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KAS-CUT&Tag for direct mapping of transcription bubbles

Wu, W.; Greene, J. E.; Ahmad, K.; Henikoff, S.

2026-05-19 genomics
10.64898/2026.05.15.725569 bioRxiv
Show abstract

Transcription by RNA Polymerase II (Pol II) generates dynamic transcription bubbles as it moves forward, but existing methods map transcriptional activity in vivo only indirectly. We introduce KAS-CUT&Tag, a method that combines N3-kethoxal labeling of exposed guanines with CUT&Tag to directly map transcription bubbles. We find that bubble density varies across Pol II-bound genes at transcription start sites, gene bodies, and termination sites. Transcription bubbles are enriched at genes marked by the H3K36me3 histone modification and chromatin-bound splicing factor U2AF2, but the highest enrichment is at replication-coupled histone genes throughout the cell cycle. KAS-CUT&Tag also detects colocalization of the histone gene transcription factor NPAT with Pol II at the Histone Locus Body, suggesting NPAT is juxtaposed to transcription bubbles via interaction with Pol II. Together, KAS-CUT&Tag enables direct mapping of Pol II and regulatory interactions at transcription bubbles, providing a powerful tool for precise analysis of transcriptional activity.

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