Investigating the Mechanism of B-myb Dependent Gene Expression

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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.