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 细胞周期是一个精心控制的细胞过程，维持生物体生长的完整性。 细胞周期的失调导致细胞死亡或不规则的细胞生长，这是癌症中常见的特征。 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 基因表达受多种转录因子调控。最近，MuvB复合物已被鉴定， 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E2 F4-p130结合来抑制转录。在进入细胞周期后，MuvB 与E2 F4-p130解离，并在S期与B-Myb结合以激活有丝分裂基因。B-Myb表达于 所有增殖细胞和功能丧失导致有丝分裂基因表达减少， 小鼠的致死率。B-Myb的过表达与乳腺癌、肺癌和结肠癌有关。B-Myb的ChIP数据 显示许多有丝分裂基因是B-Myb-MuvB（MMB）复合物的直接靶基因，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 作为一种与MBS相互作用的序列特异性转录因子，几条证据表明B-Myb是 通过MuvB结合到启动子元件而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偏移 我已经确定B-Myb通过其N-末端DNA与重组的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 结合结构域，以在细胞周期基因启动子处稳定MuvB，从而募集共激活因子p300/CBP，并且 活性通过多位点磷酸化调节。在目的1中，我将分析B-Myb- 通过解决高分辨率冷冻EM结构以及它如何调节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， 与共激活剂p300/CBP。这些目标的完成将增加我们对B-Myb如何激活 细胞周期依赖基因