Regulation of Gene Expression by PML

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DESCRIPTION (provided by applicant): The promyelocytic leukemia PML gene is consistently disrupted by the nonrandom chromosomal translocation t(15; 17) in acute promyelocytic leukemia. PML is a protein with multiple functions involves in regulation of apoptosis, cell cycle progression, gene expression, genome stability, and cellular senescence. How PML involves in such diverse multiple regulatory functions remains unknown. When fused downstream of the GAL4-DNA binding domain, PML acts as a transcriptional repressor by recruiting histone deacetylases. PML also represses transcription by interacting with different transcription factors including Sp 1, Nur77, and NF-kappaB and disrupt their binding to the DNA target sites. PML activates fos-mediated AP-1 transactivation. PML also mediates transactivation by recruiting coactivator CBP has been reported by several groups, strongly supporting a role of PML in transcription activation. PML interacts with p53, forms a PML/p53/CBP complex and upregulates p53 mediated transcription. The objectives of this proposal are to understand the mechanisms of gene expression regulated by PML. Based on the results accomplished in the preliminary studies, our main hypothesis is that PML regulates transcription by (1) interaction and sequestration of transcription factors; (2) recruits CBP/HDACs to the target promoters to achieve transcriptional activation and repression. The following three specific aims will be pursued to support the hypothesis: 1. We will study the mechanism and functional significance of transcriptional repression by PML. Our hypothesis predicts that PML interacts and sequesters transcription factors and limiting their accessibility to the DNA binding sites. We will test the hypothesis by CHIP assay, double color immunofluorescence staining, co-purification of PML and its associated proteins in the nuclear matrix, and to study how PML regulates the availability of Spl during G1/S cell cycle transition. 2. We will study the functional significance of different PML isoforms in mediating expression of target genes. We hypothesize that alternative splicing of the primary PML transcript produces various isoforms with different C-terminals regulating different target genes. We will study the transcriptional regulatory functions of different PML isoforms; study the expression pattern, cellular distribution. 3. We will identify and characterize the PML target genes mediated through HDACs and CBP. Our hypothesis predicts that PML recruits corepressors HDACs and coactivator CBP to the target promoters and represses/activates transcription. We will identify PML target genes by construction and screening of a chromatin immunoprecipitated DNA library, identify the PML target genes by the genome-wide location technology, and to characterize the PML target genes. Finally we will investigate whether these PML target genes are deregulated in acute promyelocytic leukemia.