Differential tissue specific, temporal and spatial expression patterns of the Aggrecan gene is modulated by independent enhancer elements.

The transcriptional mechanism through which chondrocytes control the spatial and temporal composition of the cartilage tissue has remained largely elusive. The central aim of this study was to identify whether transcriptional enhancers played a role in the organisation of the chondrocytes in cartilaginous tissue. We focused on the Aggrecan gene (Acan) as it is essential for the normal structure and function of cartilage and it is expressed developmentally in different stages of chondrocyte maturation. Using transgenic reporter studies in mice we identified four elements, two of which showed individual chondrocyte developmental stage specificity. In particular, one enhancer (-80) distinguishes itself from the others by being predominantly active in adult cartilage. Furthermore, the -62 element uniquely drove reporter activity in early chondrocytes. The remaining chondrocyte specific enhancers, +28 and -30, showed no preference to chondrocyte type. The transcription factor SOX9 interacted with all the enhancers in vitro and mutation of SOX9 binding sites in one of the enhancers (-30) resulted in a loss of its chondrocyte specificity and ectopic enhancer reporter activity. Thus, the Acan enhancers orchestrate the precise spatiotemporal expression of this gene in cartilage types at different stages of development and adulthood.

Multiple enhancer regions govern the transcription of CCN2 during embryonic development.

CCN2 is a critical matricellular protein that is expressed in several cells with major implications in physiology and different pathologies. However, the transcriptional regulation of this gene remains obscure. We used the Encyclopaedia of DNA Elements browser (ENCODE) to visualise the region spanning from 300 kb upstream to the CCN2 start site in silico in order to identify enhancer regions that regulate transcription of this gene. Selection was based on three criteria associated with enhancer regions: 1) H3K4me1 and H3K27ac histone modifications, 2) DNase I hypersensitivity of chromatin and 3) inter-species conservation. Reporter constructs were created with sequences spanning each of the regions of interest placed upstream of an Hsp68 silent proximal promoter sequence in order to drive the expression of ß-galactosidase transgene. Each of these constructs was subsequently used to create transgenic mice in which reporter gene production was assessed at the E15.5 developmental stage. Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints. The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. These findings suggest that the global expression of CCN2 during development results from the activity of several tissue-specific enhancer regions in addition to proximal regulatory elements that have previously been demonstrated to drive transcription of the gene during development.

Characterization of Mesenchymal-Fibroblast Cells Using the Col1a2 Promoter/Enhancer.

Excessive deposition of extracellular matrix (ECM) is a common hallmark of fibrotic diseases in various organs. Chiefly among this ECM are collagen types I and III, secreted by local fibroblasts, and other mesenchymal cells recruited for repair purposes. In the last two decades, the search for a fibroblast-specific promoter/enhancer has intensified in order to control the regulation of ECM in these cells and limit the scarring of the fibrotic process. In our previous work, we characterized an enhancer region 17 kb upstream of the Col1a2 gene transcription start site. This enhancer in transgenic mice is expressed mainly in mesenchymal cells during development and in adults upon injury. When driving transgenes such as beta-galactosidase or luciferase, this construct acts as an informative reporter of collagen transcription and is predictive of collagen type I deposition. In this chapter, we provide detailed protocols for identifying similar enhancers and using the sequence to generate a construct for transfection and producing transgenic animals. We also provided information on the use of luminescence in transgenic mice, tissue processing, as well as using cre/lox system to obtain conditional gain and loss of function in mice.