The Primary Study On The Mechanism How The Long-range Sonic Hedgehog Enchancer ZRS Regulates Gene Expression

Sonic Hedgehog works like a morphogen during the animal individual development. It functions in many organs, such as brain, spinal cord, thalamus, limb bud and epithelial cells. Recently reports show that in different tissues, different tissue specificity enhancers regulate shh expression. During limb bud shaping, the conserved ZRS sequence locating IMbp upstream of the shh proximal promoter regulates shh expression as a long-range enhancer. And point mutation in this ZRS sequence would cause the preaxial polydactyly(PPD), a severe genetic disease. Also, the DNA duplication of ZRS would may lead to the PPD.ZRS is ultra conserved across vertebrates including Human, mouse, rat, Chicken and Fugu. More than 10 point mutations have been linked to the PPD. This work successfully screening a novel point mutation from the patient sample. We amplify 1065bp and 214bp DNA sequence including the ZRS point mutation and the wild type. After excluding the shh exons'abnormality, the point mutation might be the cause of the disease. We conform that in HEK-293A cells, the 1065bp and 214bp ZRS sequence shows the enhancer activity. The mutant sequence significantly-increased luciferase activity compared with the wild type sequence.The biotinylated DNA protein precipitation pull down assay and bioinformation analysis show that the biotin DNA may specially bind to transcription factor F0X03a.This could may give some explanations and methods to the molecular mechanism of how the 1Mbp long-range enhancer regulates shh expression.