Demonstration of the enhancing effect of a putative regulatory region of the RSPO4 gene predicted to play role in susceptibility to periodontitis

Periodontitis is one of the complex diseases characterized by the destruction of tooth-specific supporting structures in which genetic, environmental and lifestyle factors play a role, and the genetic mechanisms associated with its pathogenesis and susceptibility have not yet been fully elucidated. RSPO4 is a gene that is involved in bone formation and mature skeleton, as well as has role in mechanisms related to nail and extremity development. Findings showing increased RSPO4 expression as a result of RNA-seq in specific mouse lines resistant to Porphyromonas gingivalis (major periodontopathogen) infection suggested that RSPO4 gene regulation plays a role in resistance to periodontal destruction. According to the genome-wide association studies, ENCODE data and ChIP-seq analyses in various cell types; a haplotype block located in the 3'UTR region of RSPO4, which has transcription factor binding sites, H3K4Me1 and H3K27Ac histone modifications, DNAse I hypersensitivity, and single nucleotide polymorphisms showing strong linkage disequilibrium suggested that the downstream gene expression of a haplotype block is a putative enhancer region. Accordingly, it was thought that the relevant chromosome region containing regulatory elements that have a function in the etiology of periodontitis. The aim of the study is to confirm that these putative regulatory elements enhance gene expression. For this purpose, the region was amplified and confirmed by DNA sequencing, cloned into the firefly luciferase reporter vector pgl4.24, and then transfected into cells where RSPO4 was strongly expressed after it was expressed at a high level in competent bacteria, and the effect of the putative sequence on the promoter activity was carried out. The effect was examined with the luciferase assay. As a result, it was determined that the sequence decreased the promoter activity revealing that the region is a repressor region.