3D genome organization and its study in livestock breeding

Eukaryotic genomes are hierarchically packaged into cell nucleus,affecting gene regulation.The genome is organized into multiscale structural units,including chromosome territories,compartments,topologically associating domains(TADs),and DNA loops.The identification of these hierarchical structures has benefited from the development of experimental approaches,such as 3C-based methods(Hi-C,ChIA-PET,etc.),imaging tools(2D-FISH,3D-FISH,Cryo-FISH,etc.)and ligation-free methods(GAM,SPRITE,etc.).In recent two decades,numerous studies have shown that the 3D organization of genome plays essential roles in multiple cellular processes via various mechanisms,such as regulating enhancer activity and promoter-enhancer interactions.However,there are relatively few studies about the 3D genome in livestock species.Therefore,studies for exploring the function of 3D genomes in livestock are urgently needed to provide a more comprehensive understanding of potential relationships between the genome and production traits.In this review,we summarize the recent advances of 3D genomics and its biological functions in human and mouse studies,drawing inspiration to explore the 3D genomics of livestock species.We then mainly focus on the biological functions of 3D genome organization in muscle development and its implications in animal breeding.

Reorganization of 3D genome architecture across wild boar and Bama pig adipose tissues

Background:A growing body of evidence has revealed that the mammalian genome is organized into hierarchical layers that are closely correlated with and may even be causally linked with variations in gene expression.Recent studies have characterized chromatin organization in various porcine tissues and cell types and compared them among species and during the early development of pigs.However,how chromatin organization differs among pig breeds is poorly understood.Results:In this study,we investigated the 3D genome organization and performed transcriptome characterization of two adipose depots(upper layer of backfat[ULB]and greater omentum[GOM])in wild boars and Bama pigs;the latter is a typical indigenous pig in China.We found that over 95%of the A/B compartments and topologically associating domains(TADs)are stable between wild boars and Bama pigs.In contrast,more than 70%of promoterenhancer interactions(PEIs)are dynamic and widespread,involving over a thousand genes.Alterations in chromatin structure are associated with changes in the expression of genes that are involved in widespread biological functions such as basic cellular functions,endocrine function,energy metabolism and the immune response.Approximately 95%and 97%of the genes associated with reorganized A/B compartments and PEIs in the two pig breeds differed between GOM and ULB,respectively.Conclusions:We reported 3D genome organization in adipose depots from different pig breeds.In a comparison of Bama pigs and wild boar,large-scale compartments and TADs were mostly conserved,while fine-scale PEIs were extensively reorganized.The chromatin architecture in these two pig breeds was reorganized in an adipose depotspecific manner.These results contribute to determining the regulatory mechanism of phenotypic differences between Bama pigs and wild boar.

Allopolyploidization increases genetic recombination in the ancestral diploid D genome during wheat evolution

Genetic recombination produces new allelic combinations,thereby introducing variation for domestication.Allopolyploidization has increased the evolutionary potential of hexaploid common wheat by conferring the advantages of heterosis and gene redundancy,but whether a relationship exists between allopolyploidization and genetic recombination is currently unknown.To study the impact of allopolyploid ization on genetic recombination in the ancestral D genome of wheat,we generated new synthetic hexaploid wheats by crossing tetraploid Triticum turgidum with multiple diploid Aegilops tauschii accessions,with subsequent chromosome doubling,to simulate the evolutionary hexaploidization process.Using the DArT-Seq approach,we determined the genotypes of two new synthetic hexaploid wheats with their parents,F;plants in a diploid population(2 x,D_(1)D_(1)×D_(2)D_(2))and its new synthetic hexaploid wheatderived population(6 x,AABBD_(1)D_(1)×AABBD_(2)D_(2)).About 11%of detected SNP loci spanning the D genome of Ae.tauschii were eliminated after allohexaploidization,and the degree of segregation distortion was increased in their hexaploid offspring from the F_(1) generation.Based on codominant genotypes,the mean genetic interval length and recombination frequency between pairs of adjacent and linked SNPs on D genome of the hexaploid F;population were 2.3 fold greater than those in the diploid F_(2) population,and the recombination frequency of Ae.tauschii was increased by their hexaploidization with T.turgidum.In conclusion,allopolyploidization increases genetic recombination of the ancestral diploid D genome of wheat,and DNA elimination and increased segregation distortion also occur after allopolyploidization.Increased genetic recombination could have produced more new allelic combinations subject to natural or artificial selection,helping wheat to spread rapidly to become a major global crop and thereby accelerating the evolution of wheat via hexaploidization.

Unraveling the 3D Genome Architecture in Plants:Present and Future

The eukaryotic genome has a hierarchicalthree-dimensional(3D)organization with functional implications for DNA replication,DNA repair,and transcriptional regulation.Over the past decade,scientists have endeavored to elucidate the spatial characteristics and functions of plant genome architecture using high-throughput chromatin conformation capturing technologies such as Hi-C,ChlA-PET,and HiChIP.Here,we systematically review current understanding of chromatin organization in plants at multiple scales.We also discuss the emerging opinions and concepts in 3D genome research,focusing on state-of-the-art 3D genome techniques,RNA-chromatin interactions,liquid-liquid phase separation,and dynamic chromatin alterations.We propose the application of single-cell/single-molecule multi-omics,multiway(DNA-DNA,DNA-RNA,and RNA-RNA interactions)chromatin conformation capturing methods,and proximity ligation-independent 3D genome-mapping technologies to explore chromatin organization structure and function in plants.Such methods could reveal the spatial interactions between trait-related SNPs and their target genes at various spatiotemporal resolutions,and elucidate the molecular mecha-nisms of the interactions among DNA elements,RNA molecules,and protein factors during the formation of key traits in plants.

Architectural proteins for the formation and maintenance of the 3D genome

Eukaryotic genomes are densely packaged into hierarchical three-dimensional(3D) structures that contain information about gene regulation and many other biological processes. With the development of imaging and sequencing-based technologies, 3D genome studies have revealed that the high-order chromatin structure is composed of hierarchical levels, including chromosome territories, A/B compartments, topologically associated domains, and chromatin loops. However, how this chromatin architecture is formed and maintained is not completely clear. In this review, we introduce experimental methods to investigate the 3D genome, review major architectural proteins that regulate 3D chromatin organization in mammalian cells, such as CTCF(CCCTC-binding factor), cohesin, lamins, and transcription factors, and discuss relevant mechanisms such as phase separation.

Toward precise CRISPR DNA fragment editing and predictable 3D genome engineering

Ever since gene targeting or specific modification of genome sequences in mice was achieved in the early 1980s,the reverse genetic approach of precise editing of any genomic locus has greatly accelerated biomedical research and biotechnology development.In particular,the recent development of the CRISPR/Cas9 system has greatly expedited genetic dissection of 3D genomes.CRISPR gene-editing outcomes result from targeted genome cleavage by ectopic bacterial Cas9 nuclease followed by presumed random ligations via the host double-strand break repair machineries.Recent studies revealed,however,that the CRISPR genomeediting system is precise and predictable because of cohesive Cas9 cleavage of targeting DNA.Here,we synthesize the current understanding of CRISPR DNA fragment-editing mechanisms and recent progress in predictable outcomes from precise genetic engineering of 3D genomes.Specifically,we first briefly describe historical genetic studies leading to CRISPR and 3D genome engineering.We then summarize different types of chromosomal rearrangements by DNA fragment editing.Finally,we review significant progress from precise ID gene editing toward predictable 3D genome engineering and synthetic biology.The exciting and rapid advances in this emerging field provide new opportunities and challenges to understand or digest 3D genomes.

Integrative Analysis of Genome,3D Genome,and Transcriptome Alterations of Clinical Lung Cancer Samples

Genomic studies of cancer cell alterations,such as mutations,copy number variations(CNVs),and translocations,greatly promote our understanding of the genesis and development of cancers.However,the 3D genome architecture of cancers remains less studied due to the complexity of cancer genomes and technical difficulties.To explore the 3D genome structure in clinical lung cancer,we performed Hi-C experiments using paired normal and tumor cells harvested from patients with lung cancer,combining with RNA sequenceing analysis.We demonstrated the feasibility of studying 3D genome of clinical lung cancer samples with a small number of cells(1×10^(4)),compared the genome architecture between clinical samples and cell lines of lung cancer,and identified conserved and changed spatial chromatin structures between normal and cancer samples.We also showed that Hi-C data can be used to infer CNVs and point mutations in cancer.By integrating those different types of cancer alterations,we showed significant associations between CNVs,3D genome,and gene expression.We propose that 3D genome mediates the effects of cancer genomic alterations on gene expression through altering regulatory chromatin structures.Our study highlights the importance of analyzing 3D genomes of clinical cancer samples in addition to cancer cell lines and provides an integrative genomic analysis pipeline for future larger-scale studies in lung cancer and other cancers.

Dynamic chromatin architectures provide insights into the genetics of cattle myogenesis

Background Sharply increased beef consumption is propelling the genetic improvement projects of beef cattle in China.Three-dimensional genome structure is confirmed to be an important layer of transcription regulation.Although genome-wide interaction data of several livestock species have already been produced,the genome structure states and its regulatory rules in cattle muscle are still limited.Results Here we present the first 3D genome data in Longissimus dorsi muscle of fetal and adult cattle(Bos taurus).We showed that compartments,topologically associating domains(TADs),and loop undergo re-organization and the structure dynamics were consistent with transcriptomic divergence during muscle development.Furthermore,we annotated cis-regulatory elements in cattle genome during myogenesis and demonstrated the enrichments of promoter and enhancer in selection sweeps.We further validated the regulatory function of one HMGA2 intronic enhancer near a strong sweep region on primary bovine myoblast proliferation.Conclusions Our data provide key insights of the regulatory function of high order chromatin structure and cattle myogenic biology,which will benefit the progress of genetic improvement of beef cattle.

3D genome organization in the central nervous system,implications for neuropsychological disorders

Chromosomes in eukaryotic cell nuclei are highly compacted and finely organized into hierarchical threedimensional(3 D) configuration. In recent years, scientists have gained deeper understandings of 3 D genome structures and revealed novel evidence linking 3 D genome organization to various important cell events on the molecular level. Most importantly, alteration of 3 D genome architecture has emerged as an intriguing higher order mechanism that connects disease-related genetic variants in multiple heterogenous and polygenic neuropsychological disorders, delivering novel insights into the etiology. In this review, we provide a brief overview of the hierarchical structures of 3 D genome and two proposed regulatory models,loop extrusion and phase separation. We then focus on recent Hi-C data in the central nervous system and discuss 3 D genome alterations during normal brain development and in mature neurons. Most importantly,we make a comprehensive review on current knowledge and discuss the role of 3 D genome in multiple neuropsychological disorders, including schizophrenia, repeat expansion disorders, 22 q11 deletion syndrome, and others.

Evidence of constraint in the 3D genome for trans-splicing in human cells

Fusion transcripts are commonly found in eukaryotes, and many aberrant fusions are associated with severe diseases, including cancer. One class of fusion transcripts is generated by joining separate transcripts through trans-splicing. However, the mechanism of trans-splicing in mammals remains largely elusive. Here we showed evidence to support an intuitive hypothesis that attributes trans-splicing to the spatial proximity between premature transcripts. A novel trans-splicing detection tool(TSD) was developed to reliably identify intra-chromosomal trans-splicing events(i TSEs) from RNA-seq data. TSD can maintain a remarkable balance between sensitivity and accuracy, thus distinguishing it from most state-of-the-art tools. The accuracy of TSD was experimentally demonstrated by excluding potential false discovery from mosaic genome or template switching during PCR. We showed that i TSEs identified by TSD were frequently found between genomic regulatory elements, which are known to be more prone to interact with each other. Moreover, i TSE sites may be more physically adjacent to each other than random control in the tested human lymphoblastoid cell line according to Hi-C data. Our results suggest that trans-splicing and 3 D genome architecture may be coupled in mammals and that our pipeline, TSD, may facilitate investigations of trans-splicing on a systematic and accurate level previously thought impossible.

Developing biolmaging and quantitative methods to study 3D genome

The recent advances in chromosome configuration capture （3C）-based series molecular methods and optical super- resolution （SR） techniques offer powerful tools to investigate three dimensional （3D） genomic structure in prokaryotic and eukaryotic cell nucleus. In this review, we focus on the progress during the last decade in this exciting field. Here we at first introduce briefly genome organization at chromosome, domain and sub-domain level, respectively; then we provide a short introduction to various super-resolution microscopy techniques which can be employed to detect gcnome 3D structure. We also reviewed the progress of quantitative and visualization tools to evaluate and visualize chromatin interactions in 3D genome derived from Hi-C data. We end up with the discussion that imaging methods and 3C-based molecular methods are not mutually exclusive -- actually they arc complemental to each other and can be combined together to study 3D genome organization.

On the evolution and genetic diversity of the bread wheat D genome

Bread wheat(Triticum aestivum)became a globally dominant crop after incorporating the D genome from the donor species Aegilops tauschii,but the evolutionary history that shaped the D genome during this process remains to be clarified.Here,we propose a renewed evolutionary model linking Ae.tauschii and the hexaploid wheat D genome by constructing an ancestral haplotype map covering 762 Ae.tauschii and hexaploid wheat accessions.We dissected the evolutionary trajectories of Ae.tauschii lineages and reported a few independent intermediate accessions,demonstrating that low-frequency intersublineage gene flow had enriched the diversity of Ae.tauschii.We discovered that the D genome of hexaploid wheat was inherited from a unified ancestral template,but with a mosaic composition that was highly mixed and derived mainly from three Ae.tauschii L2 sublineages located in the Caspian coastal region.This result suggests that early agricultural activities facilitated innovations in D-genome composition and finalized the success of hexaploidization.We found that the majority(51.4%)of genetic diversity was attributed to novel mutations absent in Ae.tauschii,and we identified large Ae.tauschii introgressions from various lineages,which expanded the diversity of the wheat D genome and introduced beneficial alleles.This work sheds light on the process of wheat hexaploidization and highlights the evolutionary significance of the multi-layered genetic diversity of the bread wheat D genome.

The Genomic Characterization of Enterovirus D68 from 2011 to 2015 in Beijing, China

A retrospective surveillance study on enterovirus D68 was performed in Beijing, China, following the largest and most widespread EV-D68 infection, which occurred in the USA. From January 2011 to July 2015, EV-D68 was identified in 12 individuals with respiratory infections in Beijing, China. The phylogenetic relationships based on the genomic sequence alignment showed that there were two lineages circulating in Beijing from 2011 to 2015. Eight EV-D68 strains belonged to group 1 and four belonged to group 3. All EV-D68 strains from Beijing in 2014 were separately clustered into subgroup II of group 1. Based on these results, we concluded that the Beijing EV-D68 strains had little association with the EV-D68 strains circulating in the 2014 USA outbreak.

Topological reorganization and functional alteration of distinct genomic components in gallbladder cancer

Altered three-dimensional architecture of chromatin influences various genomic regulators and subsequent gene expression in human cancer.However,knowledge of the topological rearrangement of genomic hierarchical layers in cancer is largely limited.Here,by taking advantage of in situ Hi-C,RNA-sequencing,and chromatin immunoprecipitation sequencing(ChIP-seq),we investigated structural reorganization and functional changes in chromosomal compartments,topologically associated domains(TADs),and CCCTC binding factor(CTCF)-mediated loops in gallbladder cancer(GBC)tissues and cell lines.We observed that the chromosomal compartment A/B switch was correlated with CTCF binding levels and gene expression changes.Increased inter-TAD interactions with weaker TAD boundaries were identified in cancer cell lines relative to normal controls.Furthermore,the chromatin short loops and cancer unique loops associated with chromatin remodeling and epithelial–mesenchymal transition activation were enriched in cancer compared with their control counterparts.Cancer-specific enhancer–promoter loops,which contain multiple transcription factor binding motifs,acted as a central element to regulate aberrant gene expression.Depletion of individual enhancers in each loop anchor that connects with promoters led to the inhibition of their corresponding gene expressions.Collectively,our data offer the landscape of hierarchical layers of cancer genome and functional alterations that contribute to the development of GBC.

A chromosome-scale genome assembly of Dasypyrum villosum provides insights into its application as a broad-spectrum disease resistance resource for wheat improvement

Dasypyrum villosum is one of the most valuable gene resources in wheat improvement,especially for disease resistance.The mining of favorable genes from D.villosum is frustrated by the lack of a whole genome sequence.In this study,we generated a doubled-haploid line,91C43^(DH),using microspore culture and obtained a 4.05-GB high-quality,chromosome-scale genome assembly for D.villosum.The assembly contains39727 high-confidence genes,and 85.31% of the sequences are repetitive.Two reciprocal translocation events were detected,and 7VS-4VL is a unique translocation in D.villosum.The prolamin seed storage protein-coding genes were found to be duplicated;in particular,the genes encoding low-molecular-weight glutenin at the Glu-V3 locus were significantly expanded.RNA sequencing(RNA-seq)analysis indicated that,after Blumeria graminearum f.sp tritici(Bgt)inoculation,there were more upregulated genes involved in the pattern-triggered immunity and effector-triggered immunity defense pathways in D.villosum than in Triticum urartu.MNase hypersensitive sequencing(MH-seq)identified two Bgt-inducible MH sites(MHSs),one in the promoter and one in the 3'terminal region of the powdery mildew resistance(Pm)gene NLR1-V.Each site had two subpeaks and they were termed MHS1(MHS1.1/1.2)and MHS2(MHS2.1/2.2).Bgt-inducible MHS2.2 was uniquely present in D.villosum,and MHS1.1 was more inducible in D.villosum than in wheat,suggesting that MHSs may be critical for regulation of NLR1-V expression and plant defense.In summary,this study provides a valuable genome resource for functional genomics studies and wheat-D.villosum introgression breeding.The identified regulatory mechanisms may also be exploited to develop new strategies for enhancing Pm resistance by optimizing gene expression in wheat.

Many facades of CTCF unified by its coding for three-dimensional genome architecture

CCCTC-binding factor(CTCF)is a multifunctional zinc finger protein that is conserved in metazoan species.CTCF is consistently found to play an important role in many diverse biological processes.CTCF/cohesin-mediated active chromatin‘loop extrusion’architects three-dimensional(3D)genome folding.The 3D architectural role of CTCF underlies its multifarious functions,including developmental regulation of gene expression,protocadherin(Pcdh)promoter choice in the nervous system,immunoglobulin(Ig)and T-cell receptor(Tcr)V(D)J recombination in the immune system,homeobox(Hox)gene control during limb development,as well as many other aspects of biology.Here,we review the pleiotropic functions of CTCF from the perspective of its essential role in 3D genome architecture and topological promoter/enhancer selection.We envision the 3D genome as an enormous complex architecture,with tens of thousands of CTCF sites as connecting nodes and CTCF proteins as mysterious bonds that glue together genomic building parts with distinct articulation joints.In particular,we focus on the internal mechanisms by which CTCF controls higher order chromatin structures that manifest its many fa?ades of physiological and pathological functions.We also discuss the dichotomic role of CTCF sites as intriguing3D genome nodes for seemingly contradictory‘looping bridges’and‘topological insulators’to frame a beautiful magnificent house for a cell’s nuclear home.

3D disorganization and rearrangement of genome provide insights into pathogenesis of NAFLD by integrated Hi-C, Nanopore, and RNA sequencing

The three-dimensional(3D)conformation of chromatin is integral to the precise regulation of gene expression.The 3D genome and genomic variations in non-alcoholic fatty liver disease(NAFLD)are largely unknown,despite their key roles in cellular function and physiological processes.Highthroughput chromosome conformation capture(Hi-C),Nanopore sequencing,and RNA-sequencing(RNA-seq)assays were performed on the liver of normal and NAFLD mice.A high-resolution 3D chromatin interaction map was generated to examine different 3D genome hierarchies including A/B compartments,topologically associated domains(TADs),and chromatin loops by Hi-C,and whole genome sequencing identifying structural variations(SVs)and copy number variations(CNVs)by Nanopore sequencing.We identified variations in thousands of regions across the genome with respect to 3D chromatin organization and genomic rearrangements,between normal and NAFLD mice,and revealed gene dysregulation frequently accompanied by these variations.Candidate target genes were identified in NAFLD,impacted by genetic rearrangements and spatial organization disruption.Our data provide a high-resolution 3D genome interaction resource for NAFLD investigations,revealed the relationship among genetic rearrangements,spatial organization disruption,and gene regulation,and identified candidate genes associated with these variations implicated in the pathogenesis of NAFLD.The newly findings offer insights into novel mechanisms of NAFLD pathogenesis and can provide a new conceptual framework for NAFLD therapy.

Mapping Multi-factor-mediated Chromatin Interactions to Assess Dysregulation of Lung Cancer-related Genes

Studies on the lung cancer genome are indispensable for developing a cure for lung cancer.Whole-genome resequencing,genome-wide association studies,and transcriptome sequencing have greatly improved our understanding of the cancer genome.However,dysregulation of longrange chromatin interactions in lung cancer remains poorly described.To better understand the three-dimensional(3D)genomic interaction features of the lung cancer genome,we used the A549 cell line as a model system and generated high-resolution chromatin interactions associated with RNA polymerase II(RNAPII),CCCTC-binding factor(CTCF),enhancer of zeste homolog 2(EZH2),and histone 3 lysine 27 trimethylation(H3K27me3)using long-read chromatin interaction analysis by paired-end tag sequencing(ChIA-PET).Analysis showed that EZH2/H3K27me3-mediated interactions further repressed target genes,either through loops or domains,and their distributions along the genome were distinct from and complementary to those associated with RNAPII.Cancer-related genes were highly enriched with chromatin interactions,and chromatin interactions specific to the A549 cell line were associated with oncogenes and tumor suppressor genes,such as additional repressive interactions on FOXO4 and promoter–promoter interactions between NF1 and RNF135.Knockout of an anchor associated with chromatin interactions reversed the dysregulation of cancer-related genes,suggesting that chromatin interactions are essential for proper expression of lung cancer-related genes.These findings demonstrate the 3D landscape and gene regulatory relationships of the lung cancer genome.

Large-scale data-driven and physics-based models offer insights into the relationships among the structures,dynamics,and functions of chromosomes

The organized three-dimensional chromosome architecture in the cell nucleus provides scaffolding for precise regulation of gene expression.When the cell changes its identity in the cell-fate decision-making process,extensive rearrangements of chromo-some structures occur accompanied by large-scale adaptations of gene expression,underscoring the importance of chromosome dynamics in shaping genome function.Over the last two decades,rapid development of experimental methods has provided unprecedented data to characterize the hierarchical structures and dynamic properties of chromosomes.In parallel,these enormous data offer valuable opportunities for developing quantitative computational models.Here,we review a variety of large-scale polymer models developed to investigate the structures and dynamics of chromosomes.Different from the underlying modeling strategies,these approaches can be classified into data-driven(‘top-down’)and physics-based(‘bottom-up’)categories.We discuss their contributions to offering valuable insights into the relationships among the structures,dynamics,and functions of chromosomes and propose the perspective of developing data integration approaches from different experimental technologies and multidisciplinary theoretical/simulation methods combined with different modeling strategies.

The 3D organization of genome in the nucleus: from the nucleosome to the 4D nucleome

The genetic information,stored in the linear sequences of DNA,encodes all of the genes that a living organism uses to produce proteins and RNAs essential for various cellular functions.In eukaryotes,however,genomic DNA is hierarchically and efficiently packaged into multiple-level chromatin structures within the nucleus,including the nucleosome,30 nm fibers,chromatin loops,topology associated domains(TADs).