Presentation Information
[II-TKIS-P-5]Design of the epitope-tagged PITX2C for chromatin profiling based on structural prediction with AlphaFold3
○Kana Kitazaki1, Masaki Shigeta1, Shinichiro Sakaki1,2, Sou Miyanishi1, Rie Saba1, Kenta Yashiro1 (1.Anatomy & Developmental Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2.Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan)
Keywords:
Transcription factor,Epitope-tagged proteins,Protein structure prediction
The cardiac outflow tract (OFT) undergoes complex rotational morphogenesis during the heart development, and disruption of this process leads to severe congenital heart defects such as transposition of the great arteries (TGA). Dysregulation of the transcription factor PITX2C has been shown to lead to TGA in mice; however, the underlying molecular mechanisms remain unclear.
To elucidate the regulatory role of PITX2C in OFT development, we aimed to identify its direct genomic targets. Chromatin profiling approaches such as CUT&RUN are suitable for mapping transcription factor binding sites. However, studies of PITX2C-dependent gene regulation have been limited by the lack of ChIP-grade antibodies, thus far.
To overcome this limitation, we generated epitope-tagged PITX2C constructs for tag-based chromatin profiling. Since epitope insertion may disrupt protein structure and function, we first screened multiple tagged constructs by AlphaFold3-based structural predictions.
Based on these predictions, we selected three constructs showing minimal predicted conformational changes, including N- or C-terminal FLAG tags and an internal PA tag inserted into a non-structured region, for experimental analysis. Among them, immunofluorescence analysis indicated that only the internally PA-tagged PITX2C exhibited stable expression and clear nuclear localization in mouse embryonic stem cells.
These results highlight the importance of structure-guided epitope tag design for chromatin profiling and provide a rational framework for optimizing tagged transcription factor constructs.
To elucidate the regulatory role of PITX2C in OFT development, we aimed to identify its direct genomic targets. Chromatin profiling approaches such as CUT&RUN are suitable for mapping transcription factor binding sites. However, studies of PITX2C-dependent gene regulation have been limited by the lack of ChIP-grade antibodies, thus far.
To overcome this limitation, we generated epitope-tagged PITX2C constructs for tag-based chromatin profiling. Since epitope insertion may disrupt protein structure and function, we first screened multiple tagged constructs by AlphaFold3-based structural predictions.
Based on these predictions, we selected three constructs showing minimal predicted conformational changes, including N- or C-terminal FLAG tags and an internal PA tag inserted into a non-structured region, for experimental analysis. Among them, immunofluorescence analysis indicated that only the internally PA-tagged PITX2C exhibited stable expression and clear nuclear localization in mouse embryonic stem cells.
These results highlight the importance of structure-guided epitope tag design for chromatin profiling and provide a rational framework for optimizing tagged transcription factor constructs.
