Motoki Project

项目总结。项目5，M.TAKAKU。 先锋转录因子能够与封闭的染色质结合，并诱导其开放以驱动细胞 重新编程。先锋因素的作用机制在很大程度上仍不清楚。使用一个模型 在人类乳腺癌细胞间充质向上皮转化(MET)过程中，我们发现了一种先驱 因子GATA3只能在其结合位点子集中打开染色质。我们的中心假设是GATA3 需要适当的染色质环境来实现GATA3介导的细胞所必需的基因激活 重新编程。这项资助的总体目标是确定染色质结构和GATA3辅助因子 GATA3驱动的染色质开放和细胞重新编程所需的。核心假设将通过以下方式进行检验 追求两个具体目标：(1)确定GATA3驱动的MET所需的特定染色质结构；以及 (2)明确GATA3辅助因子在上皮性癌细胞重编程中的作用。我们将在体外和体内使用 体内乳腺癌细胞系统来解决这些特定的目标。在第一个目标下，多西环素可诱导 我们建立的GATA3在MDA-MB-231细胞中的表达系统将用于鉴定GATA3的作用 GATA3介导的MET过程中的核小体定位和连接子组蛋白(H1.3和H1.5)。基因组学 CHIP-SEQ、ATAC-SEQ、RNA-SEQ和MNase-SEQ以及CRISPR-Cas9基因组技术 将使用编辑和shRNA敲除方法。对于第二个目的，同样的可诱导的GATA3 表达系统和GATA3突变体T47D细胞系(本课题组建立)将用于鉴定 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 诱导上皮向间充质转化(EMT)。理解GATA3共同的作用非常重要。 在GATA3突变细胞环境中的因素，因为GATA3最近被发现是最常见的 乳腺癌中的突变基因。在这两个目标下，将使用小鼠异种移植瘤模型在体内识别 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。建议数 研究在两个方面具有创新性：首先，它的特点是提出了新的假设，这将推动 转录、染色质和癌症生物学，其次，有多种创新技术， 提议，其中大部分是由项目负责人制定的。这项拟议的研究具有重要意义，因为它 有望为理解转录因子的功能提供一个新的框架，染色质 癌细胞重编程中的重塑因子和染色质结构。归根结底，这些知识已经 为乳腺癌治疗提供新的治疗策略的潜力。

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.