MBP-FoxP3 pulldown-seq on naked DNA
To re-evaluate FoxP3 sequence specificity, we performed an unbiased pull-down of genomic DNA with recombinant FoxP3 protein. Use of genomic DNA, as opposed to synthetic DNA oligos, enables testing sequence specificity in the context of naturally existing repertoire of sequences. De novo motif analysis showed a strong enrichment of TnG repeats (n=2-5) by FoxP3 pull-down, using either MBP alone pull-down or input as a control. Overall design: We isolated genomic DNA from mouse EL4 cells, fragmented it to approximately 100-200 bp in size, and then incubated it with purified MBP-tagged mouse FoxP3. Subsequently, we conducted an MBP pull-down, followed by next-generation sequencing (NGS) of the co-purified DNA, a process referred to as FoxP3 PD-seq. We utilized the enriched peaks from this dataset to perform motif analysis, shedding light on the DNA binding patterns of FoxP3 on naked genomic DNA.