The overall goal of this proposal is to understand how factors that mediate the balance between differentiation and proliferation maintain cells in a normal versus tumorigenic state. Our approach is to analyze the relationship between the cell cycle regulator p21 and the erythroid-specific transcription factor GATA-1 in murine erythroleukemia (MEL) cells. MEL cells are blocked from differentiating due to direct binding and repression of GATA-1 by PU.1, a myeloid transcription factor activated by viral integration. p21 expression is low in undifferentiated MEL cells but is significantly upregulated as PU.1 declines during differentiation. Furthermore, exogenous GATA-1 and p21 can overcome this block and trigger terminal differentiation. Preliminary data indicates that GATA-1 can induce p21 gene transcription. We will test the hypothesis that the p21 gene is a direct transcriptional target of GATA-1 by pursuing three specific aims: 1) To identify DNA sequences in the p21 promoter required for GATA- 1 stimulation using reporter assays and electrophoretic mobility shifts (EMS A' s); 2) To determine if the p21 gene is a GATA-1 target in differentiating MEL cells by testing for GATA- 1 occupancy of the endogenous p21 promoter using chromatin immunoprecipitation; and 3) To determine whether the p21 gene is a target for PU.1-mediated repression of GATA-1 in undifferentiated MEL cells. We will examine whether reducing PU.1 levels with short interfering RNA (siRNA) derepresses p21 gene transcription and test for occupancy of the p21 promoter by PU.1 and transcriptional corepressors such as Rb. These studies will elucidate how proliferation and differentiation are coordinated in erythroid cells and how this coordination is disrupted during oncogenesis.
| Papetti, Michael; Wontakal, Sandeep N; Stopka, Tomas et al. (2010) GATA-1 directly regulates p21 gene expression during erythroid differentiation. Cell Cycle 9:1972-80 |
| Papetti, Michael; Skoultchi, Arthur I (2007) Reprogramming leukemia cells to terminal differentiation and growth arrest by RNA interference of PU.1. Mol Cancer Res 5:1053-62 |
| Yu, Yung-Luen; Chiang, Yun-Jung; Chen, Yu-Chun et al. (2005) MAPK-mediated phosphorylation of GATA-1 promotes Bcl-XL expression and cell survival. J Biol Chem 280:29533-42 |
| Stopka, Tomas; Amanatullah, Derek F; Papetti, Michael et al. (2005) PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure. EMBO J 24:3712-23 |
