The t(15;17) translocation accounts for over 95 percent of all human acute promyelocytic leukemia (APL) cases. This translocation fuses the PML gene with the nuclear receptor for retinoic acid (RARa) and creates an oncogenic fusion protein, PML-RARa. It is thought that PML-RARa may interfere with the function of wild-type PML and RARa. Immunocytochemistry studies reveal the localization of PML at discrete nuclear domains known as PML oncogenic domains (PODs), Kr bodies, ND10, or nuclear bodies. While the exact function of the PODs remains unclear, its structure is disrupted in t(15;17)-translocation APL cells, suggesting an important roles for the PODs in APL. To investigate the function of the PODs, they have screened for PML-interacting factors using the yeast two-hybrid system. They have isolated three specific interacting proteins including a previously demonstrated PML-modifier, SUMO-1, and two novel proteins that are implicated in transcriptional regulation. In this project, they will investigate the interactions of PML with these two novel cofactors. Standard techniques including the yeast two-hybrid assay, GST pull down, Far-Western blot, and immunofluorescence microscopy will be utilized to establish the interaction of PML with these cofactors. Based on their preliminary studies, they propose that these two cofactors are transcriptional repressors. Experiments will be conducted to determine the mechanism of transcriptional repression by these proteins. They propose that the roles of PML and the PODs are to recruit these cofactors to the restricted PML domains by protein-protein interaction with PML. They further propose that localization of these cofactors to the PODs will lead to inactivation of their transcriptional repressor function. These hypotheses will be tested by a combination of transient transfection and immunocytochemistry studies. In addition, they will investigate the role of these PML cofactors in APL, based on observations that these two proteins also interact with the APL oncogenic fusion protein, PML-RARa. Together, these studies should shed new lights into the function of PML and its nuclear domains, as well as providing new insights to the function and regulation of the new cofactors. A better understanding of mechanism of APL at the molecular and cellular levels should lead to a more effective diagnosis and treatment of this disease, as well as providing implications for other malignancies.
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