<i>SMAD5</i> Regulates Hainan Black Goat <i>ATG16L2</i> Gene Transcription in Primary Goat Fibroblasts

Autophagy, a fundamental cellular process, is indispensable for maintaining cellular homeostasis through the regulated degradation and recycling of intracellular components, including damaged organelles and invading pathogens. ATG16L2 is a key autophagy gene essential for autophagosome formation and maturation. Unraveling the intricate transcriptional regulation of such autophagy-related genes is paramount for optimizing livestock germplasm and enhancing breeding programs. This study aimed to comprehensively elucidate the transcriptional regulation of the goat ATG16L2 gene. Tissue-specific qPCR analysis revealed the highest ATG16L2 mRNA expression in the goat spleen among key tissues examined (liver, spleen, lung, kidney, fat, rumen, and small intestine). Subsequently, to characterize the ATG16L2 promoter, we cloned a 1,304 bp upstream sequence (from -1,080 to + 224 relative to the transcription start site, TSS). Truncated promoter constructs derived from this fragment were evaluated via dual-luciferase reporter assays to identify the core promoter region. To investigate the transcriptional regulation of goat ATG16L2, small interfering RNAs (siRNA) were used to target specific transcription factors. Dual-luciferase reporter assays demonstrated that deleting the identified SMAD5 binding site significantly diminished promoter activity. This was further supported by siRNA-mediated knockdown of SMAD5, which led to reduced luciferase activity in transfected goat primary fibroblasts. In conclusion, we identified the core promoter region of the goat ATG16L2 gene that maintains basal transcription, and demonstrated the role of SMAD5 in its transcriptional regulation, laying the foundation for future studies on the function of goat ATG16L2.