Fig. 6: Functional Validation of a Distal Enhancer Regulating the PSE gene MTSS1 in Pig Muscle.
From: Mechanism of parent-of-origin effects revealed by multi-omic data in euro-chinese hybrid pigs
a The MTSS1 is regulated by the PSHM within the loop-mediated regulatory region in LD. b Plasmid construction for luciferase reporter assay (top). The fluorescence intensity of reporter genes was used to examine the activity of the regulatory element. Bar plots, from left to right, represent the following groups: no vector, control plasmid, cells transfected with a recombinant plasmid containing the promoter sequence, and cells transfected with a recombinant plasmid containing both the enhancer and promoter sequences (bottom). c The 3C-qPCR assay for MTSS1 was designed to include genomic regions predicted by Hi-C to interact, restriction enzyme sites, histone modification peaks identified by Cut&Tag, and the positions of dual-luciferase reporter insertion sequences (top). The PCR detection model is illustrated in the lower-left panel. Through ligation reactions, crosslinking, and subsequent reverse crosslinking steps, the two distal genomic regions were brought into proximity, reducing the intervening sequence to less than 300 bp, thereby enabling detection by qPCR (lower-left). The PCR results are presented as a bar chart in the lower-right panel. gDNA input and IgG-enriched DNA served as positive and negative controls, respectively, using primers targeting the GAPDH promoter region. To further validate the experimental results, the proximal flanking sequence of the detected region was amplified as an internal control. The detection of ligation products in the +ligase group confirmed the successful ligation of distal regions following reverse crosslinking (lower-right). d CRISPR/Cas9-mediated deletion strategy and sgRNA design targeting the enhancer of MTSS1. e Relative expression of MTSS1 (mean ± SD) following disruption of enhancers separately by sgRNAs. In all bar graphs related to functional validation, each data point represents technical replicates, with a consistent sample size (n = 3). Two-tailed t-tests were performed to calculate the corresponding P-values. Source data are provided as a Source Data file.
