Title : Bioengineering of identifying transcriptional elements driving MMP13 gene in skeletal development
Abstract:
MMP13 is a primary catabolic factor involved in cartilage degradation through its ability to cleave type II collagen. Transcriptionally, MMP13 is regulated by 2 main elements: proximal prompter and distal enhancers by both molecular and epigenetic factors. The aim of this study is to identify transcriptional elements that regulate the MMP13 gene in order to control the substantial rise in MMP13 expression observed in Osteoarthritis and other diseases. Identification of MMP13 novel Enhancers In silico was by using the Encyclopaedia of DNA Elements (ENCODE), Based on RUNX2 peaks and VDR, Histone modifications (Limb H3K4ME1 and Limb H3k27AC), fibroblast coverage, Chondrocyte and Embryonic limb regulatory elements, Regulatory regions from Public ChIP- Seq data for transcriptional regulators based on osteoblast cells and evolutionarily conserved sequence. along to Main MMP13 Enhancer Based on Encode Mouse (GRCm38/mm10) of candidate cisregulatory elements that exists at chr9:7,250,844-7,251,224 (381bp). All MMP13 Enhancers sequences went through 3 different analysis software: CIIIDER, TRANSFAC, and JASPAR used in the prediction of Transcription factors binding sites. Construction of MMP13 Enhancers in expression Vector was by cloning each Enhancer upstream of the HSP68 Silenced promoter and LacZ gene to create a ß-galactosidase reporter construct. Each of these constructs has been Identified with R.Es. And then linearized and tested In Vivo (Transgenic embryos at E15.5 days). Genotyping was carried out embryo's tail to verify DNA integration. Constructs Also, were transfected In Vitro to detect the ß-galactosidase activity from the cell lysate of Mice Pre-Osteoblast (MC3T3E1), NIH3T3 Adult Mice Fibroblast, Human Chondrocyte (SW1353) and Primary extracted cells from OA patient (Human articular cartilage) (P69 p.2). In Vivo, From 7 tested regions of MMP13 Enhancer; the skeletal elements expressing in Transgenic Embryos at E15.5 were detected in the 5th Intron, Proximal Promoter, and the distal enhancers at -10, -19.4, and -21.4kb. In addition, expression was also seen in other cell types such as developing skin, tendons, muscles, and fibroblasts in other types of tissues. In contrast, the sequence overlapping with the highest peak of Runx2 at -29kb and -32.5kb did not show a significant expression. Sections of Whole mount reporter from each sequence of intron5', Proximal promoter, -10, -19.4, and -21.4kb showed high localization expression in Chondrocyte and Osteoblast cells in specific limbs expression. The Expression ratio vs. Genotyping ratio showed the Enhancer region at -21.4 to -21.07kb has the highest and equal level of expression as conformation to the main enhancer of MMP13 in the early stages of the skeletal development, matching with the main distal enhancer at around -21.6 to -21.2 kb in ENCODE Mouse (GRCm38/mm10) of cisregulatory elements where expression goes beyond skeletal elements. The Transgenic Embryos results showed that the MMP13 is regulated at the level of transcription in at least four different regions, which coincides with Runx2 peak activity. The expression is not located only at the hypertrophic zone but in proliferating chondrocytes. Intronic 5 sequences that respond to IL-1 showed an exclusively skeletal expression. In vitro, MMP13 Enhancers were transfected in Mice Pre-Osteoblast (MC3T3E1), NIH3T3 Adult Mice Fibroblast, Human Chondrocyte (SW1353), and Primary OA Human articular cartilage to detect the ß-alactosidase activity from the cell lysate using Glacton-Plus substrate. The cleaved enzyme activity and triggering light emission of MMP13 Enhancers for skeletal development showed that -10 kb and -29 kb from the MMP13 gene are major enhancers in chondrocyte cells in adult mice and Human cells. Comparisons of potential enhancers' ability in mouse embryos supported -10 kb enhancers but contradicted -29 and -32.5 kb and instead provided evidence for the -21.4 kb region as an MMP13 enhancer. Disagreements were speculated to be caused by complexities of gene regulation in whole organisms compared with isolated ones. The preservation of MMP13 in tissue development and repair while relieving osteoarthritis symptoms requires drugs that can selectively block MMP13 activity. As a key to controlling the development of selective drugs; the effect of -21.4kb enhancer in the early stages of skeletal development to the late stages at -29.3kb, can be used as a targeted drug that controls the substantial rise in MMP13 expression observed in osteoarthritis either to suppress or to minimize the secretion of MMP13 in the ECM.
