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

通过激活 B-Myb 进行细胞周期依赖性基因转录

• 批准号: 10313815
• 负责人: Tilini Wijeratne
• 金额: $ 3.84万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313815
• 关键词: Affinity; B-MYB Protein; Binding; Binding Sites; Biochemical; Biological Assay; Biological Markers; CREBBP gene; CRISPR/Cas technology; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell Line; Cell Proliferation; Cell physiology; Cells; Chromatin; Colon Carcinoma; Complement Factor B; Complex; Consensus; Cryoelectron Microscopy; Cyclin-Dependent Kinases; DNA; DNA Binding; DNA Binding Domain; DNA Structure; Data; EP300 gene; Elements; Embryo; Engineering; Event; Fluorescence Polarization; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Growth; Hallmark Cell; Image; Individual; Knowledge; Laboratories; Learning; Length; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Mediating; Mitosis; Mitotic; Modeling; Molecular; Mus; Mutation; N-terminal; Nucleosome Binding Domain; Nucleosomes; Oncogenic; PLK1 gene; Periodicity; Phosphorylation; Phosphorylation Site; Positioning Attribute; Proliferating; Proteins; Protocols documentation; Regulation; Resolution; Role; S Phase; Sampling; Scientist; Site; Structure; Techniques; Tertiary Protein Structure; Testing; Training; Transcriptional Activation; Transcriptional Regulation; base; biophysical properties; cancer cell; cdc Genes; cell growth; improved; loss of function; malignant breast neoplasm; mutant; nanomolar; overexpression; programs; promoter; reconstitution; recruit; therapeutic target; tool; trait; transcription factor

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.

项目概要 细胞周期是一个精心控制的细胞过程，可维持生物体生长的完整性。 细胞周期失调会导致细胞死亡或细胞生长不规则，这是癌症中常见的特征。 细胞周期的标志之一是细胞周期基因的周期性表达。细胞周期依赖性的时间 基因表达受多种转录因子调控。最近，MuvB 复合物被发现 调节数百个细胞周期依赖性基因的表达。在非增殖细胞中， MuvB 核心复合物通过与 E2F4-p130 结合来抑制转录。进入细胞周期后，MuvB 从 E2F4-p130 解离并在 S 期与 B-Myb 结合以激活有丝分裂基因。 B-Myb 表达为 所有增殖细胞和功能丧失都会导致有丝分裂基因表达减少和早期胚胎发育 对小鼠的致死率。 B-Myb 的过度表达与乳腺癌、肺癌和结肠癌有关。 B-Myb 的 ChIP 数据 表明许多有丝分裂基因是 B-Myb-MuvB (MMB) 复合物的直接目标，然而，典型的 Myb 结合位点（MBS）在细胞周期启动子中并不常见。即使 B-Myb 最初被描述为 与 MBS 相互作用的序列特异性转录因子，多项证据表明 B-Myb 是 通过与 CHR 启动子元件结合的 MuvB 募集到有丝分裂基因。在这种情况下，B-Myb 可能宁愿 以非序列特异性方式接触 DNA。通过荧光偏振和电迁移率位移 我已确定 B-Myb 通过其 N 末端 DNA 与重建的 Widom 核小体结合 结合域（DBD）。因此，我的工作假设是 B-Myb 通过其 DNA 与核小体结合 结合域稳定细胞周期基因启动子处的 MuvB，以招募共激活子 p300/CBP， 活性通过多位点磷酸化进行调节。在目标1中，我将分析B-Myb-的关联模式 通过解决高分辨率冷冻电镜结构及其如何调节 MMB 占据来研究核小体复合物 细胞周期启动子。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 与共激活剂 p300/CBP。完成这些目标将增强我们对 B-Myb 如何激活的了解 细胞周期依赖性基因。