The Role of Microphthalmia Transcription Factor (Mitf) in Osteoclast Gene Regulation

Meadows, Nicholas Axel (2006). The Role of Microphthalmia Transcription Factor (Mitf) in Osteoclast Gene Regulation PhD Thesis, , University of Queensland.

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Author Meadows, Nicholas Axel
Thesis Title The Role of Microphthalmia Transcription Factor (Mitf) in Osteoclast Gene Regulation
Institution University of Queensland
Publication date 2006
Thesis type PhD Thesis
Supervisor Dr. Alan Cassady
Abstract/Summary Microphthalmia transcription factor (Mitf) has been shown to be a key regulator of osteoclast function by activating genes like TRAP and cathepsin K. Mitf is a member of the helix-loop-helix leucine zipper family of transcription factors and regulates its target genes by binding as a homo- or heterodimer to an E-box consensus sequence termed the M-Box (T/CANNTG/A). In order to identify the suite of genes that are regulated by Mitf in macrophages and osteoclasts, the level of Mitf activity has been modulated in the pre-osteoclastic macrophage cell line, RAW/C4. Stable transfectant cell lines expressing the Mitf-A isoform (RAW/C4-Mitf-A/pEF6) and the dominant negative isoform, mi (RAW/C4-mi/pEF6), under the control of the EF1a promoter, were prepared. Both the expression and function of the exogenous genes were validated using western blotting, immunocytochemical staining, Q-PCR and promoter-reporter transfection analysis. The genes differentially regulated by the alteration of the level of Mitf in these cells were expression profiled by microarray analysis. The 22 000 element murine Compugen array was hybridized with RNA prepared from the RAW/C4-Mitf/pEF6 and RAW/C4-mi/pEF6 cell lines before and after induction of osteoclast differentiation with RANKL. Genes identified include those either up- or down-regulated during osteoclast differentiation, as well as genes with differential expression as a result of overexpression of Mitf protein. A bioinformatic analysis was undertaken in parallel to identify candidate M-box-containing genes that may be Mitf targets. A large-scale Blast technique was developed to download 2 kb of DNA sequence 5’ of the translation start site for all the genes on the Compugen array. These promoter regions were searched for the presence of M-boxes and a subset of potential Mitf target genes was compiled and compared with targets generated from the microarray analysis. This approach identified eight genes that were indicated to be Mitf regulated based on their expression profile and the presence of a M-box within their proximal promoter region. The expression of these genes was validated using Q-PCR and following a literature review of their functional biology, five genes remained. Ccl9, Clcn7, Eos, Fra1 and Ostm1 all met the stringent criteria necessary for selection for further investigation. A bioinformatic analysis of the promoter regions for these genes was undertaken to determine mouse-human evolutionary conservation and establish a common mechanism for transcriptional regulation of each gene. Common conserved transcription factor binding sites were identified and candidate M-boxes within their promoter regions were assayed for Mitf binding and transcription activation using a series of in vitro techniques on the cell lines. Electrophoretic mobility shift and promoter-reporter luciferase assays were performed on RAW/C4 cells to demonstrate binding site specificity and promoter activation by Mitf. Consequently osteopetrosis associated protein 1, Ostm1 and chloride channel 7, Clcn7 were identified as specific targets for Mitf transcriptional regulation. Primary cells were also used for Q-PCR and chromatin immunoprecipitation assays, which confirmed Mitf regulation of Clcn7. Both Ostm1 and Clcn7 have known mutations that can cause a failure of osteoclast activity and severe osteopetrosis, a phenotype characteristic of particular Mitf mutations. These findings validate the combined bioinformatic and microarray approach used to identify novel Mitf targets during osteoclastogenesis. The identification of two genes, whose expression appears to be regulated by Mitf, that are directly involved with osteoclast resorption suggests the role of Mitf in controlling bone resorption might be more significant than previously perceived. These results indicate that Mitf may be a master regulator of osteoclast bone resorption and genes regulated by Mitf during osteoclast activation may be considered therapeutic targets for the treatment of bone resorption disorders.

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Created: Fri, 21 Nov 2008, 14:21:47 EST