Analyses of Transcription Co-repressor Complexes Involved in Differentiation Block of Leukemia Cells, and Development of a New Molecular Targeting Therapy.

PML-RAR a是一种与急性早幼粒细胞白血病密切相关的嵌合转录因子。PML-RARα在野生型视黄酸受体靶基因的异常转录抑制中发挥重要作用，募集N-CoR/SMRT（核受体共抑制因子/类视黄酸和甲状腺激素受体沉默介质）共抑制蛋白复合物。在本研究项目中，我们证明了N-CoR转录抑制复合物的一个组成部分HDAC3是PML-RARα在体内转录抑制的关键调节因子。通过免疫沉淀，我们证明了PML-RAR a在没有配体的情况下与N-CoR/HDAC3相互作用。接下来，利用染色质免疫沉淀（ChIP）实验，通过PML-RARα将N-CoR/HDAC3共抑制复合物募集到内源性目标启动子（RARβ和CYP26）上。邻近的组蛋白去乙酰化，基因表达受到抑制。在pml - rar α-表达细胞中，通过RNA干扰敲低HDAC3蛋白，内源靶基因被显著激活，启动子荧光素酶报告基因实验也证实了这一点。这些结果表明，N-CoR/HDAC3共抑制因子复合物参与了pml - rar α-表达细胞的异常转录调控。我们还利用一种显性阴性蛋白（DNP）在体内干扰N-CoR与HDAC3的相互作用。在DNP存在的情况下，PML-RARα可以与N-CoR相互作用，但不能与HDAC3相互作用。我们的数据表明，DNP可能作为一种新的分子靶向治疗策略，专注于白血病中HDAC3的功能。

PML-RAR a is a chimeric transcription factor tightly associated with acute promyelocytic leukemia. PML-RARα plays an important role in the aberrant transcription repression on the target genes of wild-type retinoic acid receptors, recruiting N-CoR/SMRT (nuclear receptor co-repressor / silencing mediator for retinoid and thyroid hormone receptors) co-repressor protein complexes. In this research project, we demonstrated that HDAC3, one component of the N-CoR transcription repressor complex, is a key regulator of the transcription repression by PML-RARα in vivo. Using immunoprecipitation, we demonstrated that PML-RAR a interacts with N-CoR/HDAC3 in vivo without ligand. Next, using chromatin immunoprecipitation (ChIP) assay, this N-CoR/HDAC3 co-repressor complex was recruited to the endogenous target promoters (RARβ and CYP26) through PML-RARα. The neighboring histones were de-acetylated and gene expression was repressed. When HDAC3 protein was knocked down by RNA interference in PML-RARα-expressing cells, the endogenous target genes were significantly activated, which was also confirmed by promoter-luciferase reporter assay. These results provide evidence to show that the N-CoR/HDAC3 co-repressor complex is involved in the aberrant transcription regulation in PML-RARα-expressing cells. We also utilize a dominant-negative protein (DNP) that interfere N-CoR interaction with HDAC3 in vivo. In the presence of DNP, PML-RARα can interact with N-CoR but not with HDAC3. Our data suggest that DNP may be introduced as a new strategy for the molecular targeting therapy focusing on HDAC3 function in leukemia.