Cell-cycle dependent gene transcription through activation of B-Myb

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PROJECT SUMMARY The cell cycle is a carefully controlled cellular process that maintains the integrity of organismal growth. Deregulation of the cell cycle leads to cell death or irregular cell growth which is a common trait seen in cancer. One hallmark of the cell cycle is the periodic expression of cell-cycle genes. Timing of cell cycle-dependent gene expression is regulated by multiple transcription factors. Recently, MuvB complexes had been identified to regulate the expression of several hundred cell-cycle dependent gene expression. In non-proliferating cells, the MuvB core complex represses transcription by binding to E2F4-p130. Upon entering the cell cycle, MuvB dissociates from E2F4-p130 and binds to B-Myb in S phase to activate mitotic genes. B-Myb is expressed in all proliferating cells and loss of function leads to reduced mitotic gene expression and to early embryonic lethality in mice. Overexpression of B-Myb is implicated in breast, lung and colon cancer. ChIP data of B-Myb show that many mitotic genes are direct targets of the B-Myb-MuvB (MMB) complex, however, canonical Myb binding site (MBS) are not commonly found in cell-cycle promoters. Even if B-Myb was originally described as a sequence- specific transcription factor interacting with MBS, several lines of evidence imply that B-Myb is recruited to mitotic genes through MuvB binding to CHR promoter elements. In this case, B-Myb may rather contact the DNA in a non-sequence-specific manner. By fluorescence polarization and electromobility shift assays I have determined that B-Myb binds to reconstituted Widom nucleosomes through its N-terminal DNA binding domain (DBD). Thus, my working hypothesis is that B-Myb binds to nucleosomes through its DNA binding domain to stabilize MuvB at cell-cycle gene promoters to recruit the co-activator p300/CBP and this activity is regulated through multi-site phosphorylation. In aim 1, I will analyze the association modes of B-Myb- nucleosome complex by solving the high-resolution cryo-EM structure and how it regulates MMB occupancy at cell-cycle promoters. In aim 2, I will determine how phosphorylation regulates its auto-inhibitory state to bind with co-activator p300/CBP. Completion of these aims will enhance our knowledge on how B-Myb can activate cell-cycle dependent genes.