epigenomics intermediate

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

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

Nature Communications · Aug 5, 2026 · 2 min read

TL;DR

By developing a new microfluidic assay called meSMiLE-seq, researchers identified DNA binding motifs for 99 previously uncharacterized human transcription factors and discovered that 14 of them specifically prefer binding to methylated DNA.

Problem / question

The DNA binding specificities for about 400 of the roughly 1600 human transcription factors remain unknown, partly because standard profiling methods struggle to capture how epigenetic modifications like CG methylation alter binding affinities.

Methods

The authors used SMiLE-seq to screen 284 uncharacterized human transcription factors and developed meSMiLE-seq to simultaneously expose 114 transcription factors to barcoded methylated and unmethylated DNA libraries in a single microfluidic enrichment step. They analyzed the sequencing data using the ProBound Suite to generate position-specific affinity matrices and validated findings using EMSA, circular dichroism spectroscopy, and by intersecting motifs with ChIP-seq and whole-genome bisulfite sequencing data from HEK293/T cells.

Key findings

SMiLE-seq identified binding motifs for 74 transcription factors, while meSMiLE-seq characterized 48, yielding a total of 99 new motifs. Among the 48 meSMiLE-seq profiled proteins, 14 were classified as methyl plus (preferring methylated DNA, like PRDM13), 13 as methyl minus (repelled by methylation, like USF3), and 21 showed little effect. Additionally, the assay revealed that ZHX2 specifically enriches methylated CG repeats, suggesting it acts as a putative binder of the non-canonical Z-DNA conformation.

Why it matters

This study significantly expands the known catalog of human transcription factor binding sites and provides a streamlined, single-step platform to uncover how DNA methylation directs gene regulation and transcription factor recruitment.

Limitations

The single-round enrichment approach is sensitive to nucleotide synthesis biases, which initially limited the motif discovery success rate to 26 percent. Furthermore, the evidence for ZHX2 binding Z-DNA is correlational and lacks definitive structural confirmation, and some transcription factors that preferred methylated DNA in vitro bound unmethylated regions in living cells, likely due to chromatin inaccessibility.

Takeaway

meSMiLE-seq successfully maps the DNA binding preferences of uncharacterized transcription factors, revealing that many rely on DNA methylation or non-canonical DNA structures for specific genomic targeting.