Development and application of a new Silent reporter system to quantitate the activity of enhancer elements in the type II Collagen Gene

• We developed a novel reporter assay system to quantify enhancer activities.
• The system is based on a silent reporter gene integrated in a chromosome.
• Activities of different enhancer elements were systematically compared.
• Col2a1 enhancers E1 and E2 exhibit additive and synergistic effect, respectively.

Type II collagen is a major component of cartilage, which provide structural stiffness to the tissue. As a sufficient amount of type II collagen is critical for maintaining the biomechanical properties of cartilage, its expression is tightly regulated in chondrocytes. Therefore, it is essential to elucidate in detail the transcriptional mechanism that controls expression of type II collagen, in particular by two enhancer elements we recently discovered. To systematically analyze and compare enhancer activities, we developed a novel reporter assay system that exploits site-specific integration of promoter and enhancer elements to activate a transcriptionally silent reporter gene. Using this system, we found that the enhancer elements have distinct characteristics, with one exhibiting additive effects and the other exhibiting synergistic effects when repeated in tandem.