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<p>Transcription factors (TFs) are key players in eukaryotic gene regulation, but the DNA binding specificity of many TFs remains unknown. Here, we assay 284 mostly uncharacterized putative human TFs using selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq), revealing 74 new DNA binding motifs. To investigate whether TFs lacking detectable motifs preferably bind epigenetically modified DNA, we develop methylation-sensitive SMiLE-seq (meSMiLE-seq), a microfluidic assay that simultaneously probes binding to methylated and unmethylated DNA. Using meSMiLE-seq, we assay 114 TFs and identify DNA-binding models for 48 proteins, including known methylation-sensitive binding modes for POU5F1 and RFX5. 11 TFs prefer methylated DNA or display alternative methylation-dependent motifs (e.g. PRDM13), while 13 show aversion to methylated sequences (e.g. USF3). Finally, we identify ZHX2 as a putative Z-DNA binder. Altogether, our study significantly expands the human TF codebook, while providing a versatile platform to quantitatively assay the impact of DNA modifications on TF binding.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年8月5日

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  • Antoni J. Gralak,
  • Katerina Faltejskova,
  • Ally W. H. Yang,
  • Clemence Steiner,
  • Julie Russeil,
  • Nadia Grenningloh,
  • Sachi Inukai,
  • Mustafa Demir,
  • Riccardo Dainese,
  • Cooper Owen,
  • Eugenia V. Pankevich,
  • Philipp Bucher,
  • Oriol Fornes,
  • Jan Grau,
  • Ivo Grosse,
  • Arttu Jolma,
  • Fedor A. Kolpakov,
  • Vsevolod J. Makeev,
  • Mihai Albu,
  • Marjan Barazandeh,
  • Alexander Brechalov,
  • Zhenfeng Deng,
  • Ali Fathi,
  • Chun Hu,
  • Samuel A. Lambert,
  • Kaitlin U. Laverty,
  • Zain M. Patel,
  • Sara E. Pour,
  • Rozita Razavi,
  • Mikhail Salnikov,
  • Isaac Yellan,
  • Hong Zheng,
  • Georgy Meshcheryakov,
  • Giovanna Ambrosini,
  • Marie-Luise Plescher,
  • Semyon Kolmykov,
  • Ivan Yevshin,
  • Nikita Gryzunov,
  • Ivan Kozin,
  • Mikhail Nikonov,
  • Vladimir Nozdrin,
  • Arsenii Zinkevich,
  • Pavel Kravchenko,
  • Sergey Abramov,
  • Alexandr Boytsov,
  • Vasilii Kamenets,
  • Dmitry Penzar,
  • Anton Vlasov,
  • Ilya E. Vorontsov,
  • Aldo Hernandez-Corchado,
  • Hamed S. Najafabadi,
  • Quaid Morris,
  • Xiaoting Chen,
  • Matthew T. Weirauch,
  • Timothy R. Hughes,
  • Ivan V. Kulakovskiy,
  • Judith F. Kribelbauer-Swietek,
  • Guido van Mierlo,
  • Bart Deplancke

摘要

Transcription factors (TFs) are key players in eukaryotic gene regulation, but the DNA binding specificity of many TFs remains unknown. Here, we assay 284 mostly uncharacterized putative human TFs using selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq), revealing 74 new DNA binding motifs. To investigate whether TFs lacking detectable motifs preferably bind epigenetically modified DNA, we develop methylation-sensitive SMiLE-seq (meSMiLE-seq), a microfluidic assay that simultaneously probes binding to methylated and unmethylated DNA. Using meSMiLE-seq, we assay 114 TFs and identify DNA-binding models for 48 proteins, including known methylation-sensitive binding modes for POU5F1 and RFX5. 11 TFs prefer methylated DNA or display alternative methylation-dependent motifs (e.g. PRDM13), while 13 show aversion to methylated sequences (e.g. USF3). Finally, we identify ZHX2 as a putative Z-DNA binder. Altogether, our study significantly expands the human TF codebook, while providing a versatile platform to quantitatively assay the impact of DNA modifications on TF binding.