Identification and characterization of GHR gene promoter of yak

Animal growth and development are regulated by the growth hormone receptor (GHR). To elucidate the transcriptional regulation mechanism of the yak GHR gene, we cloned its proximal promoter region and performed bioinformatic analyses. Deletion constructs were generated to produce fragments of the proximal promoter, and the fragment exhibiting the highest activity was identified using a dual-luciferase reporter assay system. Moreover, the functionality of the transcription factor binding sites was confirmed via site-directed mutagenesis and electrophoretic mobility shift assay (EMSA). Bioinformatics analysis revealed two transcriptional initiation sites were located in the upstream regulation region at positions - 277/ - 327 and - 1734/ - 1784 bp, and CpG islands were located at - 552/ - 654 and - 82/ - 289 bp upstream. We identified the - 160 to + 27 bp region as the minimal core promoter of the yak GHR gene. The transcription factor KLF5, which is involved in muscle growth and development, was predicted to bind the proximal minimum core promoter region of the GHR gene. Using site-directed mutagenesis and EMSA, we demonstrated that KLF5 is a critical transcription regulator of the GHR gene. Our findings establish a theoretical basis for investigating the regulatory mechanisms of GHR gene expression.