Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• DNA sequence dictates protein structure and expression timing via transcription factors (TFs).
• The DNA-binding specificity of most human TFs remains largely unknown.
• Current methods for TF binding analysis are limited in throughput and scope.

Purpose of the Study:

• To develop a high-throughput method for analyzing transcription factor DNA-binding specificity.
• To characterize binding specificities for a large number of human TFs in parallel.
• To identify novel DNA-binding modes and improve understanding of gene regulation.

Main Methods:

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) coupled with massively parallel sequencing.
• Utilized affinity-tagged proteins, barcoded oligonucleotides, and multiplexed sequencing for high throughput.
• Developed a bioinformatic platform for data analysis, quality control, and motif generation.

Main Results:

• The method enables high-throughput, parallel analysis of TF DNA-binding specificities, yielding longer binding profiles than existing techniques.
• Validated the method by characterizing 14 TF classes and confirming specificities for NFATC1 and RFX3 via ChIP-seq.
• Discovered unexpected dimeric binding modes for several TFs previously thought to bind as monomers.

Conclusions:

• The developed SELEX-sequencing technology significantly enhances the throughput and accuracy of TF binding specificity determination.
• This platform allows for the analysis of full-length proteins and those requiring post-translational modifications.
• The findings reveal new insights into TF binding mechanisms, including novel dimeric interactions, crucial for understanding gene regulation.