Investigating The Role Of Tbx3 On Runx2 Activity & Function

The skeleton is an integral part of human body; it provides shape, structure and rigidity. Skeleton composed of bone is subject to continuous modifications throughout the life span of organism. During the process of intramembranous ossification mesenchymal cells differentiate directly into the bones whereas during endochondral ossification a cartilage model of the future bone is first produced. Both these process are critical for the survival of the organism and are strictly regulated at the transcriptional level. A network of transcription factors and proteins take part in the process. Runx2 is one major transcription factor which acts as a major switch to osteoblast differentiation. Tbx3, a Tbox containing transcription factor was recently shown to be expressed in bone in a growth hormone induced condition and negatively regulate Runx2 and osteoblastogenesis. The present thesis work was done to understand the mechanism of Tbx3 induced repression on Runx2 activity and function.Runx2 is the master regulator of osteoblast differentiation. Runx2 overexpressing C3h10t1/2 cell-line was generated to study the functions of Runx2 and factors influencing Runx2 function. Various reports have shown that Runx2 recruits co-activators such as HATs or co-repressors like HDACs to regulate transcription of its target genes. We chose HDAC 1, 5 and p300 (HAT) to analyze the localization of these acetylase and deacetylases in presence or absence of Runx2. Immunofluorescence results indicated that all the three regulatory proteins have a nuclear localization and doesn’t shuttle between the nuclear compartments regardless of the presence or absence of Runx2.Role of Tbx3 in regulating Runx2 induced osteoblast differentiation was studied. Stable C3h10t1/2 cell-line co-expressing Tbx3 alongwith Runx2 were generated. Dual luciferase assay results showed that Tbx3 interferes in the Runx2 mediated activation of osteopontin promoter by abrogating Runx2 activity. Immunofluorescence results demonstrated that Runx2 was simultaneously localized in the nucleus and the cytoplasm of Tbx3 overexpressing cells. These results confirmed that Tbx3 abrogates Runx2 activity and elevated levels of Tbx3 in the cells can result in mislocalization of Runx2.HDACs are an important group of proteins recruited during various cellular processes by transcription factors. The role of HDAC1 in Tbx3 mediated influence on Runx2 activity was studied. Reverse transcription PCR, Western Blotting and ALP staining was used to study the expression and effects of HDAC1 on Tbx3. HDAC1 mRNA levels in C3h10t1/2 cells overexpressing Tbx3 alongwith Runx2 were increased dramatically to two folds during proliferation of the cells whereas differentiating cells showed more than three fold increase in HDAC1 expression as compared to cells containing Runx2 only. Protein levels of HDAC1 were also remarkably higher during both the stages. Cells, with suppressed Runx2 mediated alkaline phosphatase activity (ALP), showed ALP positive staining in the presence of HDAC inhibitor TSA.Overall this thesis examines the role of Tbx3 on Runx2 activity and function. Tbx3 potently represses Runx2 and affects the nuclear localization. Tbx3 upregulates HDAC1 to influence Runx2 activity and function, inhibiting HDAC1 may relax the repressive effects of Tbx3 on Runx2.