In Vitro Functional Characterization of the -143A>C Variant in the Porcine DLD Promoter and Its Modulation of KLF2-Dependent Repression and Myoblast Phenotypes

Meat quality in pigs is fundamentally shaped by the relative abundance and metabolic properties of distinct skeletal muscle fiber types. Dihydrolipoyl dehydrogenase (DLDH), an indispensable catalytic subunit of mitochondrial dehydrogenase complexes, has been documented to link with myocyte development; nevertheless, the effects of the causal regulatory variants governing transcription of its encoding gene DLD on the myogenic fate remain largely uncharacterized. In this study, comparative polymorphism analysis between Large White and Min pigs uncovered a regulatory single nucleotide polymorphism (SNP) at position -143A>C within the porcine DLD promoter. Mechanistic dissection using dual-luciferase reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays revealed that the C allele strengthens Krüppel-like factor 2 (KLF2) DNA-binding affinity and potentiates KLF2-mediated transcriptional repression, leading to significantly reduced DLD expression. Functional characterization in porcine primary myoblasts demonstrated that DLD promotes myoblast proliferation by accelerating G2/M transition and upregulating cell-cycle regulators while suppressing terminal differentiation. Furthermore, DLD overexpression inhibited slow-twitch (myosin heavy chain (MYH) 7-positive) myogenic differentiation and suppressed the expression of myogenic regulatory factors MYOD and MYOG, whereas DLD knockdown produced converse effects. Taken together, these multiple lines of evidence suggest that the −143 A>C variant may reinforce transcriptional repression of DLD by increasing KLF2-binding occupancy. Because reduced DLD expression favors MYH7-positive myotube formation in our myoblast assays, this variant is hypothesized to influence myotube subtype balance. These findings may provide a potential molecular mechanism contributing to pork-quality variation and suggest the −143 A>C variant on the DLD promoter as a candidate marker for future genomic-selection studies.