Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to ...comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.
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•ChIA-PET is inclusive in mapping 3D genome at multi-scale and nucleotide resolution•CTCF foci spatially arrange RNAPII transcription concordant in CTCF-motif direction•SNPs alter haplotype chromatin topology and function that link to disease risks•3D genome models elucidate topological framework for transcriptional regulation
Advanced ChIA-PET shows that CTCF/cohesin and RNA polymerase II arrange spatial organization for coordinated transcription. Haplotype variants exhibit allelic effects on chromatin topology and transcription that link disease susceptibility.
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin ...immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.
The genomes of multicellular organisms are extensively folded into 3D chromosome territories within the nucleus
. Advanced 3D genome-mapping methods that combine proximity ligation and ...high-throughput sequencing (such as chromosome conformation capture, Hi-C)
, and chromatin immunoprecipitation techniques (such as chromatin interaction analysis by paired-end tag sequencing, ChIA-PET)
, have revealed topologically associating domains
with frequent chromatin contacts, and have identified chromatin loops mediated by specific protein factors for insulation and regulation of transcription
. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C
potentially overcomes this issue, this method may be limited by the sparsity of data that is inherent to current single-cell assays. Recent advances in microfluidics have opened opportunities for droplet-based genomic analysis
but this approach has not yet been adapted for chromatin interaction analysis. Here we describe a strategy for multiplex chromatin-interaction analysis via droplet-based and barcode-linked sequencing, which we name ChIA-Drop. We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with single-molecule precision, which has not been possible using methods based on population-level pairwise contacts. By applying ChIA-Drop to Drosophila cells, we show that chromatin topological structures predominantly consist of multiplex chromatin interactions with high heterogeneity; ChIA-Drop also reveals promoter-centred multivalent interactions, which provide topological insights into transcription.
EndoC-βH1 is emerging as a critical human β cell model to study the genetic and environmental etiologies of β cell (dys)function and diabetes. Comprehensive knowledge of its molecular landscape is ...lacking, yet required, for effective use of this model. Here, we report chromosomal (spectral karyotyping), genetic (genotyping), epigenomic (ChIP-seq and ATAC-seq), chromatin interaction (Hi-C and Pol2 ChIA-PET), and transcriptomic (RNA-seq and miRNA-seq) maps of EndoC-βH1. Analyses of these maps define known (e.g., PDX1 and ISL1) and putative (e.g., PCSK1 and mir-375) β cell-specific transcriptional cis-regulatory networks and identify allelic effects on cis-regulatory element use. Importantly, comparison with maps generated in primary human islets and/or β cells indicates preservation of chromatin looping but also highlights chromosomal aberrations and fetal genomic signatures in EndoC-βH1. Together, these maps, and a web application we created for their exploration, provide important tools for the design of experiments to probe and manipulate the genetic programs governing β cell identity and (dys)function in diabetes.
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•Comprehensive multiomic maps of EndoC-βH1 human β cell line and primary islets•Sequence motifs enriched in EndoC-specific enhancers reflect its precursor state•Identification of regulatory hubs preserved between EndoC-βH1 and human islets•Identified SNP alleles (including T2D GWAS) altering cis-regulatory signatures
EndoC-βH1 is becoming an important cellular model to study genes and pathways governing human β cell identity and function, but its (epi)genomic similarity to primary human islets is unknown. Lawlor et al. complete and compare extensive EndoC and primary human islet multiomic maps to identify shared and distinct genomic circuitry.
The genomes of multicellular organisms are extensively folded into 3D chromosome territories within the nucleus. Advanced 3D genome-mapping methods that combine proximity ligation and high-throughput ...sequencing (such as chromosome conformation capture, Hi-C), and chromatin immunoprecipitation techniques (such as chromatin interaction analysis by paired-end tag sequencing, ChIA-PET), have revealed topologically associating domains with frequent chromatin contacts, and have identified chromatin loops mediated by specific protein factors for insulation and regulation of transcription. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C potentially overcomes this issue, this method may be limited by the sparsity of data that is inherent to current single-cell assays. Recent advances in microfluidics have opened opportunities for droplet-based genomic analysis but this approach has not yet been adapted for chromatin interaction analysis. Here we describe a strategy for multiplex chromatin-interaction analysis via droplet-based and barcode-linked sequencing, which we name ChIA-Drop. We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with single-molecule precision, which has not been possible using methods based on population-level pairwise contacts. By applying ChIA-Drop to Drosophila cells, we show that chromatin topological structures predominantly consist of multiplex chromatin interactions with high heterogeneity; ChIA-Drop also reveals promoter-centred multivalent interactions, which provide topological insights into transcription.
Remarkably, cell types sharing the same linear genome sequence express different genes and have distinct functions. 3D genomic arrangement has been demonstrated to play a critical role in this ...process. Fine scale organization of genes and regulatory elements within active and inactive domains underlie gene expression and disruption of this process has been shown to influence development and disease. Yet, the precise dynamics of cell type specific 3D genomic interactions mediating mammalian gene expression, such as those between enhancers and promoters, remain lacking on a genome wide level. Neurons represent a specialized cell type known to respond to a myriad of physiological stimuli by changes in transcription of activity dependent (AD) genes. Neuron specific enhancer activation has been identified genome wide in mouse neurons during AD gene transcription, yet we lack genome wide 3D connectivity information allowing assignment of AD enhancer gene targets. Many questions about chromatin structure and how 3D structural changes influence cell type specificity and function through changes in gene expression remain unanswered due to technological limitations and the nature of biological samples required. This thesis specifically addresses such limitations focusing on application to mammalian genomes.
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