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.

到目前为止，已经使用不同的动物模型对TBX5的功能进行了深入的研究。这些数据在人体模型中的翻译仍然缺失。因此，在应用项目中，我们打算使用基于Holt-Oram综合征(HOS)的人iPS模型来研究TBX5在人类心脏发育中的作用。在我们之前的工作中，我们能够在一名具有严重表型的HOS患者中发现一种新的TBX5 Pro85Thr de nevo功能丧失突变。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，使用无整合重编程的HIPS系(野生型与突变型)，我们可以在自发分化过程中定义第一个体外表型。在应用项目中，我们将使用基于iPS系的下一代测序(RNA-Seq)来揭示心脏分化过程中转录组的差异，iPS系分别携带野生型TBX5基因(TBX5 IPSWT)或TBX5 Pro85Thr突变(TBX-iPSMUT)。HIPS细胞系的分化将使用定向心脏分化方案(心肌细胞纯度为80%-90%)进行。这一策略保证了对低丰度表达基因的检测。此外，还将产生携带特定FLAG序列的转基因株系(TBX5-iPSWT/FLAG和TBX5-iPSMUT/FLAG)，以确保通过特定FLAG抗体检测到TBX5蛋白。利用转基因株系检测TBX5是必不可少的，因为到目前为止，还没有能直接检测到TBX5的高特异性抗体。CRISPR/Cas技术将用于产生携带外显子9和TBX5基因终止密码子之间的标志序列的转基因品系。为了突出TBX5WT和TBX5MUT的结合基序和结合动力学中的差异，将应用使用转基因系的CHIP-SEQ技术。结合分析特定的转录组数据(定向分化的RNA-Seq)和高度特异的ChIP-Seq数据(标志下拉)，可以可靠地分析表达谱、选定的结合基序和结合动力学，以及识别可能的脱靶效应，特别是在HOS表型背景下。结果将进一步验证使用的髋关节系产生的HOS患者没有TBX5突变。基于患者特定iPS模型研究TBX5在HOS中的作用的拟议项目是独一无二的。这项研究将结合CHIP-SEQ的表达分析和高度特异的蛋白质检测，对TBX5蛋白在人类心脏发育过程中的作用获得详细的见解。此外，该应用项目可能会对进一步的研究产生开创性的影响，在患者特有的人类iPS模型中研究低丰度表达的基因(如转录因子)在人类发育过程中的功能。