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

PML-RARA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在野生型维甲酸受体靶基因的异常转录抑制中起重要作用，募集N-COR/SMRT(核受体共抑制/沉默维甲酸和甲状腺激素受体)共抑制蛋白复合体。在本研究项目中，我们证明了N-COR转录抑制复合体中的HDAC3是体内PML-RARα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在体内与N-COR/HDAC3相互作用而不需要配基。接下来，通过染色质免疫沉淀(ChIP)实验，将N-COR/HDAC3共阻遏复合体通过PML-RARβ与内源性目的启动子(RARα和CYP26)结合。相邻的组蛋白被去乙酰化，基因表达受到抑制。在pML-RARα表达细胞中，当hDAC3蛋白被干扰后，内源性目的基因被显著激活，启动子-荧光素酶报告实验也证实了这一点。这些结果表明，N-COR/HDAC3共抑制物参与了PML-RARα表达细胞的异常转录调控。我们还在体内利用了一种显性负蛋白(DNP)来干扰N-COR与HDAC3的相互作用。在DNP存在下，PML-RARα能与N-COR相互作用，但不能与HDAC3相互作用。提示DNP可作为白血病HDAC3功能分子靶向治疗的一种新策略。

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.