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

迄今为止，TBX 5的功能已经使用不同的动物模型进行了深入研究。这些数据在人类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）的人类iPS模型来研究TBX 5在人类心脏发育中的作用。在我们之前的工作中，我们能够在具有严重表型的HOS患者中鉴定出一种新的TBX 5 Pro 85 Thr从头功能丧失突变。我们可以显示突变的TBX 5蛋白的核定位，并将功能丧失的机制与TBX 5蛋白的构象变化联系起来。此外，使用无整合重编程hiPS系（野生型与突变体），我们可以在自发分化期间定义第一体外表型。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TBX 5基因（TBX 5 iPSWT）或TBX 5 Pro 85 Thr突变（TBX-iPSMUT）。hiPS系的分化将使用定向心脏分化的方案（心肌细胞的纯度为80-90%）进行。这种策略保证了低丰度表达基因的检测。此外，将产生携带特异性FLAG序列的转基因系（TBX 5-iPSWT/flag和TBX 5-iPSMUT/flag），以确保通过特异性FLAG抗体检测TBX 5蛋白。使用转基因品系进行TBX 5检测是必不可少的，因为到目前为止，还没有高度特异性的抗体来直接检测TBX 5。CRISPR/Cas技术将用于产生在TBX 5基因的外显子9和终止密码子之间携带FLAG序列的转基因株系。为了突出TBX 5 WT和TBX 5 MUT的结合基序和结合动力学内的差异，将应用使用转基因系的ChiP-Seq技术。特异性转录组数据（定向分化的RNA-Seq）和高度特异性ChIP-Seq数据（FLAG下拉）的组合分析允许对表达谱、选择的结合基序和结合动力学进行可靠分析，以及鉴定可能的脱靶效应，特别是在HOS表型背景中。将使用从无TBX 5突变的HOS患者产生的hiPS系进一步验证结果。基于HOS患者特异性iPS模型研究TBX 5作用的拟议项目是独一无二的。该研究将结合表达分析和ChIP-Seq的高度特异性蛋白检测，详细了解TBX 5蛋白在人类心脏发育过程中的作用。此外，该应用项目可能对进一步的研究产生开创性影响，在患者特异性人类iPS模型中研究人类发育过程中必不可少的低丰度表达基因（例如转录因子）的功能。

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.