Regulation of Gene Expression by PML

描述（由申请人提供）：早幼粒细胞白血病PML基因在急性早幼粒细胞白血病中持续被非随机染色体易位t破坏（15;17）。PML是一种具有多种功能的蛋白，参与调控细胞凋亡、细胞周期进程、基因表达、基因组稳定性和细胞衰老。PML如何参与如此多样的多种调节功能尚不清楚。当融合到GAL4-DNA结合域的下游时，PML通过募集组蛋白去乙酰化酶作为转录抑制因子。PML还通过与不同的转录因子（包括Sp 1、Nur77和NF-kappaB）相互作用来抑制转录，并破坏它们与DNA靶位点的结合。PML激活fos介导的AP-1反激活。PML还通过招募共激活因子CBP介导转录激活，这有力地支持了PML在转录激活中的作用。PML与p53相互作用，形成PML/p53/CBP复合物，上调p53介导的转录。本研究旨在了解PML调控的基因表达机制。基于前期研究的结果，我们的主要假设是PML通过(1)转录因子的相互作用和隔离来调节转录；(2)将CBP/ hdac招募到目标启动子，实现转录激活和抑制。为了支持这一假设，我们将追求以下三个具体目标：我们将研究PML转录抑制的机制和功能意义。我们的假设预测PML相互作用和隔离转录因子，并限制它们对DNA结合位点的可及性。我们将通过CHIP实验、双色免疫荧光染色、核基质中PML及其相关蛋白的共纯化来验证这一假设，并研究PML在G1/S细胞周期转变过程中如何调节Spl的可用性。2. 我们将研究不同PML亚型在介导靶基因表达中的功能意义。我们假设PML初级转录物的选择性剪接产生具有不同c端调控不同靶基因的各种同种异构体。我们将研究不同PML亚型的转录调控功能；研究表达模式，细胞分布。3. 我们将鉴定和表征通过hdac和CBP介导的PML靶基因。我们的假设预测PML将共抑制因子hdac和共激活因子CBP招募到目标启动子并抑制/激活转录。我们将通过构建和筛选染色质免疫沉淀DNA文库来鉴定PML靶基因，通过全基因组定位技术鉴定PML靶基因，并对PML靶基因进行表征。最后，我们将研究这些PML靶基因在急性早幼粒细胞白血病中是否失调。

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.