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.

Public Health Relevance

B-myb, a regulator of the cell cycle, is overexpressed in multiple cancer types with poor outcomes. This study seeks to further investigate the mechanism by B-myb promotes cell cycle gene expression and understand whether B-myb functions similarly in cancer-like situations. Successful completion of this proposal will provide insight as to why B-myb is associated with poor outcomes as well as further our understanding of the cell cycle.

Agency
National Institute of Health (NIH)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31CA189328-01
Application #
8778414
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcguirl, Michele
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115