Transcriptional regulation of the Ccn2 gene

Abstract

Cellular Communication Network Factor 2 (CCN2) is a matricellular protein which functions in many tissues, and is most notably expressed by chondrocytes, with knockout of the Ccn2 gene expression causing severe chondrodysplasia in mice. Regulation at the prerequisite stage of transcription by non-coding genomic elements is fundamental in the expression of every gene. Until now, the capacity for cis-acting regulatory modules to control the expression of Ccn2 has been obscure. The current project sought to delineate the capacity for cis-acting enhancer regions to regulate Ccn2 transcription within highly specific temporospatial contexts. A 300 kilobase intergenic genomic region upstream of Ccn2 was examined in silico in order to identify putative enhancer regions based on chromatin characteristics of enhancers; histone modification, DNase I hypersensitivity and interspecies evolutionary conservation of DNA sequence. Transgenic mice were created using constructs consisting of each putative enhancer region driving the expression of a LacZ reporter gene in conjunction with a silent Hsp68 minimal promoter. Expression of the protein product of LacZ, β-galactosidase was assayed at several developmental time-points in order to determine whether candidate enhancers were able to control gene transcription. Five enhancer regions of murine Ccn2 expression, located -230kb, -198kb, -148kb, -137kb and -102kb upstream of the gene each drove LacZ expression in a tissue-specific manner at embryonic day E15.5. -230kb drove transgene activity within osteoblasts, whereas -198kb, -148kb and -137kb all exhibited function within chondrocytes. The -102kb enhancer was active within the superficial vasculature. More comprehensive examination of the - 148kb enhancer revealed function in adulthood, and that a truncated region of this sequence is also capable of enhancing gene transcriptional output. Enhancer sequences were also examined in vitro using Electrophoretic Mobility Shift Assay in order to test the capacity of transcription factors of interest to bind to enhancers and modulate function. Master regulator of chondrocyte physiology, the transcription factor SOX9 was found to bind to sequences within the -137kb, -148kb and -230kb enhancers. The findings described herein are the first to characterise the role of cis-acting enhancer regions in the transcription of Ccn2 within the murine genome. Understanding the mechanisms that underpin temporospatial control of Ccn2 expression will inform both the characterisation and amelioration of profound pathological conditions that result from loss of CCN2 transcriptional regulation such as osteoarthritis, fibrosis and cancer in humans.