RUNX3 단백질의 조절인자로서의 BRD 단백질의 동정 = Identification of BRD as a Novel Regulator of RUNX3

다국어 초록 (Multilingual Abstract)

The RUNX gene family has attracted broad interest in recent years because these genes encode transcription factors that are involved in cell lineage determination during development and various forms of cancer. Of the three known RUNX family members, RUNX3 has been shown to be involved in T-cell differentiation and tumorigenesis of gastric epithelium. However, the molecular mechanisms for the regulation of RUNX family members have been poorly understood. BRD proteins containing bromo-domains which recognize acetylated lysines, include mammalian Brd2, Brd3, Brd4, Brdt and Drosophila Fsh, yeast Bdf1, Bdf2. BRD proteins are novel protein kinases. In this study, Drosophila mutant library screening showed that BRD proteins of BET (bromo-domains and extraterminal) family antagonize function of RUNX3 protein. Immunoprecipitation followed by immuno-blotting analysis revealed that BRD2/3 interacted with RUNX3 and decreased the level of RUNX3 protein in mammalian cells. Coincidently, BRD2/3 knock-down increased RUNX3 protein level. Further studies revealed that the first bromo-domain of BRD2/3 and runt-domain of RUNX3 were critical for the interaction. Increase of RUNX3 acetylation facilitates RUNX3-BRD2/3 interaction, suggesting that BRD2/3 proteins associated with acetylated RUNX3 through bromo-domain. These results demonstrate that BRD2/3 proteins recognize acetylated RUNX3 and inhibit function of RUNX3 protein.

목차 (Table of Contents)

• Introduction 1
• Materials and Methods 4
• 1. Reagents 4
• 2. Cell culture, Transient Transfection 4
• 3. Plasmids 5
• 4. Antibodies 5
• 5. Immunoprecipitation and Immunoblotting 5
• 6. Fly strains 6
• 7. Plasmid construction and generation of transgenic flies 6
• 8. Scanning Electron Microscopy (SEM) 6
• Results 7
• 1. BRD antagonizes RUNX3 protein in Drosophila. 7
• 2. Interaction of BRD2/3 proteins with RUNX3 protein. 7
• 3. si-BRD2/3 increases expression level of RUNX3. 8
• 4. BRD2/3 protein binds to runt-domain in RUNX3 protein. 8
• 5. RUNX3 protein binds to first bromo-domain in BRD2/3 protein. 9
• 6. Interaction between RUNX3 and BRD2/3 is acetylation dependent. 9
• Discussion 29
• Reference 31