Identification of methylation-sensitive human transcription factors using meSMiLE-seq

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Transcription factors (TFs) are crucial for gene regulation, but their DNA binding specificities are often unknown.
• Understanding TF binding is essential for deciphering complex gene regulatory networks.

Purpose of the Study:

• To identify novel DNA binding motifs for poorly characterized human transcription factors.
• To investigate the impact of DNA methylation on transcription factor binding preferences.
• To develop and apply microfluidics-based assays for quantitative analysis of TF-DNA interactions.

Main Methods:

• Selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq) was used to assay DNA binding motifs for 284 human TFs.
• Methylation-sensitive SMiLE-seq (meSMiLE-seq) was developed to simultaneously probe TF affinity to methylated and unmethylated DNA.
• 114 TFs were assayed using meSMiLE-seq to identify methylation-aware binding sites.

Main Results:

• 72 new DNA binding motifs were identified for human TFs using SMiLE-seq.
• meSMiLE-seq identified DNA-binding models for 48 TFs, including 11 showing preference for methylated DNA and 13 showing aversion.
• A potential role for ZHX2 in binding Z-DNA, which is associated with CpG methylation, was uncovered.

Conclusions:

• This study significantly expands the human TF codebook by identifying DNA binding motifs for 98 TFs.
• The developed meSMiLE-seq platform provides a versatile tool to quantitatively assess how DNA modifications influence TF binding.
• Findings offer new insights into epigenetic regulation and TF-mediated gene control.