Bou-Gharios, GeorgeAlSalhi, Sara Ibrahim2024-11-242024-08AlSalhi, S.I., 2024. Bioengineering of transcriptional elements driving MMP13 gene in skeletal development. PhD thesis. Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, Faculty of Health and Life Sciences, University of Liverpool. Available via Symplectic (part of Digital Science). https://elements.liverpool.ac.uk/viewobject.html?cid=1&id=770893ID: 1ECE7F0E-FC6E-4654-94B5-DE96AF34C254https://hdl.handle.net/20.500.14154/73689This study investigates the transcriptional regulation of Mmp13, a primary enzyme in bone development and in adults, and a catabolic factor implicated in cartilage degradation. Through a combination of in silico analyses and in vitro/in vivo experiments, the research identifies and characterizes multiple enhancer regions associated with the Mmp13 gene, aiming to unravel their roles in gene expression and activity during embryonic development. The study employs a comprehensive approach, utilizing data from ENCODE, RUNX2 peaks, VDR, histone modifications, fibroblast coverage, chondrocyte, and embryonic limb regulatory elements, as well as public ChIP-Seq data. This study breaks new ground by integrating these diverse data sources and using three different analysis software tools (CIIIDER, TRANSFAC, JASPAR) to predict transcription factor binding sites, thus identifying several enhancer regions and discovering skeletal element expression. The construction of Mmp13 enhancers in an expression vector, followed by in vivo testing on transgenic embryos and in vitro transfections in various cell types, provides a thorough understanding of the regulatory landscape. From seven tested regions of the Mmp13 enhancer, skeletal elements expressing chondrocytes in transgenic embryos at E15.5 were detected in the 5th Intron, Proximal Promoter, and the distal enhancers at -10, -19.4, and -21.4 kb. Additionally, hypertrophic chondrocyte and osteoblast expression were detected in the -21.9 to -21.1 kb hotspot overlapping region between Runx2 and Sox9, where Runx2 is considered a master TF for osteogenesis, and Sox9 is a key transcription factor in chondrogenesis. Along with expressions in various tissues and organs observed in multiple regions. In contrast, the sequence overlapping with the highest peak of Runx2 at -29 kb and -32.5 kb did not show significant expression. The persistent expression in the same anatomical locations indicates that the enhancer's regulatory function is not solely dependent on genotyping efficiency but also involves specific interactions and regulatory elements within the shared sequence. In vitro experiments confirm the significance of the -10 kb and -29 kb enhancers in chondrocyte cells of both mouse and human cells. However, discrepancies between mouse embryos and isolated cells underscore the complexities of gene regulation in whole organisms. The study unveils a detailed map of Mmp13 enhancer regions, each playing distinct roles during embryonic development. Mmp13 enhancers provide insights into their regulatory functions in musculoskeletal organs and tissues in the early embryonic stage at E15.5, some were considered at E13.5 and E14.5 to address the constant skeletal element expression at -21.4 kb, in addition to the advanced stage in vitro cell line and primary extracted OA cell. This nuanced understanding enhances our knowledge of Mmp13-related pathologies, offering potential targets for therapeutic interventions. Building on this, future studies could explore the interaction between these enhancers and other transcription factors beyond Runx2 and Sox9 and investigate the regulatory mechanisms in other developmental stages or in response to environmental or mechanical stressors. This study's findings are directly relevant to human health, especially by considering how some regions on or upstream of Mmp13 that might have a clear role in bone development and suggest several targeting to control cartilage degradation. The insights gained into the regulation of Mmp13 not only enhance our understanding of developmental biology but also have direct implications for treating and managing diseases that affect the musculoskeletal system, such as osteoarthritis. The translational potential of these findings could lead to novel strategies for preventing or reversing the damage caused by excessive Mmp13 activity.Mmp13 is a primary catabolic factor involved in cartilage degradation through its ability to cleave collagen type II and other cellular components. In addition to being necessary for the formation of various cells, organs, and tissues, Mmp13 expression is regulated transcriptionally by two main elements: the proximal promoter and distal enhancers. This study aims to identify the transcriptional elements that regulate Mmp13 gene expression. Identification of novel Mmp13 enhancers was conducted in silico using the Encyclopaedia of DNA Elements (ENCODE), based on histone modifications (Limb H3K4me1 and Limb H3K27Ac), fibroblast coverage, chondrocyte, and embryonic limb regulatory elements from public ChIP-Seq data, and evolutionarily conserved sequences, in addition to transcription factor profiles of Runx2 and vitamin D. All prospective Mmp13 enhancer sequences were analysed using three different software tools: CIIIDER, TRANSFAC, and JASPAR, for the prediction of transcription factor binding sites. Each enhancer sequence was cloned upstream of the Hsp68 silenced promoter and lacZ gene to create a β-galactosidase reporter construct, which was then used to generate transgenic mice. Embryos were collected at E15.5 and tested for lacZ gene expression and tissue expression analysis. Constructs were also transfected in vitro into pre-osteoblasts (MC3T3- E1), NIH3T3 mouse embryo fibroblasts, human chondrocytes (SW1353), and primary chondrocytes extracted from OA patients. Among the seven tested Mmp13 enhancer regions, strong skeletal element expression was detected in the region from -21.9 to -21.1 kb, which overlaps with Runx2 and Sox9 binding sites. Other enhancers revealed some skeletal element activity but were not as prominent. Expression in various tissues and organs, including skin, was observed in multiple regions. In contrast, sequences aligning with the highest peaks of Runx2 at -29 kb and -32.5 kb did not show significant expression. In vitro, Mmp13-transfected enhancer sequences demonstrated enzyme activity, with the highest responses observed in chondrocyte and human cells at -10 kb and -29 kb regions, along with -21.4 kb that indicate a potential regulatory influence. Comparisons of potential enhancers in mouse embryos highlighted the sequences in the intronic 5', -10 kb, -19.2 kb, and -21.4 kb regions as significant Mmp13 enhancers regulating gene expression but not -29 and -32.5 kb regions. Identifying these specific enhancers could lead to targeted therapeutic strategies to modulate MMP13 activity, potentially slowing or preventing cartilage degradation in degenerative diseases.324enCIIIDERENCODEExpressionGenotypingHistone modificationHSP68-LacZIn silicoIn vitroIn vivoJASPARMC3T3E1MMP13MMP13 EnhancersMulti-Mmp13 enhancerNIH3T3OsteoarthritisRUNX2skeletal developmentskeletal elementsSOX9ß-galactosidase activitySW1353Transcriptional elementsTRANSFACTransgenic embryosThesis