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.

项目摘要 细胞周期是经过精心控制的细胞过程，可维持生物生长的完整性。 细胞周期的失调导致细胞死亡或不规则细胞生长，这是癌症中的常见特征。 细胞周期的一个标志是细胞周期基因的周期性表达。细胞周期依赖性的时间 基因表达受多种转录因子调节。最近，已经确定了MUVB复合物 调节数百个细胞周期依赖基因表达的表达。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分离，并在S期与B-MYB结合以激活有丝分裂基因。 B-myb表示 所有增殖细胞和功能丧失都会导致有丝分裂基因表达和早期胚胎 小鼠的致死性。 B-MYB的过表达与乳腺癌，肺癌和结肠癌有关。 B-MYB的芯片数据 表明许多有丝分裂基因是B-Myb-MUVB（MMB）复合物的直接靶标 结合位点（MB）在细胞周期启动子中通常没有发现。即使B-Myb最初被描述为 序列特异性转录因子与MBS相互作用，几行证据表明B-Myb是 通过MUVB与CHR启动子元素结合，招募到有丝分裂基因。在这种情况下，b-myb可能宁愿 以非序列特异性方式与DNA联系。通过荧光偏振和电动性偏移 测定我已经确定B-Myb通过其N末端DNA结合了重构的宽核小体 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 结合结构域以稳定在细胞周期基因启动子处MUVB以募集共激活器p300/cbp，这 活性通过多位磷酸化来调节。在AIM 1中，我将分析B-Myb-的关联模式 核小体复合物通过求解高分辨率的冷冻EM结构以及如何调节MMB占用率 细胞周期启动子。在AIM 2中，我将确定磷酸化如何调节其自动抑制状态以结合 与共激活器P300/CBP。这些目标的完成将增强我们对B-MYB如何激活的了解 细胞周期依赖基因。