MethMotif 2025 Protocol Output Files and Markdown

Abstract:

This protocol presents a comprehensive guide for using the MethMotif platform, which includes the MethMotif database, TFregulomeR R package, and a new R library, Forked-TF, designed specifically for analyzing leucine zipper transcription factors (TFs) that bind DNA as dimers. The MethMotif platform integrates transcription factor binding site (TFBS) motifs with DNA methylation profiles, providing an in-depth analysis of how methylation modulates TF binding across different cell types and conditions. The protocol is organized into three main workflows: (1) Exploration of transcription factor dimerization partners, (2) Visualization of methylation-specific TF motifs using TFregulomeR, and (3) Characterization of leucine zipper TF binding patterns with a focus on dimerization. Using the platform's MethMotif database, users can retrieve ChIP-seq and DNA methylation data, intersect TFBS peak regions, and generate TFBS methylation-informed motif logos. A case study of CEBPB in K562 cells is included to demonstrate the use of the platform, showing how to identify TF dimers, analyze their co-binding behaviour, and visualize the impact of DNA methylation on binding specificity. The protocol also provides step-by-step instructions for software installation, data input formats, and interpretation of results, making it accessible to researchers with varying levels of computational expertise. Through this protocol, users can uncover how DNA methylation and TF dimerization influence gene regulatory networks, with a focus on leucine zipper TFs in a cell-type-specific context.

Data Structure:

This repository contains essential resources from the MethMotif suite, including:

Protocol Output Files (protocol_output.zip): A compressed archive containing the results generated from the MethMotif suite, including processed data, intermediate outputs, and final reports.

Markdown Script (methmotif_protocol_markdown.md): A structured markdown document outlining the MethMotif suite protocol, including methodology, key parameters, and interpretation of results.

R Script (methmotif_protocol_rscript.R): A script written in R used to execute parts of the MethMotif analysis, including data preprocessing, motif discovery, and visualization.

These files facilitate reproducibility and provide a comprehensive guide for running and analyzing data with the MethMotif suite.