Investigating the Mechanism of B-myb Dependent Gene Expression

描述(申请人提供)：B-MYB是一种序列特异性转录因子，已被发现在多种癌症类型中过度表达，预后不良。在MEF中过表达B-MYB可抑制癌基因诱导的衰老，支持B-MYB在肿瘤发生中的作用。B-MYB的反式激活结构域与抑制衰老有关，表明B-MYB的转录活性在这些表型中起作用。B-MYB以细胞周期蛋白A/CDK2依赖的方式被磷酸化，从而提高B-MYB的转录活性。CDK2的磷酸化可能有助于c末端自抑制结构域的失活，因为该结构域的缺失增加了转录活性，降低了CDK2磷酸化的需要。B-MYB参与了细胞周期晚期基因表达的调控。在G2/M波的表达中，B-MYB促进了数百个基因的表达。DeCaprio实验室最近的研究表明，在S晚期和G2期，B-myb与高度保守的细胞周期调控复合体MuvB和另一种细胞周期转录因子FOXM1在周期晚期的启动子上形成复合体。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FOXM1对B-MYB/MuvB(MMB)复合体的特异性募集。我认为CDK2对B-MYB的磷酸化作用取代了自身抑制的C末端结构域，从而使FOXM1与B-MYB/MuvB(MMB)复合体相互作用，促进细胞周期晚期基因的表达，抑制癌基因诱导的衰老。首先，我将确定B-MYB对MMB复合体-FOXM1相互作用的要求。我将使用C-末端截断和相互免疫沉淀来确定FOXM1相互作用所需的B-MYB上的最低结构域。我还将评估B-MYB磷酸化对FOXM1结合的作用。我将在B-MYB磷酸化位点突变体中引入C-末端截断，以确定C-末端截断是否可以挽救B-MYB磷酸化突变体中FOXM1的结合。接下来，我将确定B-MYB磷酸化和FOXM1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。CHIP-qPCR和CHIP-ReChIP检测B-MYB磷酸化位点突变体和B-MYB FOXM1-相互作用突变体是否能够招募FOXM1到G2/M细胞周期基因启动子。我将评估RT-qPCR在B-MYB磷酸化位点突变和FOXM1结合缺陷的B-MYB突变中对G2/M期细胞周期基因表达的诱导作用。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HRasV12，以评估B-MYB磷酸化在抑制OIS中的作用。MuvB组分和FOXM1将耗竭或被抑制，以确定MuvB和FOXM1在B-MYB介导的OIS抑制中的贡献。

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.