Chromosome Conformation Capture Research Articles

The JAK2 46/1 haplotype influences PD-L1 expression.

Although described more than a decade ago, the mechanism by which the JAK2 46/1 haplotype increases the risk of developing JAK2-mutated myeloproliferative neoplasms (MPN) remains unexplained. Inflammation and immunity are linked to MPN development and thus could be relevant to the mechanism by which 46/1 mediates its effect. Here, we show that PD-L1 expression is elevated in 46/1 haplotype both in healthy carriers and CD34+ cells from MPN patients. Using circular chromosome conformation capture (4C-seq) we observed that PD-L1 and the neighboring PD-L2 loci physically interact with JAK2 in a manner that differs between 46/1 and non-risk haplotypes. CRISPR/Cas9 genome editing identified a region within JAK2 intron 2 that influences both JAK2 and PD-L1 expression. We suggest that increased PD-L1 expression may be relevant to the mechanism by which 46/1 leads to an increased inherited risk of developing MPN.

Deletion of an evolutionarily conserved TAD boundary impacts spermatogenesis in mice†.

Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8. We then used CRISPR-Cas9 to generate a mouse line where 26kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting mild increase in the DNA-DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, higher frequency of histological abnormalities, a drop of 47-52% in efficiency of meiosis, a 15-18% reduction in efficiency of spermatogenesis, and consistently, a 12-28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.

Exploring the interplay between enhancer-promoter interactions and transcription.

Enhancers in metazoan genomes are known to activate their target genes across both short and long genomic distances. Recent advances in chromosome conformation capture assays and single-cell imaging have shed light on the underlying chromatin contacts and dynamics. Yet the relationship between 3D physical enhancer-promoter (E-P) interactions and transcriptional activation remains unresolved. In this brief review, we discuss recent studies exploring this relationship across scales: from developmental stages to the minutes surrounding transcriptional activation and from the tissue level to single-allele subcellular dynamics. We discuss how seemingly contradictory observations might be reconciled and contribute to a refined causal relationship between E-P interactions and transcription, with mutual influences.

Structural Basis of Differential Gene Expression at eQTLs Loci from High-Resolution Ensemble Models of 3D Single-Cell Chromatin Conformations.

Techniques such as high-throughput chromosome conformation capture (Hi-C) have provided a wealth of information on nucleus organization and genome important for understanding gene expression regulation. Genome-Wide Association Studies (GWASs) have identified numerous loci associated with complex traits. Expression quantitative trait loci (eQTL) studies have further linked the genetic variants to alteration in expression levels of associated target genes across individuals. However, the functional roles of many eQTLs in non-coding regions remain unclear. Current joint analyses of Hi-C and eQTLs data lack advanced computational tools, limiting what can be learned from these data. We developed a computational method for simultaneous analysis of Hi-C and eQTL data, capable of identifying a small set of non-random interactions from all Hi-C interactions. Using these non-random interactions, we reconstructed large ensembles (×105) of high-resolution single-cell 3D chromatin conformations with thorough sampling, accurately replicating Hi-C measurements. Our results revealed many-body interactions in chromatin conformation at the single-cell level within eQTL loci, providing a detailed view of how 3D chromatin structures form the physical foundation for gene regulation, including how genetic variants of eQTLs affect the expression of associated eGenes. Furthermore, our method can deconvolve chromatin heterogeneity and investigate the spatial associations of eQTLs and eGenes at subpopulation level, revealing their regulatory impacts on gene expression. Together, ensemble modeling of thoroughly sampled single-cell chromatin conformations combined with eQTL data, helps decipher how 3D chromatin structures provide the physical basis for gene regulation, expression control, and aid in understanding the overall structure-function relationships of genome organization. It is available at https://github.com/uic-liang-lab/3DChromFolding-eQTL-Loci. Supplementary data are available at Bioinformatics online.

Three-dimensional genome architecture in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a common primary hepatic tumors with a 5-year survival rate of less than 20%. Therefore, it is crucial to elucidate the molecular mechanisms of ICC. Recently, the advance of high-throughput chromosome conformation capture (Hi-C) technology help us look insight into the three-dimensional (3D) genome structure variation during tumorigenesis. However, its function in ICC pathogenesis remained unclear. Hi-C and RNA-sequencing were applied to analyze 3D genome structures and gene expression in ICC and adjacent noncancerous hepatic tissue (ANHT). Furthermore, the dysregulated genes due to 3D genome changes were validated via quantitative real-time PCR and immunohistochemistry. Primarily, the intrachromosomal interactions of chr1, chr2, chr3, and chr11 and the interchromosomal interactions of chr1-chr10, chr13-chr21, chr16-chr19, and chr19-chr22 were also significantly distinct between ANHT and ICC, which may potentially contribute to the activation of cell migration and invasion via the upregulation of WNT10A, EpCAM, S100A3/A6, and MAPK12. Interestingly, 56 compartment regions from 23 chromosomes underwent A to B or B to A transitions during ICC oncogenesis, which attenuated the complement pathway through the downregulation of C8A/C8B, F7, F10, and F13B. Notably, topologically associated domain (TAD) rearrangements were identified in the region containing HOPX (chr4: 57,514,154-57,522,688) and ACVR1 (chr2:158,592,958-158,732,374) in ICC, which may contribute to the hijacking of remote enhancers that were previously outside the TAD and increased expression of HOPX and ACVR1. This study reveals relationship between 3D genome structural variations and gene dysregulation during ICC tumorigenesis, indicating the molecular mechanisms and potential biomarkers.

SMC translocation is unaffected by an excess of nucleoid associated proteins in vivo

Genome organization is important for DNA replication, gene expression, and chromosome segregation. In bacteria, two large families of proteins, nucleoid-associated proteins (NAPs) and SMC complexes, play important roles in organizing the genome. NAPs are highly abundant DNA-binding proteins that can bend, wrap, bridge, and compact DNA, while SMC complexes load onto the chromosome, translocate on the DNA, and extrude DNA loops. Although SMC complexes are capable of traversing the entire chromosome bound by various NAPs in vivo, it is unclear whether SMC translocation is influenced by NAPs. In this study, using Bacillus subtilis as a model system, we expressed a collection of representative bacterial and archaeal DNA-binding proteins that introduce distinct DNA structures and potentially pose different challenges for SMC movement. By fluorescence microscopy and chromatin immunoprecipitation, we observed that these proteins bound to the genome in characteristic manners. Using genome-wide chromosome conformation capture (Hi-C) assays, we found that the SMC complex traversed these DNA-binding proteins without slowing down. Our findings revealed that the DNA-loop-extruding activity of the SMC complex is unaffected by exogenously expressed DNA-binding proteins, which highlights the robustness of SMC motors on the busy chromatin.

Chromosome-scale and haplotype-resolved genome assembly of Populus trichocarpa

Abstract Populus trichocarpa, a pivotal model organism for woody transgenic research, not only garners substantial scientific interest but plays an integral role in forestry economics. Previous genomic assemblies of P. trichocarpa predominantly treated its heterozygous genome as homozygous, thereby neglecting crucial haplotypic diversity. Leveraging the high fidelity (HiFi) sequencing capabilities of PacBio sequencing and the chromosome conformation capture insights provided by Illumina's Hi-C technique, this study is the first to achieve a near telomere-to-telomere assembly of both paternal and maternal haplotypes in P. trichocarpa. Comparative genomic analysis between these haplotypes has uncovered several allelic variants and pathways critical for trait determination through allele-specific expression. Furthermore, utilizing RNA-seq data from multiple tissues, this investigation has detailed the tissue-specific expression patterns of the leucine-rich repeat (LRR) gene family, which are essential in mediating plant signal transduction and developmental regulation. Our results not only illuminate the functional genomics landscape of P. trichocarpa but also provide invaluable theoretical underpinnings for the genetic improvement of woody plants and a robust framework for exploring genetic variability and allelic expression disparities in arboreal species.

Comparative genomic analysis of Fusarium oxysporum f. sp. lycopersici reveals telomeric duplications of a lineage-specific region carrying SIX8 and PSL1 and genome-wide expansion of Foxy transposable elements.

Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, is a soil-borne, vascular-colonizing fungal pathogen that severely impacts tomato production in most growing regions worldwide. Despite the availability of over thirty Fol genome sequences in public databases, only one chromosome-scale assembly exists, comprising the low sequence-coverage Fol4287 reference genome generated using Sanger sequencing. Thus, genome structural variation and comparative genomics analyses of Fol remain largely unexplored. Here, using third generation Nanopore long-read sequencing in combination with high-throughput chromosome conformation capture (Hi-C) data, we have independently constructed a high-quality assembly and annotation of the Fol007 race 2 genome that consists of 15 pseudochromosomes with 25 telomeres, including 10 telomere-to-telomere chromosomes. The Fol007 genome is predicted to contain 29,148 protein-encoding genes, including all known SIX genes except for SIX4, which is absent in Fol race 2. Compared to the genome of Fusarium verticillioides, the Fol007 genome contains four complete lineage-specific (LS) chromosomes, including chromosome 5 (Chr5), chromosome 13 (Chr13), and two small chromosomes, Chr14 and Chr15. Comparative genomic analysis between the newly assembled Fol007 genome and Fol4287_2010 deposited in the GenBank database reveals that the completely assembled Chr13 of Fol007 carrying all known SIX genes (except SIX4 and SIX8) and three novel candidate effector genes is relatively stable and corresponds to pathogenicity chromosome Chr14 in Fol4287_2010. It also reveals a telomeric duplication of an LS region carrying SIX8 and PSL1 on core chromosomes Chr6, Chr7 and Chr11, and LS chromosome Chr15, as well as the absence of segmental duplications on LS chromosomes of the Fol007 genome. Furthermore, compared to the genomes of Fol race 1 and non-pathogenic Fo strains, an active and specific Foxy transposable element, responsible for the inactivation of a second copy of SIX13, was identified and found to have expanded in the genomes of Fol race 2 and 3 strains. This element may contribute to Fusarium oxysporum genome evolution and has potential as a genetic marker for studying phylogenetic relationships among formae speciales of Fusarium oxysporum. These findings provide a basis for further genetic and genomic understanding of Fol evolution and virulence mechanisms employed by Fol and contribute another reference genome for Fusarium study more broadly.

Identification of a distal enhancer of Ucp1 essential for thermogenesis and mitochondrial function in brown fat

Uncoupling protein 1 (UCP1) is a crucial protein located in the mitochondrial inner membrane that mediates nonshivering thermogenesis. However, the molecular mechanisms by which enhancer–promoter chromatin interactions control Ucp1 transcriptional regulation in brown adipose tissue (BAT) are unclear. Here, we employed circularized chromosome conformation capture coupled with next-generation sequencing (4C-seq) to generate high-resolution chromatin interaction profiles of Ucp1 in interscapular brown adipose tissue (iBAT) and epididymal white adipose tissue (eWAT) and revealed marked changes in Ucp1 chromatin interaction between iBAT and eWAT. Next, we identified four iBAT-specific active enhancers of Ucp1, and three of them were activated by cold stimulation. Transcriptional repression of the Ucp1-En4 or Ucp1-En6 region significantly downregulated Ucp1 and impaired mitochondrial function in brown adipocytes. Furthermore, depletion of the cohesin subunit RAD21 decreased the interaction intensity between Ucp1-En4 and the Ucp1 promoter and downregulated Ucp1. EBF2 cooperated with the acetyltransferase CBP to regulate Ucp1-En4 activity and increase Ucp1 transcriptional activity. In vivo, lentivirus-mediated repression of Ucp1-En4 was injected into iBAT, resulting in impacted iBAT thermogenic capacity and impaired iBAT mitochondrial function under cold acclimation conditions. Studying the functional enhancers regulating Ucp1 expression in iBAT will provide important insights into the regulatory mechanisms of BAT activity.

The haplotype-resolved and chromosome-level reference genome assembly of Diospyros deyangensis provides insights into the evolution and juvenile growth of persimmon

Abstract The Diospyros genus, which includes both wild and cultivated species such as Diospyros lotus and Diospyros kaki, represents a diverse genetic pool with significant agricultural value. In this study, we present a high-quality, haplotype-resolved, chromosome-level genome assembly for D. deyangensis (hereinafter referred to as “Deyangshi”), an autotetraploid wild species notable for its short juvenile phase, by integrating high-fidelity single-molecule, nanopore sequencing, and high-throughput chromosome conformation capture techniques. The assembled genome size is approximately 3.01 Gb, anchored onto 60 pseudo-chromosomes. Comparative genomic analysis revealed that the D. deyangensis genome underwent an additional whole-genome duplication event following the eudicots shared ancient hexaploidy event. Resequencing and clustering on 63 samples representing 11 geographically diverse Diospyros accessions revealed significant genetic differentiation between D. deyangensis and D. kaki, as well as between D. kaki and other Diospyros species using population genomic analyses, suggesting that D. kaki followed an independent evolutionary pathway. Additionally, we identified DdELF4 (EARLY FLOWERING 4) from the “Deyangshi” backcross population using bulked segregant RNA sequencing (BSR-seq) with 50 early flowering and 50 non-early flowering individuals. Overexpression of DdELF4 in Arabidopsis resulted in delayed flowering and down-regulation of FT gene expression, indicating its role as a flowering repressor. This high-quality genome assembly of “Deyangshi” provides an essential genomic resource for the Diospyros genus, particularly for breeding programs focused on developing early-flowering persimmon varieties.

A chromosome-level, haplotype-resolved genome assembly and annotation for the Eurasian minnow (Leuciscidae: Phoxinus phoxinus) provide evidence of haplotype diversity.

In this study, we present an in-depth analysis of the Eurasian minnow (Phoxinus phoxinus) genome, highlighting its genetic diversity, structural variations, and evolutionary adaptations. We generated an annotated haplotype-phased, chromosome-level genome assembly (2n = 50) by integrating high-fidelity (HiFi) long reads and chromosome conformation capture data (Hi-C). We achieved a haploid size of 940 megabase pairs (Mbp) for haplome 1 and 929 Mbp for haplome 2 with high scaffold N50 values of 36.4 Mb and 36.6 Mb and BUSCO scores of 96.9% and 97.2%, respectively, indicating a highly complete genome assembly. We detected notable heterozygosity (1.43%) and a high repeat content (approximately 54%), primarily consisting of DNA transposons, which contribute to genome rearrangements and variations. We found substantial structural variations within the genome, including insertions, deletions, inversions, and translocations. These variations affect genes enriched in functions such as dephosphorylation, developmental pigmentation, phagocytosis, immunity, and stress response. In the annotation of protein-coding genes, 30,980 messenger RNAs and 23,497 protein-coding genes were identified with a high completeness score, which further underpins the high contiguity of our genome assemblies. We performed a gene family evolution analysis by comparing our proteome to 10 other teleost species, which identified immune system gene families that prioritize histone-based disease prevention over NB-LRR-related-based immune responses. Additionally, demographic analysis indicates historical fluctuations in the effective population size of P. phoxinus, likely correlating with past climatic changes. This annotated, phased reference genome provides a crucial resource for resolving the taxonomic complexity within the genus Phoxinus and highlights the importance of haplotype-phased assemblies in understanding haplotype diversity in species characterized by high heterozygosity.

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.

Using Chromosome Conformation Capture Combined with Deep Sequencing (Hi-C) to Study Genome Organization in Bacteria.

Genome organization is fundamental to all living organisms. Long DNA molecules are organized in hierarchical orders to be accommodated into eukaryotic nuclei or bacterial cells, which are thousands of folds shorter. Over the past two decades, chromosome conformation capture (3C) techniques substantially advanced our understanding of genome folding inside cells. 3C involves crosslinking and proximity ligation, and quantifies the physical contacts between two DNA regions within the genome. Coupled with high-throughput sequencing, 3C-seq and Hi-C techniques detect genome-wide DNA interactions, providing a comprehensive view of global genome organization. Here, we describe a detailed method to prepare Hi-C libraries using Bacillus subtilis, which includes procedures of crosslinking chromatin, digesting the crosslinked genome, labeling DNA ends with biotin, ligating DNA, and preparing the DNA library for sequencing using an Illumina platform.

Read Mapping for Hi-C Analysis.

Hi-C is a popular ligation-based technique to detect 3D physical chromosome structure within the nucleus using cross-linking and next-generation sequencing. As an unbiased genome-wide assay based on chromosome conformation capture, it provides rich insights into chromosome structure, dynamic chromosome folding and interactions, and the regulatory state of a cell. Bioinformatics analyses of Hi-C data require dedicated protocols as most genome alignment tools assume that both paired-end reads will map to the same chromosome, resulting in large two-dimensional matrices as processed data. Here, we outline the necessary steps to generate high-quality aligned Hi-C data by separately mapping each read while correcting for biases from restriction enzyme digests. We introduce our own custom open-source pipeline, which enables users to select an aligner of their choosing with high accuracy and performance. This enables users to generate high-resolution datasets with fast turnaround and fewer unmapped reads. Finally, we discuss recent innovations in experimental techniques, bioinformatics techniques, and their applications in clinical testing for diagnostics.

Systematic Inference of Multi-scale Chromatin Sub-compartments Using Calder2.

The compartmentalization of chromatin reflects its underlying biological activities. Inferring chromatin sub-compartments using Hi-C data is challenged by data resolution constraints. Consequently, comprehensive characterizations of sub-compartments have been limited to a select number of Hi-C experiments, with systematic comparisons across a wide range of tissues and conditions still lacking. Our original Calder algorithm marked a significant advancement in this field, enabling the identification of multi-scale sub-compartments at various data resolutions and facilitating the inference and comparison of chromatin architecture in over 100 datasets. Building on this foundation, we introduce Calder2, an updated version of Calder that brings notable improvements. These include expanded support for a wider array of genomes and organisms, an optimized bin size selection approach for more accurate chromatin compartment detection, and extended support for input and output formats. Calder2 thus stands as a refined analysis tool, significantly advancing genome-wide studies of 3D chromatin architecture and its functional implications.

A generalizable Hi-C foundation model for chromatin architecture, single-cell and multi-omics analysis across species.

Nuclear DNA is organized into a compact three-dimensional (3D) structure that impacts critical cellular processes. High-throughput chromosome conformation capture (Hi-C) is the most widely used method for measuring 3D genome architecture, while linear epigenomic assays, such as ATAC-seq, DNase-seq, and ChIP-seq, are extensively employed to characterize epigenomic regulation. However, the integrative analysis of chromatin interactions and associated epigenomic regulation remains challenging due to the pairwise nature of Hi-C data, mismatched resolution between Hi-C and epigenomic assays, and inconsistencies among analysis tools. Here we propose HiCFoundation, a Hi-C-based foundation model for integrative analysis linking chromatin structure to downstream regulatory function. HiCFoundation is trained from hundreds of Hi-C assays encompassing 118 million contact matrix submatrices. The model achieves state-of-the-art performance on multiple types of 3D genome analysis, including reproducibility analysis, resolution enhancement, and loop detection. We further demonstrate the model's generalizability through genome architecture analysis of 316 species. Notably, by enhancing low-coverage experimental Hi-C data, HiCFoundation reveals genome-wide loop loss during differentiation of hematopoietic stem and progenitor cells (HSPCs) to neutrophils. Additionally, HiCFoundation is able to predict multiple types of epigenomic activity from Hi-C input and further interprets the link between Hi-C input and epigenomic output to reveal the relationship between chromatin conformation and genome function. Finally, HiCFoundation can analyze single-cell Hi-C data, shedding light on genome structure at single-cell resolution. HiCFoundation thus provides a unified, efficient, generalizable, and interpretable foundation for genome architecture, single-cell and multi-omics analysis across species, paving the path for systematically studying genome 3D architecture and its regulatory mechanisms.

Chromosome-level genome assembly and annotation of a sea toad (Chaunax sp.)

The sea toad genus Chaunax is a group of small benthic fishes that predominantly inhabiting the deep seas of the Atlantic, Indian, and Pacific Oceans. Although they have the potential to make excellent systems for studies of evolutionary adaptation to deep-sea environments, genomic research on Chaunax has been hindered by a scarcity of high-quality genomic resources. We present a chromosome-scale genome assembly of a Chaunax specimen generated using PacBio long-read sequencing and high-throughput chromosome conformation capture technology. The size of the assembled genome was 706.94 Mb, with a contig N50 of 15.24 Mb and scaffold N50 of 29.42 Mb. Approximately 96.11% of assembled sequences were anchored and oriented onto 24 pseudo-chromosomes. The genome contained 213.47 Mb repetitive sequences, 25,280 protein-coding genes, and 5,090 non-coding RNAs. The high ratio of complete BUSCO genes (97.20%) indicates high quality of genome assembly. The chromosomal-level reference genome of Chaunax sp. provides a preliminary molecular basis for understanding deep-sea adaptation and phenotypic evolution as well as an important reference for whole-genome sequencing of related species.

ChiTaRS 8.0: the comprehensive database of chimeric transcripts and RNA-seq data with applications in liquid biopsy.

Gene fusions are nucleotide sequences formed due to errors in replication and transcription control. These errors, resulting from chromosomal translocation, transcriptional errors or trans-splicing, vary from cell to cell. The identification of fusions has become critical as key biomarkers for disease diagnosis and therapy in various cancers, significantly influencing modern medicine. Chimeric Transcripts and RNA-Sequencing database version 8.0 (ChiTaRS 8.0; http://biosrv.org/chitars) is a specialized repository for human chimeric transcripts, containing 47445 curated RNA transcripts and over 100000 chimeric sequences in humans. This updated database provides unique information on 1055 chimeric breakpoints derived from public datasets using chromosome conformation capture techniques (the Hi-C datasets). It also includes an expanded list of gene fusions that are potential drug targets, and chimeric breakpoints across 934 cell lines, positioning ChiTaRS 8.0 as a valuable resource for testing personalized cancer therapies. By utilizing text mining on a curated selection of disease-specific RNA-sequencing data from public datasets, as well as patient blood and plasma samples, we have identified novel chimeras-particularly in diseases such as oral squamous cell carcinoma and glioblastoma-now catalogued in ChiTaRS. Thus, ChiTaRS 8.0 serves as an enhanced fusion transcript repository that incorporates insights into the functional landscape of chimeras in cancers and other complex diseases, based on liquid biopsy results.

Hi-C Calibration by Chemically Induced Chromosomal Interactions.

The genome-wide chromosome conformation capture method, Hi-C, has greatly advanced our understanding of genome organization. However, its quantitative properties, including sensitivity, bias, and linearity, remain challenging to assess. Measuring these properties in vivo is difficult due to the heterogenous and dynamic nature of chromosomal interactions. Here, using Chemically Induced Chromosomal Interaction (CICI) method, we create stable intra- and inter-chromosomal interactions in G1-phase budding yeast across a broad range of contact frequencies. Hi-C analysis of these engineered cell populations demonstrates that static intra-chromosomal loops do not generate Topologically Associated Domains (TADs) and only promote 3D proximity within ∼50kb flanking regions. At moderate sequencing depth, Hi-C is sensitive enough to detect interactions occurring in 5-10% of cells. It also shows no inherent bias toward intra-versus inter-chromosomal interactions. Furthermore, we observe a linear relationship between Hi-C signal intensity and contact frequency. These findings illuminate the intrinsic properties of the Hi-C assay and provide a robust framework for its calibration.

The cells are all-right: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells.

Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.