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A High-Throughput Genome-Integrated Assay Reveals Spatial Dependencies Governing Tcf7l2 Binding
Tomasz Szczesnik1, Lendy Chu2, Joshua W K Ho3
1Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; St Vincent's Clinical School, University of New South Wales, Darlinghurst, NSW 2010, Australia; Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, Basel 4058, Switzerland.
Understanding transcription factor binding requires considering surrounding DNA sequences. This study reveals how Oct4 and Klf4 motifs influence Tcf7l2 binding in mouse stem cells, showing a helical phasing pattern.
Area of Science:
- Genomics
- Molecular Biology
- Developmental Biology
Background:
- Predicting transcription factor binding in vivo is challenging due to complex interactions.
- The Wnt pathway effector Tcf7l2's in vivo binding logic requires detailed characterization.
- In vitro DNA-binding affinity alone does not fully explain in vivo binding patterns.
Purpose of the Study:
- To develop a high-throughput screening platform for analyzing Tcf7l2 binding.
- To investigate the influence of local sequence context on Tcf7l2 in vivo binding.
- To identify specific cofactor motifs that modulate Tcf7l2 genomic occupancy.
Main Methods:
- Developed a high-throughput screening platform using synthesized DNA phrases in a specific genomic locus.
- Employed DamID (DNA adenine methyltransferase identification) to measure Tcf7l2 binding.
- Utilized controlled sequence perturbation at two genomic loci in mouse embryonic stem cells.
Main Results:
- Tcf7l2 binding correlates with in vitro motif-binding strength and local chromatin accessibility.
- Binding is significantly influenced by the surrounding 99 bp of DNA sequence.
- Oct4 and Klf4 motifs promote Tcf7l2 binding, exhibiting a specific helical phasing (10.8 bp).
Conclusions:
- Local sequence context and cofactor interactions are critical determinants of Tcf7l2 in vivo binding.
- The helical phasing of cofactor motifs relative to Tcf7l2 binding sites is a key regulatory mechanism.
- This study provides a novel platform for dissecting transcription factor binding specificity in complex genomic environments.
