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.

为重新评估FoxP3的序列结合特异性，我们利用重组FoxP3蛋白对基因组DNA开展无偏向性下拉实验（pull-down）。相较于合成DNA寡核苷酸链，采用基因组DNA可在天然序列库的背景下完成序列结合特异性检测。分别以仅含麦芽糖结合蛋白（MBP）的下拉实验和Input样品作为对照时，FoxP3下拉实验的从头基序分析结果显示，TnG重复序列（n=2-5）存在显著富集。

实验整体设计：我们从小鼠EL4细胞中提取基因组DNA，将其片段化至约100~200 bp的长度，随后与纯化的带有麦芽糖结合蛋白（MBP）标签的小鼠FoxP3蛋白共孵育。后续我们开展MBP下拉实验，并对共纯化获得的DNA进行下一代测序（next-generation sequencing, NGS），该实验流程被命名为FoxP3 PD-seq。我们借助该数据集的富集峰开展基序分析，阐明了FoxP3在裸露基因组DNA上的DNA结合模式。