A cluster of regulatory elements that drives the aggrecan gene expression in chondrocytes

Purpose: Aggrecan (Acan) is a large aggregating proteoglycan which is functionally important for the mechanical properties of cartilage. It is required for the development and maintenance of mature cartilage. The loss of Acan is considered one of the first events in the development of osteoarthritis (OA). Very little is known about transcriptional control of Acan. This study aims to characterise cis-acting elements of Acan in skeletal elements. Methods: The publically available ENCODE consortium data was used to identify possible regulatory elements of Acan. The criteria for selection included: highly evolutionary conserved sequences, marked by histone modifications such as mono-methylation of histone H3 lysine 4 (H3K4me1) and acetylation of histone H3 lysine 27 (H3K27ac). Nine cis-acting sequences were identified between −150kb to +60kb flanking the Acan gene. One of these was the already identified enhancer at −10kb by Han and Lefebvre, 2008. We cloned each of the sequences upstream of the HSP68 minimal promoter driving a LacZ reporter and generated transgenic mice. Results: We found a cluster of enhancers located −10kb to −87kb upstream of the transcription start site, comprising of 4 elements in total (−10kb, −35kb, −65kb and −87kb) and one intronic region +26kb that express in chondrocytes and no other Acan expressing cells. The −65kb is active primarily in hypertrophic regions in the limbs and skull but is absent from the intervertebral disk and Meckel’s cartilage. The −35kb and +26kb present in all chondrocytes at E15.5, irrespective of the stage of the chondrocyte, and do not favour other tissues that have been shown to contain Acan such as in the brain. The final −87kb region shows weak and scattered expression development in the limbs, ribs and skull; this progress in 8 week old mice the expression becomes more robust in articular cartilage and growth plate. In vitro analysis of the enhancers, utilising Electromobility Shift Assays (EMSAs) and conservation reveals that the transcription factor Sox9 interacts at multiple sites in the +26kb, +35kb −65kb and −87kb regions and mutations introduced into the sequences abrogates the binding. Taking the enhancer that expresses in all chondrocytes, we found the 412bp sequence contains interaction sites with Sox9 as point mutations in any of the binding sites in vivo ‘shifts’ the expression to areas other than chondrocytes. Conclusions: We have shown Acan is under the influence of multiple cis-acting elements, the apparent overlap in expression of these enhancers indicates possible phenotypical robustness in Acan transcription. We are exploring the function and importance of these enhancers in the expression of Acan during development, adulthood and in disease such as OA.