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Degree

Doctor of Philosophy, Ohio State University, Molecular, Cellular, and Developmental Biology, 2007.

Abstract

The microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor, regulates distinct target genes in several cell types including osteoclasts. Osteoclasts are terminally differentiated multinuclear cells responsible for bone resorption. CSF-1 and RANKL are two critical cytokines to induce osteoclast differentiation from bone morrow-derived precursors. In osteoclasts, MITF interacts with the Ets family transcription factor PU.1 to synergistically activate target genes like Cathepsin K (Ctsk) and Acid Phosphatase 5 (Acp5). The region of MITF required for the physical interaction with PU.1 is the bHLH-Zip domain. To finer map the interacting domain of MITF, we introduced point mutations into the loop region of MITF via in vitro site-directed mutagenesis. The properties of mutated MITF proteins were analyzed by transient transfection assays, EMSAs, and in vitro GST pulldown assays. We identified that specific amino acids including N235, D236, and W241 in the loop region of MITF are critical to mediate physical and functional interaction with PU.1. Both MITF and PU.1 are known transcriptional activators in osteoclasts. Results presented here demonstrate that they can also act as components of repressor complexes that suppress target gene expression in committed myeloid precursors. The direct interaction of MITF and PU.1 with the zinc-finger protein Eos, an Ikaros family member, appears to be necessary for repression of Ctsk and Acp5. In bone marrow-derived precursors treated with CSF-1 alone, Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of co-repressors CtBP and Sin3A. Eos expression was reduced during osteoclast differentiation initiated by combined CSF-1 and RANKL stimulation and Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited co-activators to these target promoters resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors inhibited multinuclear osteoclast formation and repressed transcription of subset of osteoclast specific genes that are regulated by MITF and PU.1. This work provides a novel mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.

Subject Headings

Biology, Molecular

Keywords

Cell differentiation; osteoclasts; transcriptional regulation

Advisor

Michael C Ostrowski

Pages

242p.

Document number: osu1166644761
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1166644761

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