Investigating the Mechanism of B-myb Dependent Gene Expression

描述（由申请人提供）：B-myb是一种序列特异性转录因子，已发现其在多种预后不良的癌症类型中过表达。MEFs中B-myb过表达抑制癌基因诱导的衰老，支持B-myb在致癌作用中的作用B-myb的反式激活结构域与衰老的抑制有关，表明B-myb的转录活性在这些表型中起作用。B- myB b以细胞周期蛋白A/Cdk 2依赖的方式磷酸化，这可以增加B- myB b的转录活性。Cdk 2磷酸化可以促进C-末端自抑制结构域的失活，因为该结构域的缺失增加转录活性并降低Cdk 2磷酸化的需要。B-myb参与细胞周期晚期基因表达的调控。B-myb在G2/M表达波期间促进数百个基因的表达。DeCaprio实验室最近的研究表明，B-myb与MuvB（一种高度保守的细胞周期调控复合物）和FoxM 1（另一种细胞周期转录因子）在晚期S期和G2期的晚期周期启动子处形成复合物。B-myb和MuvB在S期与细胞周期晚期启动子相关。B-myb促进G2/M基因表达的能力依赖于FoxM 1对B-myb/MuvB（MMB）复合物的特异性募集。 我建议，磷酸化的B-myb的Cdk 2取代的auto-promotory C-末端结构域，从而使FoxM 1与B-myb/MuvB（MMB）复合物的相互作用，以促进细胞周期晚期基因的表达和抑制癌基因诱导的衰老。首先，我将确定B-myb对MMB复合物-FoxM 1相互作用的要求。我将使用C末端截短和相互免疫沉淀来确定FOXM 1相互作用所需的B-myb上的最小结构域。我还将评估B-myb磷酸化对FoxM 1结合的作用。我将在B-myB磷酸化位点突变体中引入C-末端截短，以确定C-末端截短是否可以挽救B- myB磷酸化突变体中的FoxM 1结合。接下来，我将确定B-myb磷酸化和FoxM 1相互作用在B-myb介导的G2/M细胞周期基因反式激活中的作用。将进行ChIP-qPCR和ChIP-ReChIP，以确定B-myb磷酸化位点突变体和B-myb FoxM 1相互作用突变体是否可以募集FoxM 1至G2/M细胞周期基因启动子。我将在B-myb磷酸化位点突变体和FoxM 1结合缺陷的B-myb突变体中使用RT-qPCR评估G2/M细胞周期基因表达的诱导。最后，我将确定B-myb抑制癌基因诱导的衰老（OIS）的要求。我将在成纤维细胞系中过表达B-myb磷酸化位点突变体和HRasV 12，以评估B-myb磷酸化在抑制OIS中的作用。MuvB组分和FoxM 1将被消耗或抑制，以确定MuvB和FoxM 1对B-myb介导的OIS抑制的贡献。

DESCRIPTION (provided by applicant): B-myb is a sequence-specific transcription factor that has been found to be overexpressed in multiple cancer types with poor prognosis. B-myb overexpression in MEFs inhibits oncogene-induced senescence supporting a role for B-myb in carcinogenesis. The transactivation domain of B-myb has been implicated in the inhibition of senescence indicating that the transcriptional activity of B-myb plays a role in these phenotypes. B- myb is phosphorylated in a cyclin A/Cdk2-dependent manner that can increase the transcriptional activity of B- myb. Cdk2 phosphorylation may facilitate the inactivation of a c-terminal autoinhibitory domain since deletion of this domain increases transcriptional activity and reduces the requirement of Cdk2 phosphorylation. B-myb is involved in the regulation of late cell cycle gene expression. B-myb promotes the expression of hundreds of genes during the G2/M wave of expression. Recent studies by the DeCaprio lab show that B-myb forms a complex with MuvB, a highly conserved cell cycle regulation complex, and FoxM1, another cell cycle transcription factor, at the late cycle promoters during late S phase and G2. B-myb and MuvB associate with late cell cycle promoters during S phase. The ability of B-myb to promote G2/M gene expression is dependent on specific recruitment of FoxM1 to the B-myb/MuvB (MMB) complex. I propose that phosphorylation of B-myb by Cdk2 serves to displace the autoinhibtory C-terminal domain thereby enabling the interaction of FoxM1 with B-myb/MuvB (MMB) complex to promote late cell cycle gene expression and inhibit oncogene-induced senescence. First, I will determine the requirements of B-myb for the MMB complex-FoxM1 interaction. I will determine the minimum required domain on B-myb needed for the FOXM1 interaction using C-terminal truncations and reciprocal immunopreciptations. I will also assess the role of B-myb phosphorylation for FoxM1 binding. I will introduce C-terminal truncation into B-myb phosphorylation site mutants to determine whether C-terminal truncations can rescue FoxM1 binding in a B- myb phosphorylation mutant. Next, I will determine the role of B-myb phosphorylation and the FoxM1 interaction in B-myb mediated transactivation of G2/M cell cycle genes. ChIP-qPCR and ChIP-ReChIP will be conducted to determine whether B-myb phosphorylation site mutants and B-myb FoxM1-interaction mutants can recruit FoxM1 to G2/M cell cycle gene promoters. I will assess the induction of G2/M cell cycle gene expression with RT-qPCR in B-myb phosphorylation site mutants and B-myb mutants defective for FoxM1 binding. Finally, I will determine the requirements for the B-myb inhibition of oncogene-induced senescence (OIS). I will overexpress the B-myb phosphorylation site mutants and HRasV12 in fibroblast cell lines to assess the role of B-myb phosphorylation in the inhibition of OIS. MuvB compents and FoxM1 will deplete or inhibited to determine the contribution of MuvB and FoxM1 to B-myb mediated inhibition of OIS.