Investigation of the regulatory mechanism of gene expression of human ecogenin/CTGF, a chondrocyte-derived growth factor.

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1) Research on transcriptional control of ecogenin/CTGF gene expression : We constructed a series of chimeric reporter gene constructs, in which the human CTGF promoter and its deletion mutants were linked upstream of firefly luciferase genes. Using these constructs, we comparatively analyzed their promoter activities via transient expression assay in chondrocytic HCS-2/8 cells. Then, we found a 110-bp DNA segment located at 88 bp-upstream of the transcription initiation site as a critical determinant of the enhanced CTGF gene expression in HCS-2/8 cells. Moreover, we found two enhancer elements that were active in HCS-2/8 cells. One of them was a known TGF-β response element, whereas the other was a novel one discovered in this study. Mutation of either element resulted in drastic decrease of the promoter activity in HCS-2/8 cells. It is especially interesting that binding counterpart(s) of the latter latter element was found to be present specifically in HCS-2/8 cells.2) Research on … More post-transcriptional control of ecogenin/CTGF gene expression : We uncovered the strong repressive effect of the 1 kb-long 3'-untranslated region (UTR) of the ecogenin/CTGF gene on gene expression by comparatively evaluating the luciferase gene expression with or without the cis-linked 3'-UTR. Furthermore, we could identify an 84 base repressive cis-element by deletion analysis based on computer-associated structural prediction. Since this RNA element formed a stable secondary structure in solution, and the repressive function was highly dependent on the secondary structure forming potential, we entitled this element "cis-acting element of structure-anchored repression (CAESAR). Also recently, multiple stem-loop structure has been observed to be the structural determinant of CAESAR function. CAESAR did not display any effect outside of the transcribed region, and it did not affect the intracellular distribution of mRNA linked in cis. Therefore, CAESAR is thought to act at a step of mRNA translation without affecting the nuclear export of mRNA. Less