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

项目总结 细胞周期是一个精心控制的细胞过程，它维持着生物生长的完整性。 放松对细胞周期的调控会导致细胞死亡或细胞生长不规律，这是癌症的常见特征。 细胞周期的一个标志是细胞周期基因的周期性表达。细胞周期依赖的时序 基因表达受多种转录因子调控。最近，MuvB复合体被发现 调控数百个细胞周期相关基因的表达。在非增殖细胞中， MuvB核心复合体通过与E2F4-p130结合来抑制转录。进入细胞周期后，MuvB 解离E2F4-p130，在S期结合B-Myb激活有丝分裂基因。B-Myb在 所有增殖细胞和功能丧失导致有丝分裂基因表达减少和早期胚胎 对小鼠的致命性。B-Myb的过度表达与乳腺癌、肺癌和结肠癌有关。B-Myb的芯片数据 许多有丝分裂基因是B-Myb-MuvB(MMB)复合体的直接靶点，而典型的Myb 结合位点(MB)在细胞周期启动子中并不常见。即使B-Myb最初被描述为 一种与MBS相互作用的序列特异性转录因子，多条证据表明B-Myb是 通过MuvB与CHR启动子元件结合而被招募到有丝分裂基因。在这种情况下，B-Myb可能更愿意 以非特定序列的方式联系DNA。通过荧光偏振和电迁移率漂移 分析I已确定B-Myb通过其N-末端DNA与重组的Widom核小体结合 结合结构域(DBD)。因此，我的工作假设是B-Myb通过其DNA与核小体结合 结合结构域稳定MuvB在细胞周期基因启动子招募共激活p300/CBP和这 活性是通过多位点磷酸化来调节的。在目标1中，我将分析B-Myb-的关联模式。 核小体复合体通过解决高分辨率冷冻-EM结构以及它如何调节MMB在 细胞周期促进剂。在目标2中，我将确定磷酸化如何调节其自身抑制状态以结合 与共激活子p300/CBP结合。这些目标的完成将增强我们对B-Myb如何激活的了解 依赖细胞周期的基因。

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.