Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

Yousefi, F.; Simental, E. A.; Du, Y.; Whedon, S. D.; Trnka, M. J.; Darling, D.; Jia, S.; Panning, B.; Cole, P. A.; Halic, M.; Al-Sady, B.

doi:10.64898/2026.01.21.700667

bioRxiv · 10.64898/2026.01.21.700667

Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading

Yousefi, F.·Simental, E. A.·Du, Y.·Whedon, S. D.·Trnka, M. J.·Darling, D.·Jia, S.·Panning, B.·Cole, P. A.·Halic, M.·Al-Sady, B.

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Abstract

Repressive histone methyltransferases carry a catalytic ("write") domain and a separate domain specialized for recognizing ("reading") the reaction product. This read-write configuration acts as a positive feedback mechanism for epigenetic maintenance and the growth of repressive chromatin domains. Feedback exhibits as catalytic stimulation and is understood to act towards a proximal (trans) nucleosome. Whether this stimulation affects a specific methylation transition and whether it is restricted to trans-stimulation remains opaque. Here, we dissect the positive feedback in the heterodimeric histone 3 lysine 9 (H3K9) mono- and dimethlyase G9a-GLP, which carries two catalytic SET and two product-reading Ankyrin repeat (ANK) domains. We find that reading by both ANK domains is required for H3K9 di-, but not monomethylation on nucleosomes and for tight binding to them. As this read-writing occurs on dilute mononucleosomes, we propose that intranucleosomal feedback occurs for G9a-GLP. Swapping the ANK domains results in loss of dimethylation while maintaining nucleosome binding, indicating catalytic coupling of nucleosome methylation intermediates to reading. Crosslinking mass spectrometry reveals specific G9a surfaces that contact nucleosomal methylation intermediates. Structural approaches reveal how these surfaces position the G9a ANK domain on the methylation-intermediate nucleosome and stabilize G9a-GLP on chromatin during the reaction.

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Yousefi, F., Simental, E. A., Du, Y., Whedon, S. D., Trnka, M. J., Darling, D., Jia, S., Panning, B., Cole, P. A., Halic, M., Al-Sady, B.. 2026-01-22. Histone 3 lysine 9 dimethylation by the G9a-GLP heterodimer requires intranucleosomal product reading. https://doi.org/10.64898/2026.01.21.700667

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