Antoni J. Gralak, Kateřina Faltejsková, Ally Yang, Clemence Steiner, Julie Russeil, Nadia Grenningloh, Sachi Inukai, Mustafa Demir, Riccardo Dainese, C. C. Owen, Eugenia V. Pankevich, Philipp Bucher, Oriol Fornés, 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, G. A. 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 Hernández-Corchado, Hamed S. Najafabadi, Quaid Morris, Xiaoting Chen, Matthew T. Weirauch, Timothy R. Hughes, Ivan V. Kulakovskiy, Judith F. Kribelbauer, Guido van Mierlo, Bart Deplancke
Transcription factors (TFs) are key players in eukaryotic gene regulation, but the DNA binding specificity of many TFs remains unknown. Here, we assayed 284 mostly poorly characterized, putative human TFs using selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq), revealing 72 new DNA binding motifs. To investigate whether some of the 158 TFs for which we did not find motifs preferably bind epigenetically modified DNA (i.e. methylated CG dinucleotides), we developed methylation-sensitive SMiLE-seq (meSMiLE-seq). This microfluidic assay simultaneously probes the affinity of a protein to methylated and unmethylated DNA, augmenting the capabilities of the original method to infer methylation-aware binding sites. We assayed 114 TFs with meSMiLE-seq and identified DNA-binding models for 48 proteins, including the known methylation-sensitive binding modes for POU5F1 and RFX5. For 11 TFs, binding to methylated DNA was preferred or resulted in the discovery of alternative, methylation-dependent motifs (e.g. PRDM13), while aversion towards methylated sequences was found for 13 TFs (e.g. USF3). Finally, we uncovered a potential role for ZHX2 as a putative binder of Z-DNA, a left-handed helical DNA structure which is adopted more frequently upon CpG methylation. Altogether, our study significantly expands the human TF codebook by identifying DNA binding motifs for 98 TFs, while providing a versatile platform to quantitatively assay the impact of DNA modifications on TF binding.