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

• Applicants: Professorin Dr. Lesca Miriam Holdt; Professor Dr. Markus Krane
• Subject Area: Cardiac and Vascular Surgery ; Developmental Biology
• Term: from 2017 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 394237736

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.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Martina Dreßen