Motoki Project

本木项目

• 批准号: 10204032
• 负责人: Motoki Takaku
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-10 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204032
• 关键词: ATAC-seq; Address; Affect; Binding; Binding Sites; Biochemical; Breast Cancer Cell; Breast Cancer Treatment; CHD4 gene; CRISPR/Cas technology; Cancer Biology; Carcinoma; Cause of Death; Cell Line; Cells; Centers of Research Excellence; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; DNA; Data; Development; Disease; Doxycycline; Epigenetic Process; Epithelial; Epithelial Cells; GATA3 gene; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Goals; Grant; Histone H1; Histones; Homeostasis; Human; In Vitro; Knowledge; MDA MB 231; Malignant Neoplasms; Mammary Neoplasms; Measures; Mediating; Methods; Modeling; Mus; Mutate; Mutation; Neoplasm Metastasis; Nucleosomes; Nude Mice; Outcome; Phenotype; Play; Positioning Attribute; Process; Public Health; Research; Role; SMARCA4 gene; System; T47D; Techniques; Testing; Tissues; Tumor Suppressor Proteins; Woman; Work; Xenograft Model; activating transcription factor; aggressive breast cancer; anticancer research; base; cancer cell; epigenomics; epithelial to mesenchymal transition; genome editing; genomic locus; human disease; in vivo; innovation; innovative technologies; insight; knock-down; malignant breast neoplasm; mutant; next generation sequencing; novel; novel therapeutic intervention; small hairpin RNA; transcription factor; transcriptome sequencing; tumor growth; tumor xenograft

PROJECT SUMMARY. PROJECT 5, M.TAKAKU. Pioneer transcription factors are capable of binding to closed chromatin and inducing its opening to drive cell reprogramming. The mechanisms of pioneer factors’ actions remain largely unknown. Using a model of mesenchymal-to-epithelial transition (MET) in human breast cancer cells, we have discovered that a pioneer factor GATA3 can open chromatin only at a subset of its binding sites. Our central hypothesis is that GATA3 requires appropriate chromatin context to achieve gene activation that are essential for GATA3 mediated cell reprogramming. The overall objectives in this grant are to identify chromatin structures and GATA3 co-factors needed for GATA3-driven chromatin opening and cell reprogramming. The central hypothesis will be tested by pursuing two specific aims: (1) Define the specific chromatin structures required for GATA3 driven MET; and (2) Identify the roles of GATA3 co-factors for epithelial cancer cell reprogramming. We will use in vitro and in vivo breast cancer cell systems to address these specific aims. Under the first aim, the doxycycline inducible GATA3 expression system in MDA-MB-231 cells that we have established will be used to identify the roles of nucleosome positioning and linker histones (H1.3 and H1.5) during GATA3-mediated MET. Genomic techniques including ChIP-seq, ATAC-seq, RNA-seq and MNase-seq together with CRISPR-Cas9 genome editing and shRNA knockdown methods will be utilized. For the second aim, the same inducible GATA3 expression system and a GATA3 mutant T47D cell line (established by our group) will be used to identify the roles of chromatin remodeling factors BRG1 and CHD4 in the GATA3-mediated MET and the GATA3 mutant induced epithelial-to-mesenchymal transition (EMT). It is important to understand the roles of GATA3 co- factors in the GATA3 mutant cell context, because GATA3 was recently identified as one of the most frequently mutated genes in breast cancer. Under both aims, mouse xenograft tumor model will be used to identify in vivo roles of chromatin structures and chromatin remodeling factors in tumor growth and metastasis. The proposed research is innovative in two ways: first, it features novel hypotheses that will advance the fields of transcription, chromatin and cancer biology, and secondly, there are multiple innovative technologies that are proposed, most of which the project leader has developed. The proposed research is significant because it is expected to provide a new framework for understanding the functions of transcription factors, chromatin remodeling factors, and chromatin structures in cancer cell reprogramming. Ultimately, such knowledge has the potential to offer development of new therapeutic strategies of breast cancer treatment.

项目摘要。项目5，M.Takaku。 先锋转录因子能够与封闭的染色质结合并诱导其开口驱动细胞 重新编程。先锋因素的行为的机制在很大程度上尚不清楚。使用模型 人乳腺癌细胞中的间充质到上皮过渡（MET），我们发现了一个先锋 因子GATA3只能在其结合位点的一个子集中打开染色质。我们的中心假设是GATA3 需要适当的染色质上下文来实现GATA3介导的细胞必不可少的基因激活 重新编程。该赠款的总体目标是识别染色质结构和GATA3副因素 需要GATA3驱动的染色质开口和细胞重编程。中心假设将通过 追求两个具体目标：（1）定义GATA3驱动的Met所需的特定染色质结构；和 （2）确定GATA3辅助因子在上皮癌细胞重编程中的作用。我们将在体外和 体内乳腺癌细胞系统以解决这些特定目标。在第一个目标下，强力霉素可诱导 我们已经确定的MDA-MB-231单元中的GATA3表达系统将用于确定 GATA3介导的MET期间的核小体定位和连接器Hisons（H1.3和H1.5）。基因组 包括ChIP-Seq，Atac-Seq，RNA-Seq和MNase-Seq在内的技术以及CRISPR-CAS9基因组 将使用编辑和shRNA敲低方法。对于第二个目标，相同的诱导GATA3 表达系统和GATA3突变体T47D细胞系（由我们的组建立）将用于识别 染色质重塑因子BRG1和CHD4在GATA3介导的MET和GATA3突变体中的作用 诱导上皮到间质转变（EMT）。了解GATA3共同的作用很重要 GATA3突变细胞环境中的因素，因为GATA3最近被确定为最频繁的因素之一 乳腺癌中突变的基因。在这两个目标下，小鼠Xenographic肿瘤模型将用于识别体内 染色质结构和染色质重塑因子在肿瘤生长和转移中的作用。提议 研究在两种方面具有创新性：首先，它以新颖的假设为特征 转录，染色质和癌症生物学，其次，有多种创新技术 拟议的，大多数项目负责人已经发展了。拟议的研究很重要，因为它是 预计将提供一个新的框架来理解转录因子的功能，染色质 癌细胞重编程中的重塑因子和染色质结构。最终，这些知识有 提供开发乳腺癌治疗的新治疗策略的潜力。