Motoki Project

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，并诱导其开放，从而驱动细胞凋亡。 重新编程先驱因子的作用机制在很大程度上仍不清楚。使用一个模型， 在人类乳腺癌细胞的间充质上皮转化（MET）中，我们发现， GATA 3因子仅能在其结合位点的一部分打开染色质。我们的核心假设是GATA 3 需要适当的染色质环境来实现GATA 3介导的细胞增殖所必需的基因活化。 重新编程这项资助的总体目标是鉴定染色质结构和GATA 3辅助因子 GATA 3驱动的染色质开放和细胞重编程所需。中心假设将通过以下方式进行检验： 追求两个具体目标：（1）定义GATA 3驱动的MET所需的特定染色质结构;以及 (2)确定GATA 3辅助因子对上皮癌细胞重编程的作用。我们将在体外和体内 体内乳腺癌细胞系统，以解决这些具体的目标。在第一个目标下，多西环素诱导的 我们建立的MDA-MB-231细胞中的GATA 3表达系统将用于鉴定GATA 3在MDA-MB-231细胞中的作用。 核小体定位和接头组蛋白（H1.3和H1.5）在GATA 3介导的MET。基因组 包括ChIP-seq、ATAC-seq、RNA-seq和MNase-seq以及CRISPR-Cas9基因组的技术 将使用编辑和shRNA敲低方法。对于第二个目的，相同的诱导型GATA 3 表达系统和GATA 3突变T47 D细胞系（由我们小组建立）将用于鉴定GATA 3突变T47 D细胞系。 染色质重塑因子BRG 1和CHD 4在GATA 3介导的MET和GATA 3突变体中的作用 诱导上皮细胞向间充质细胞转化（EMT）。重要的是要了解GATA 3的作用， GATA 3突变细胞背景中的因素，因为GATA 3最近被确定为最常见的因素之一 乳腺癌的基因突变在这两个目标下，小鼠异种移植肿瘤模型将用于体内鉴定 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。拟议 研究在两个方面是创新的：第一，它以新颖的假设为特色，这些假设将推动以下领域的发展： 转录，染色质和癌症生物学，其次，有多种创新技术， 其中大部分是项目负责人开发的。这项拟议的研究意义重大，因为它是 有望为理解转录因子、染色质和蛋白质的功能提供新的框架。 重塑因子和癌细胞重编程中的染色质结构。最终，这些知识 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.