Chromatin-associated RNA-mediated Enhancer-promoter Interaction Maps Provide Insights Into Meat Production-related Traits In Porcine Gene Regulatory Networks

Pork is the main source of meat consumption in our country,and improving meat production rate and meat quality is a major goal of modern pig breeding.In the past decade researchers have identified many genetic markers associated with complex traits such as growth and meat quality through genome-wide association studies（GWAS）,but it is a major challenge to pinpoint these genetic markers to the true functional mutations based on a multi-omics strategy and then link them to the corresponding target genes.Generally,gene expression programs are intimately linked to the interplay of active cis regulatory elements mediated by chromatin contacts and associated RNAs.However,the functional interpretation of these variants remains nontrivial due to the lack of chromatin contact information or limited contact resolution.Furthermore,the distribution and role of chromatin-associated RNAs（ca RNAs）in gene expression and chromatin conformation remain poorly understood.To address this,we first present a comprehensive interaction map of 3D chromatin-chromatin interactions（H3K27ac BL-Hi ChIP）and RNA-chromatin interactions（GRID-seq）to reveal genomic variants that contribute to complex meat production traits.The main results of this study are:1.Identification of porcine skeletal ca RNAs based on GRID-seqBased on GRID-seq,we identified 1,942 and 1,488 ca RNAs in LW and MS skeletal muscle tissues,respectively.Furthermore,we systematically investigated the type,interaction mode and binding regions of these ca RNAs,and constructed a high-resolution RNA-chromatin interaction map.We found that RNA-chromatin interaction map was able to contribute breed-specificity on transcriptional regulation.2.Constructing“CREs-genes”chromatin interaction map based on BL-Hi ChIPBased on BL-Hi ChIP,we constructed a 5 kb resolution chromatin interaction map against histone modification H3K27ac in LW and MS skeletal muscle tissues.We found that about 75%of the enhancers chose to skip the nearest gene to regulate their target genes,further demonstrating the importance of using high-resolution chromatin interactions maps to explore the regulatory relationships between regulatory elements and target genes.3.Different chromatin interaction patterns affect gene expression levelsBased on chromatin interaction maps,we analyzed the interaction patterns of cis-regulatory elements with 18,166 target genes.We compared the expression patterns of genes with and without active promoter（P）and enhancer（E）regulation.We found that genes with P regulation exhibited higher expression levels than those without P regulation,while genes with E regulation also exhibited higher expression levels than those without E regulation.Moreover,the expression levels of genes with only P regulation were significantly higher than those of genes with only E regulation,revealing that P as a proximal cis-regulatory element may exert a stronger influence on gene expression levels than a single E as a distal cis-regulatory element.4.Differential loop interactions contribute to identifying breed-specific transcription regulationA total of 665 genes were differentially regulated by chromatin interactions,of which328 genes were significantly up-regulated in LW,and 337 genes were significantly up-regulated in MS（|log₂FC|>1.5,P-value<0.05）.These loop-differentially regulated genes exhibited a clear variety of functional characteristics:LW up-regulated genes were mainly enriched in muscle development-related entries,whereas MS up-regulated genes were mainly enriched in energy metabolism-related entries.In addition,TF motif analysis indicated potential synergistic regulation roles of chromatin loops and TF in gene transcription machinery.LW loop anchors of up-regulated genes were strongly enriched in LW selective sweep regions and not in MS selective sweep regions,and vice versa for MS.Taken together,high-resolution chromatin interaction can provide a breed-specific explanation for genomic variation.5.ca RNA-mediated RNA-chromatin interactions might be orchestrated with chromatin-chromatin interactions for regulating gene transcriptionWe performed differential RNA-chromatin analysis,a total of 11,397 genes regulated by ca RNA were identified,of which 828 were up-regulated in LW and 1,238 were up-regulated in MS（|log₂FC|>2,P-value<0.05）.We evaluated the chromatin-chromatin interaction levels on these ca RNA-differentially-regulated promoters and associated loop anchors,the chromatin interaction change at these representative loci were in high concordance with the change of ca RNA interaction,which indicates that nascent ca RNA-mediated RNA-chromatin interactions might be orchestrated with chromatin-chromatin interactions for regulating gene transcription.6.Enhancer-promoter interactions link candidate variants to target genesWe provided fine mapping and gene prioritization for GWAS signals of 15 muscle-related traits（including FCR,ADG,AGE,breeding value of FCR and AGE,body weight,meat color score,conductivity,drip loss,marbling score,moisture,p H,IMF,LMP and LMD）.223 candidate causal variants and 54 target genes previously not detected were identified.The effects of three functional variants regulating KLF6（related to Days to 100Kg）,MXRA8（related to lean meat percentage）,and TAF11（related to loin muscle depth）were observed in two pig populations.Moreover,we further provided luciferase enzyme assay to validate the effects of these three SNPs.7.TTN exhibited extensive trans interaction across the genomeWe found extensive TTN trans interaction across the genome.In addition,we investigated major ca RNAs that bound to the loop anchors of aforementioned 54 target genes,and we found that the composition and proportion of ca RNAs differed in LW and MS,in contrast to other ca RNAs,TTN was the most abundant in these 54 genes,implying an extensive functional trans interaction in porcine skeletal muscle.8.Functional networks of chromatin-chromatin and TTN-mediated RNA-chromatin interactionsTTN-mediated RNA-chromatin and chromatin-chromatin interactions can form a three-dimensional regulatory network within the nucleus,and within this three-dimensional regulatory network,genes in the same community might be functionally related.In addition,target genes jointly identified by GWAS can serve as the core genes for exploring peripheral weak-or moderate-effect genes for related traits within the same network.In this study,we first present a comprehensive interaction map of 3D chromatin-chromatin interactions（H3K27ac BL-Hi ChIP）and RNA-chromatin interactions（GRID-seq）to reveal genomic variants that contribute to complex skeletal muscle traits.We provide systematic fine mapping and gene prioritization related to 15 complex pig skeletal muscle traits.Our high-resolution chromatin interaction maps are valuable for identifying candidate target genes and corresponding cis-regulatory elements.The method and data presented in this study provide rich resources for functional genomics studies related to complex traits in farm animals.