Investigation of TBX5-depending regulatory processes during human cardiac embryonic development using a patient-specific Holt-Oram syndrome iPS model

使用患者特异性 Holt-Oram 综合征 iPS 模型研究人类心脏胚胎发育过程中的 TBX5 依赖性调节过程

• 批准号: 394237736
• 负责人: Professorin Dr. Lesca Miriam Holdt
• 金额: --
• 依托单位: Institut für Laboratoriumsmedizin (Großhadern)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/394237736?language=en
• 关键词: Investigation; TBX5; depending; regulatory; processes

To date TBX5 function has been intensively investigated using different animal models. A translation of these data in a human model is still missing. Therefore, in the applied project we intend to study the role of TBX5 during human cardiac development using a human iPS model based on the Holt-Oram syndrome (HOS). In our previous work, we were able to identify a novel TBX5 Pro85Thr de novo loss-of-function mutation in a HOS patient with a severe phenotype. We could show a nuclear localisation of the mutated TBX5 protein and linked the mechanism for loss-of-function to a conformational change of the TBX5 protein. Additionally, using integration free reprogrammed hiPS lines (wildtype vs. mutant) we could define a first in vitro phenotype during spontaneous differentiation. In the applied project we will unravel differences in the transcriptome during cardiac differentiation using next generation sequencing (RNA-Seq) based on the iPS lines, carrying either a wildtype TBX5 gene (TBX5 iPSWT) or a TBX5 Pro85Thr mutation (TBX-iPSMUT), respectively. Differentiation of the hiPS lines will be carried out using a protocol for directed cardiac differentiation (purity of cardiomyocytes of 80-90%). This strategy guarantees the detection of genes with a low abundance expression. Moreover transgenic lines (TBX5-iPSWT/flag and TBX5-iPSMUT/flag) carrying a specific FLAG sequence will be generated to ensure the detection of the TBX5 protein via the specific FLAG antibody. The use of transgenic lines for TBX5 detection is indispensable, because up to now, no highly specific antibody to directly detect TBX5 is available. The CRISPR/Cas technology will be used to generate the transgenic lines carrying the FLAG sequence between exon 9 and the stop codon of the TBX5 gene. To highlight differences within the binding motifs and the binding dynamics of TBX5WT and TBX5MUT, ChiP-Seq technology using the transgenic lines will be applied. The combined analysis of specific transcriptome data (RNA-Seq of the directed differentiation) and highly specific ChIP-Seq data (FLAG pull-down) allows a reliable analysis of expression profile, selected binding motifs and binding dynamics as well as the identification of possible off-target effects especially in the HOS phenotype background. Results will further be validated using a hiPS line being generated from a HOS patient without TBX5 mutation. The proposed project to investigate the role of TBX5 based on a patient-specific iPS model for HOS is unique. The research will gain detailed insights in the role of the TBX5 protein during human cardiac development combining expression analysis and highly specific protein detection for ChIP-Seq. Furthermore, the applied project could have a pioneering impact on further studies, investigating the function of low abundance expressed genes (e.g. transcription factors) essential during human development in patient specific human iPS models.

迄今为止，使用不同的动物模型对TBX5功能进行了深入研究。在人类模型中的这些数据的翻译仍然缺少。因此，在应用项目中，我们打算使用基于Holt-Oram综合征（HOS）的人类IPS模型来研究TBX5在人类心脏发展过程中的作用。在我们以前的工作中，我们能够鉴定出一种新型的TBX5 Pro85ThR de从重点丧失功能突变中，患有严重表型的HOS患者。我们可以显示突变的TBX5蛋白的核定位，并将功能丧失的机制与TBX5蛋白的构象变化联系​​起来。此外，使用自发分化期间，我们可以使用自由重编程的臀部线（Wildtype vs.突变体）定义第一个体外表型。在应用项目中，我们将使用基于IPS线的下一代测序（RNA-SEQ）在心脏分化过程中的转录组差异，分别携带WildType TBX5基因（TBX5 IPSWT）或TBX5 Pro85THR突变（TBX-IPSMUT）。臀部线的分化将使用定向心脏分化方案（心肌细胞的纯度为80-90％）进行。该策略保证了以低丰度表达的基因检测。此外，将生成带有特定标志序列的转基因线（TBX5-IPSWT/FLAG和TBX5-IPSMUT/FLAG），以确保通过特定的标志抗体检测TBX5蛋白。转基因线在TBX5检测中的使用是必不可少的，因为到目前为止，尚无高度特异性抗体直接检测到TBX5。 CRISPR/CAS技术将用于生成带有外显子9和TBX5基因的终止密码子之间的载有标志序列的转基因线。为了强调结合基序内的差异以及TBX5WT和TBX5MUT的结合动力学，将应用使用转基因线的CHIP-SEQ技术。对特定转录组数据（定向分化的RNA-seq）和高度特定的芯片序列数据（标志下拉）的组合分析允许对表达曲线，选定的结合基序和结合动力学以及鉴定可能的非目标效应的可靠分析，尤其是在HOS表型中。结果将使用没有TBX5突变的HOS患者产生的臀部线进一步验证。提出的基于患者特异性IPS模型的HOS的TBX5的作用的拟议项目是独一无二的。这项研究将获得有关TBX5蛋白在人类心脏发育中的作用的详细见解，结合了表达分析和高度特异性的蛋白质检测。此外，应用项目可能会对进一步的研究产生开创性的影响，研究低丰度表达的基因（例如转录因子）在人类发育中必不可少的患者特定于人类IPS模型的功能。