNA fragility–associated chromatin structural unit

Abstract: DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, and they can cause cancer if improperly repaired. Recent chromosome conformation capture techniques, such as Hi-C, have enabled the identification of relationships between the 3D chromatin structure and DSBs, but little is known about how to explain these relationships, especially from global contact maps, or their contributions to DSB formation.Here, we propose a framework that integrates graph neural network (GNN) to unravel the relationship between 3D chromatin structure and DSBs using an advanced interpretable technique GNNExplainer. We identify a new chromatin structural unit named the DNA fragility-associated chromatin interaction network (FaCIN). FaCIN is a bottleneck-like structure, and it helps to reveal a universal form of how the fragility of a piece of DNA might be affected by the whole genome through chromatin interactions. Moreover, we demonstrate that neck interactions in FaCIN can serve as chromatin structural determinants of DSB formation.Our study provides a more systematic and refined view enabling a better understanding of the mechanisms of DSB formation under the context of the 3D genome.

Topological constraints and finite-size effects in quantitative polymer models of chromatin organization

Abstract: Polymer physics simulations have provided a versatile framework to quantitatively explore the complex mechanisms driving chromosome organization. However, simulating whole chromosomes over biologically-relevant timescales at high resolution often constitutes a computationally-intensive task — while genes or other regions of biological interest may typically only span a small fraction of the full chromosome length. Conversely, only simulating the sub-chromosomal region of interest might provide an over-simplistic or even wrong description of the mechanism controlling the 3D organization. In this work, we characterize what should be the minimal length of chromosome to be simulated in order to correctly capture the properties of a given restricted region. In particular, since the physics of long, topologically-constrained polymers may significantly deviate from those of shorter chains, we theoretically investigate how chromosomes being a long polymer quantitatively affects the structure and dynamics of its sub-segments. We show that increasing the total polymer length impacts on the topological constraints acting on the system and thus affects the compaction and mobility of sub-chains. Depending on the entanglement properties of the system, we derive a phenomenological relation defining the minimal total length to account for to maintain a correct topological regime. We finally detail the implications of these conclusions in the case of several specific biological systems.

LPAD: using network construction and label propagation to detect topologically associating domains from Hi-C data

Abstract: With the development of chromosome conformation capture technique, the study of spatial conformation of a genome based on Hi-C technique has made a quantum leap. Previous studies reveal that genomes are folded into hierarchy of three-dimensional (3D) structures associated with topologically associating domains (TADs), and detecting TAD boundaries is of great significance in the chromosome-level analysis of 3D genome architecture. In this paper, we propose a novel TAD identification method, LPAD, which first extracts node correlations from global interactions of chromosomes based on the random walk with restart and then builds an undirected graph from Hi-C contact matrix. Next, LPAD designs a label propagation-based approach to discover communities and generates TADs. Experimental results verify the effectiveness and quality of TAD detections compared with existing methods. Furthermore, experimental evaluation of chromatin immunoprecipitation sequencing data shows that LPAD performs high enrichment of histone modifications remarkably nearby the TAD boundaries, and these results demonstrate LPAD's advantages on TAD identification accuracy.

Spatial chromatin accessibility sequencing resolves high-order spatial interactions of epigenomic markers

Abstract: As the genome is organized into a three-dimensional structure in intracellular space, epigenomic information also has a complex spatial arrangement. However, most epigenetic studies describe locations of methylation marks, chromatin accessibility regions, and histone modifications in the horizontal dimension. Proper spatial epigenomic information has rarely been obtained. In this study, we designed spatial chromatin accessibility sequencing (SCA-seq) to resolve the genome conformation by simultaneously capturing the epigenetic information in single-molecular resolution. Using SCA-seq, we simultaneously disclosed spatial interaction of chromatin accessibility (e.g. enhancer-promoter contacts), CpG island methylation, and spatial insulating functions of the CCCTC-binding factor. We demonstrate that SCA-seq paves the way to explore the mechanism of epigenetic interactions and extends our knowledge in 3D packaging of DNA in the nucleus.

3D genomics and its applications in precision medicine

Abstract: Three-dimensional (3D) genomics is an emerging discipline that studies the three-dimensional structure of chromatin and the three-dimensional and functions of genomes. It mainly studies the three-dimensional conformation and functional regulation of intranuclear genomes, such as DNA replication, DNA recombination, genome folding, gene expression regulation, transcription factor regulation mechanism, and the maintenance of three-dimensional conformation of genomes. Self-chromosomal conformation capture (3C) technology has been developed, and 3D genomics and related fields have developed rapidly. In addition, chromatin interaction analysis techniques developed by 3C technologies, such as paired-end tag sequencing (ChIA-PET) and whole-genome chromosome conformation capture (Hi-C), enable scientists to further study the relationship between chromatin conformation and gene regulation in different species. Thus, the spatial conformation of plant, animal, and microbial genomes, transcriptional regulation mechanisms, interaction patterns of chromosomes, and the formation mechanism of spatiotemporal specificity of genomes are revealed. With the help of new experimental technologies, the identification of key genes and signal pathways related to life activities and diseases is sustaining the rapid development of life science, agriculture, and medicine. In this paper, the concept and development of 3D genomics and its application in agricultural science, life science, and medicine are introduced, which provides a theoretical basis for the study of biological life processes.

An upgraded method of high-throughput chromosome conformation capture (Hi-C 3.0) in cotton (Gossypium spp.)

Abstract: High-throughput chromosome conformation capture (Hi-C) technology has been applied to explore the chromatin interactions and shed light on the biological functions of three-dimensional genomic features. However, it remains challenging to guarantee the high quality of Hi-C library in plants and hence the reliable capture of chromatin structures, especially loops, due to insufficient fragmentation and low efficiency of proximity ligations. To overcome these deficiencies, we optimized the parameters of the Hi-C protocol, principally the cross-linking agents and endonuclease fragmentation strategy. The double cross-linkers (FA+DSG) and double restriction enzymes ( Dpn II+ Dde I) were utilized. Thus, a systematic in situ Hi-C protocol was designed using plant tissues embedded with comprehensive quality controls to monitor the library construction. This upgraded method, termed Hi-C 3.0, was applied to cotton leaves for trial. In comparison with the conventional Hi-C 2.0, Hi-C 3.0 can obtain more than 50% valid contacts at a given sequencing depth to improve the signal-to-noise ratio. Hi-C 3.0 can furthermore enhance the capturing of loops almost as twice as that of Hi-C 2.0. In addition, Hi-C 3.0 showed higher efficiency of compartment detection and identified compartmentalization more accurately. In general, Hi-C 3.0 contributes to the advancement of the Hi-C method in plants by promoting its capability on decoding the chromatin organization.

Abstract 5239: Integrative analysis of 3D chromatin organization at GWAS loci identifies RAPGEF1 as a melanoma susceptibility gene

Abstract: Many GWAS loci occur in non-coding regions and often overlap with gene regulatory elements such as distant enhancers, making functional interpretation and target gene discovery challenging. The 3D chromatin organization brings enhancers in spatial proximity with a promoter to regulate target gene expression. Therefore, to map chromatin interactions between GWAS variants and target gene promoters we performed region-focused chromatin conformation capture assay (Capture-C) in primary human melanocytes. We baited the entire region of association for all 68 independent signals from the recent melanoma GWAS, and Capture-C interactions were called using CHiCAGO tool. Integrative analysis of Capture-C interactions with melanocyte- and melanoma-specific ATAC-sequencing, massive-parallel reporter assay (MPRA), ROADMAP chromatin imputed state model, and gene expression datasets helped prioritize target genes for functional follow-up. Capture-C assays identified physical chromatin interactions between fine-mapped risk variants and candidate causal gene (CCG) promoters at 90% of the GWAS loci; For 84% of the 68 loci, we observed at least one variant-to-gene promoter interaction longer than 100 kb, and for 20% of loci we found interactions beyond 1 Mb. For 76% of the 68 loci, the CCG-interacting variant was in annotated melanocyte or melanoma enhancer regions consistent with CCG regulation via an enhancer-promoter interaction. We observed at least one CCG-interacting variant in 63% and 51% of the 68 loci with distinct allele specific transcriptional activity in melanocyte and melanoma MPRA datasets respectively. A majority of the loci (60%) harbored CCG-linked risk variants in accessible chromatin regions in melanocytes and melanoma. Pathway enrichment analyses of Capture-C-nominated CCGs identified embryonic development, aryl hydrocarbon receptor signaling, and DNA repair pathways. Notably, we observed chromatin interactions between risk variants located near the 3’ of Rap Guanine Nucleotide Exchange Factor 1 (RAPGEF1) to the RAPGEF1 and UCK1 promoter regions. The risk allele of the lead variant (rs3780269) at this locus was associated with higher RAPGEF1 mRNA expression and was not associated with UCK1 expression in melanocytes. We performed a CRISPR knockout proliferation screen in immortalized melanocytes and identified RAPGEF1 as an essential gene for melanocyte growth or survival. Further, we validated the results of this screen by overexpressing RAPGEF1 in immortalized melanocytes and found that it leads to increased cellular growth. We are now characterizing its function in melanoma tumor incidence and progression in a zebrafish model. In summary, mapping GWAS loci chromatin interactions to target gene promoters and integrative analysis using cell-type specific datasets identified RAPGEF1 as a melanoma susceptibility gene. Citation Format: Rohit Thakur, Mai Xu, Alexandra Thornock, Hayley Sowards, Epring Long, Thomas Rheling, Karen Funderburk, Jinhu Yin, Rebecca Hennessey, Raj Chari, Tongwu Zhang, Lea Jessop, Timothy Myers, Matthew E. Johnson, Andrew D. Wells, Alessandra Chesi, Struan F. Grant, Mark I. Iles, Maria T. Landi, Matthew Law, Melanoma Meta-Analysis Consortium, Mitchell Machiela, Jiyeon Choi, Leonard I. Zon, Kevin M. Brown. Integrative analysis of 3D chromatin organization at GWAS loci identifies RAPGEF1 as a melanoma susceptibility gene. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5239.

Regulation of loop extrusion on the interphase genome

Abstract: Abstract In the human cell nucleus, dynamically organized chromatin is the substrate for gene regulation, DNA replication, and repair. A central mechanism of DNA loop formation is an ATPase motor cohesin-mediated loop extrusion. The cohesin complexes load and unload onto the chromosome under the control of other regulators that physically interact and affect motor activity. Regulation of the dynamic loading cycle of cohesin influences not only the chromatin structure but also genome-associated human disorders and aging. This review focuses on the recently spotlighted genome organizing factors and the mechanism by which their dynamic interactions shape the genome architecture in interphase.

Chromatin conformation dynamics during CD4+ T cell activation implicates autoimmune disease-associated genes and regulatory elements

Abstract: Abstract Genome-wide association studies (GWAS) have identified hundreds of genetic loci associated with autoimmune disease, but the causal variants and their contribution to immune dysregulation remain largely unknown. We measured the dynamics of chromosome conformation, chromatin accessibility, and gene transcription across three phases of naive human CD4+ T cell activation and co-localized active cis-regulatory elements with the 95% credible set of variants from 15 autoimmune GWAS. Reorganization of chromosome structure placed these elements in direct contact with ~1,200 protein-coding genes, at least one-third of which were dynamically regulated by stimulation. The set of implicated genes is enriched for high-throughput CRISPR screen targets that control multiple aspects of CD4+ T cell activation, and we pharmacologically validated eight novel gene products as potent regulators of T cell proliferation. These maps also allowed the identification and functional validation of a novel stretch of intergenic enhancers whose activity is required for IL2 gene expression and is influenced by autoimmune-associated genetic variation. This study represents a powerful strategy and resource for assigning physiologic relevance to autoimmune-risk variants and identifying novel genes that control T cell activation and function.

High-resolution Hi-C maps highlight multiscale chromatin architecture reorganization during cold stress in Brachypodium distachyon

Abstract: Abstract Background The adaptation of plants to cold stress involves changes in gene expression profiles that are associated with epigenetic regulation. Although the three-dimensional (3D) genome architecture is considered an important epigenetic regulator, the role of 3D genome organization in the cold stress response remains unclear. Results In this study, we developed high-resolution 3D genomic maps using control and cold-treated leaf tissue of the model plant Brachypodium distachyon using Hi-C to determine how cold stress affects the 3D genome architecture. We generated ~ 1.5 kb resolution chromatin interaction maps and showed that cold stress disrupts different levels of chromosome organization, including A/B compartment transition, a reduction in chromatin compartmentalization and the size of topologically associating domains (TADs), and loss of long-range chromatin loops. Integrating RNA-seq information, we identified cold-response genes and revealed that transcription was largely unaffected by the A/B compartment transition. The cold-response genes were predominantly localized in compartment A. In contrast, transcriptional changes are required for TAD reorganization. We demonstrated that dynamic TAD events were associated with H3K27me3 and H3K27ac state alterations. Moreover, a loss of chromatin looping, rather than a gain of looping, coincides with alterations in gene expression, indicating that chromatin loop disruption may play a more important role than loop formation in the cold-stress response. Conclusions Our study highlights the multiscale 3D genome reprogramming that occurs during cold stress and expands our knowledge of the mechanisms underlying transcriptional regulation in response to cold stress in plants.

Single-cell detection of primary transcripts, their genomic loci and nuclear factors by 3D immuno-RNA/DNA FISH in T cells

Abstract: Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture (“3C”-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages.

Profiling the Epigenetic Landscape of the Spermatogonial Stem Cell-Part 1: Epigenomics Assays.

Abstract: Epigenomics encompasses analyses of a variety of different epigenetic parameters which, collectively, make up the epigenetic programming that dictates cell fate and function. Here, protocols are provided for four different epigenomic methods including whole-genome bisulfite sequencing (WGBS) to assess DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to assess genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to assess genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to assess three-dimensional interactions among distant genomic regions, plus computational methodology to integrate data from those four methodologies using Chromatin State Discovery and Characterization (ChromHMM) to obtain the most comprehensive overall assessment of epigenetic programming.