Work

Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination

Year: 2025

Type: article

Abstract: Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis. For complete details on the use and execution of this protocol, please refer to Michael et al. 1 • Instructions for the preparation of an SeEN-seq DNA library by EcoRV digestion • Steps for purification using either the anion exchange column or Prepcell apparatus • Procedures for SeEN-seq data analysis • Guidance of TF-nucleosome complex preparation for cryo-EM analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104295

Authors: Wataru Kobayashi, Alicia K. Michael, Siwat Ruangroengkulrith, Maximilian Kümmecke, Kikuë Tachibana

Institutions: Max Planck Institute of Biochemistry, Institute of Science and Technology Austria

Language: English

Topic: Genomics and Chromatin Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,550

Funders: Max-Planck-Gesellschaft, European Research Council