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The atlas of promoter-enhancer interactions during myogenic differentiation offers novel insights into genetic variants related to meat traits in pigs

Yalong An1*, Chen Zhang1*, Zihao Ge1, Yang Li1, Chenglong Wen1, Rongrong Ding1,2, Peiyuan Han1, Yongqi Yue1, Jiangwei Wu1,2, Jianjun Jin1,2#, Xiao Li1,2#

1 Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Shaanxi, 712100

2 National Key Laboratory of Livestock Biology, Northwest A&F University, Shaanxi 712100

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摘要  

猪肉是全球第二大肉类来源,大规模的全基因组关联分析(whole genome association study, GWAS已经鉴定出许多与猪肉性状相关的遗传变异,但80%以上都于非编码区,功能未知、难以应用于育种实践。3D基因组表观基因组等多组学为非编码区的功能解析提供了有力工具。成肌分化决定产肉量和肉质的关键生物学过程,解析其中启动子-增强子互作promoter-enhancer interactions, PEIs以及关键变异位点的作用机制,对于揭示产肉性状的遗传基础具有重要意义。本研究利用猪原代骨骼肌卫星细胞并诱导分化,收集增殖期的成肌细胞和分化后的肌管,分别进行HiCuT (H3K27ac)ATAC-seqRNA-seq。并整合表达数量性状等位基因座(expression quantitative trait locus, eQTLPigGTEx)、GWASPigBiobank)、孟德尔随机化(Summary data-based mendelian randomization, SMR, PigGTEx)和结构变异(structural variations, SVs)数据筛选成肌分化中的顺式eQTLcis-eQTL)。进一步结合CRISPR干扰(CRISPR interference, CRISPRi),双荧光素酶报告基因等分子生物学实验初步验证候选cis-eQTL的功能。主要获得以下结果:分别在成肌细胞和肌管中鉴定到179,895159,255个互作loops,根据互作类型的不同,将其分为启动子-启动子互作P-P、启动子-增强子互作P-E其他-其他O-O互作,并构建了成肌分化过程中PEIs图谱。根据成肌分化中PEIs筛选到22,645cis-eQTLs,初步证实了rs326494665作为近端启动子eQTL调控CRABP2rs341971107作为远端增强子eQTL调控NUDT16L1TFAP4以及rs318959112作为启动子样增强子eQTL调控DUSP28GPR35的功能。此外,在成肌分化PEIs中筛选到39,069候选cis-SVs。进一步联合ATAC-seq和RNA-seq数据,筛选93调控成肌分化的转录因子,其中包括OLIG2ZNF341RFX5RFX6等新的转录因子,并构建了转录因子-基因表达调控网络。通过成肌分化中差异loops分析,筛选到7,491个成肌分化阶段特异性cis-eQTLs。并初步解析了成肌分化阶段特异性cis-eQTL rs325444503调控RBPJchr13_31776103调控USP19AMIGO3以及rs81437537调控MYH3表达的功能。本研究构建了成肌细胞分化过程中的PEIs图谱,进一步丰富了猪成肌分化中CREs的注释并基于PEIs筛选到22,645个与产肉性状相关的cis-eQTLs结合分子实验初步解析了产肉性状相关非编码区遗传变异的功能,为产肉性状的遗传选择提供了有效可靠候选分子标记。



Abstract  

The lack of knowledge about how the genome is regulated during skeletal myogenesis in pigs hinders the identification of genetic variants for meat traits. Here, we systematically characterized the cis-regulatory elements (CREs, promoters, enhancers and others) by using Hi-C coupled chromatin cleavage and tagmentation (HiCuT, H3K27ac), Assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA-seq to analyze primary myoblasts and in vitro well-differentiated myotubes, and generated an atlas of the promoter-enhancer interactions (PEIs) during myogenic differentiation. In total, 179,895 and 159,255 loops were identified in myoblasts and myotubes, respectively, which could be grouped into 3 categories of promoter-promoter interactions, promoter-enhancer interactions and promoter-others. Furthermore, 22,645 cis-eQTLs loci were pinpointed by integrating public genome-wide association studies (GWAS) and expression quantitative trait locus (eQTL) datasets. Notably, novel promoter-like enhancers were verified, and cis-eQTLs in promoter-like enhancers might influence gene expression over a long range. In addition, 39,069 structural variants (SVs) within the CREs were identified. A transcription factor (TF)-promoter/enhancer regulatory network for myogenesis was generated, revealing several novel TFs relevant to myogenic differentiation. This work generated a valuable resource for understanding genomic regulation in pig muscles and filtering potential variants to develop breeds with desirable traits.

Keywords:  pig       promoter-enhancer interaction       cis-eQTLs       meat traits       myogenic differentiation  
Online: 16 May 2025  
Fund: 

This work was supported by the National Key Research and Development Program of China (2021YFF1000602).

About author:  *Both authors contributed equally to this work. # E-mail: nice.lixiao@gmail.com

Cite this article: 

Yalong An, Chen Zhang, Zihao Ge, Yang Li, Chenglong Wen, Rongrong Ding, Peiyuan Han, Yongqi Yue, Jiangwei Wu, Jianjun Jin, Xiao Li. 2025. The atlas of promoter-enhancer interactions during myogenic differentiation offers novel insights into genetic variants related to meat traits in pigs. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.05.009

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