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.

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.

MicroRNA bta-miR-365-3p inhibits proliferation but promotes differentiation of primary bovine myoblasts by targeting the activin A receptor type Ⅰ

Background: MicroRNAs act as post-transcriptional regulators that repress translation or degrade mRNA transcripts.Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically btamiR-365-3 p, in muscle biology, we investigated its functions in regulating primary bovine myoblast proliferation and differentiation.Results: Firstly, we found that bta-miR-365-3 p was predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting results showed that overexpression of btamiR-365-3 p significantly reduced the expression levels of cyclin D1(CCND1), cyclin dependent kinase 2(CDK2) and proliferating cell nuclear antigen(PCNA) but stimulated the expression levels of muscle differentiation markers, i.e.,MYOD1, MYOG at both mRNA and protein level. Moreover, downregulation of bta-miR-365-3 p increased the expression of CCND1, CDK2 and PCNA but decreased the expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining results showed that increased levels of bta-miR-365-3 p suppressed cell proliferation but promoted myotube formation, whereas decreased levels of bta-miR-365-3 p resulted in the opposite consequences. Finally, we identified that activin A receptor type I(ACVR1) could be a direct target of bta-miR-365-3 p. It was demonstrated that bta-miR-365-3 p can bind to the 3'UTR of ACVR1 gene to regulate its expression based on dual luciferase gene reporter assays.Consistently, knock-down of ACVR1 was associated with decreased expressions of CDK2, CCND1 and PCNA but increased expression of MYOG and MYOD1 both at mRNA and protein level.Conclusion: Collectively, these data suggested that bta-miR-365-3 p represses proliferation but promotes differentiation of bovine myoblasts through several biological mechanisms involving downregulation of ACVR1.

The Effect of the Cyp19a1 Gene Methylation Modification on Temperature-dependent Sex Determination of Reeves' Turtle(Mauremys reevesii)

Temperature-dependent sex determination（TSD） is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature; however,the molecular mechanisms governing this process remain unknown.Aromatase,encoded by the cyp19a1 gene,which converts androgens into estrogens in animals,was considered to be the key gene for TSD.In this study,the 5＇-flanking region of the cyp19a1 gene in Reeves＇ turtle（Mauremys reevesii） was cloned,and the promoter region was identified using the luciferase reporter assay.Then the eggs of Reeves＇ turtle were incubated at different temperatures（26°C： male-biased temperature; 29°C： non-sex-biased temperature and 32°C： female-biased temperature）.During the thermosensitive period,the adrenal kidney gonad complexes（AKG） were sampled.DNA methylation analysis of the AKG samples showed that the promoter region of the cyp19a1 gene was significantly de-methylated in the female-biased temperature regime（P＆lt;0.01）.Quantitative analysis of the cyp19a1 gene and estrogen by q PCR and Elisa assay showed that the expression level of the cyp19a1 gene and estrogen content were both upregulated significantly at the female-biased temperature（P＆lt;0.01）.These results indicated that the de-methylation response of the cyp19a1 gene to incubation temperature,especially at the female-biased temperature,could lead to temperature-specific expression of aromatase and increased estrogen levels,which may further determine gonadal development in Reeves＇ turtle.These findings provide insights into the genetic mechanisms underlying TSD.

Effects of SNPs and alternative splicing within HGF gene on its expression patterns in Qinchuan cattle

Background： Identification of genetic variants, including SNPs（Single Nucleotide Polymorphisms）, CNVs（Copy Number Variations） and alternative splicing, within functional genes has received increasing attention in animal science research. HGF（Hepatocyte Growth Factor） is a very important growth factor that works as a mitogen or a morphogen during tissue growth, development and regeneration. However, to date, the functions of genetic variants within the bovine HGF gene, particularly their effects on m RNA expression, have not been determined well.Results： The present study aimed to perform association analysis between genetic variants and m RNA expression for the bovine HGF gene in Qinchuan cattle using various strategies, including PCR-RFLP（Restriction Fragment Length Polymorphism）, q PCR（Quantitative Real-time quantitative PCR）, TA cloning, DNA sequencing and bioinformatics analysis. A total of five SNPs were identified and only SV1 locus significantly affected HGF m RNA expression in fetal skeletal muscle（P 〈 0.05）. Heterozygous genotype individuals showed significantly higher HGF expression（P 〈 0.05）, which was significantly greater in the ＂CTCCAGGGTT＂ combined genotype than that in the＂CCCCGGGGTT＂ combined genotype（P 〈 0.05）. In addition, two alternative splicing variations, HGF-W and HGF-M,were identified, which resulted from alternative 3′ splice sites of exon 5, and HGF-W showed higher m RNA levels than HGF-M in all tissues.Conclusion： In summary, genetic variations within the HGF gene affected m RNA expression. These findings provide new insight into the molecular characteristics and functions of bovine HGF.

Differential expression of FOXO1 during development and myoblast differentiation of Qinchuan cattle and its association analysis with growth traits

Our previous work reported a relationship between FOXO1 mutations and growth of Qinchuan(QC) cattle. Here, we performed differential expression analysis of FOXO1 and its association analysis with growth traits in QC cattle. First, we measured the expression of the FOXO1 gene in nine tissues during three developmental stages. The results showed that FOXO1 was abundantly expressed in tissues of calves but was strongly repressed in adulthood, although there was significant transcription in skeletal muscle. FOXO1 expression showed gradual up-regulation during differentiation of primary bovine skeletal muscle cells.We also identified six SNPs of the bovine FOXO1 gene by sequencing DNA pools of samples from 488 individuals, and association analysis indicated that five SNPs were significantly associated with some growth traits in the QC population. We further analyzed four haplotype combinations of the six SNPs and found significant correlation with body length(P<0.01). In conclusion, FOXO1 participates in bovine myocyte differentiation and expression, and may be a strong candidate as a gene that affects growth traits that could be exploited in a QC cattle breeding program. More generally, our data provide a new theoretical basis for QC beef breeding and beef quality improvement.

circSVIL regulates bovine myoblast development by inhibiting STAT1 phosphorylation

Circular RNAs(circRNAs),a novel class of non-coding RNAs with a loop structure,have recently been shown to participate in various pathophysiological processes.However,the precise role of circRNAs in myoblasts remains unclear.In this report,circSVIL was screened and identified from our previous sequencing analysis;we then performed gain-and loss-of-function experiments on bovine myoblasts by CCK8,EdU,flow cytometry,qRT-PCR,and Western blotting.The results indicate that circSVIL facilitates bovine myoblast proliferation and inhibits cell apoptosis.Using mechanism assays such as bioinformatics prediction,RNA immunoprecipitation(RIP),and cytoplasmic separation,we demonstrate that circSVIL could interact with STAT1 and inhibit STAT1 phosphorylation,thereby restraining STAT1’s nuclear translocation and affecting its downstream signal cascade.Our results may elucidate a new regulatory pathway for bovine skeletal muscle development.

Insertion/deletion variants within the IGF2BP2 gene identified in reported genome-wide selective sweep analysis reveal a correlation with goat litter size

Insulin-like growth factor 2 m RNA-binding protein 2(IGF2 BP2,also called IMP2)plays an essential role in the development and maturation of germ cells and embryos and is a candidate gene for goat litter size,based on a previous genomewide selective sweep analysis.In this study,the m RNA expression level of IGF2 BP2 was found to be significantly higher in a single-lamb group than in a multi-lamb group.Insertions/deletions(indels)within the goat IGF2 BP2 gene,including P4-Ins-13 bp and P5-Del-12 bp,were verified in 918 Shaanbei White Cashmere(SBWC)female goats.The minor allelic frequencies(MAFs)of P4-Ins-13 bp and P5-Del-12 bp loci were 0.349 and 0.295,respectively.Analysis using the Chi-square(χ^(2))test showed that the genotype(χ^(2)=14.479,P=0.006)distribution of P4-Ins-13 bp was significantly different between the single-lamb and multi-lamb groups.Correlation analysis demonstrated that P4-Ins-13 bp was significantly associated with goat litter size(P=0.022),and individual goats with the homozygous deletion/deletion(DD)genotype produced more litters than other goats.Therefore,considered as a potential molecular marker significantly related to lambing traits,the P4-Ins-13 bp mutation of the goat IGF2 BP2 gene can be used in goat breeding with practical molecular marker-assisted selection(MAS)to optimize female reproduction and improve economic efficiency in the goat industry.