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PubMed · 42642513

Improved spike-in normalization clarifies the relationship between active histone modifications and transcription.

Lauren Patel·Yuwei Cao·Tianyao Xu·Eduardo Modolo·Tamar Dishon·Lingzhi Zhang·Eric Mendenhall·Sven Heinz·Itamar Simon·Christopher Benner·Alon Goren
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Abstract

Spike-in normalization enables quantitative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) signal. Here we introduce a robust dual spike-in normalization approach for ChIP-seq (ChIP-wrangler), optimize parameters and verify its accuracy in quantifying changes in ChIP-seq signal and detecting technical artifacts. We use ChIP-wrangler to revisit recent claims that active histone marks depend on transcription. We show that acute depletion of RNA polymerase II (RNAPII) has a modest impact on H3K27ac levels, with only 6% of peaks significantly changing after RNAPII depletion, indicating that histone acetylation maintenance is not entirely dependent on ongoing transcription. Promoters and enhancers are differentially affected, with 82% of decreasing acetylation peaks located at promoter-distal elements with enhancer-related motifs. ChIP-wrangler provides increased rigor and 'guardrails' for successful spike-in normalization and, as applied here, refines the understanding of crosstalk between RNAPII activity and transcription-associated histone marks.

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HistonesRNA Polymerase IITranscription, GeneticChromatin Immunoprecipitation SequencingAcetylationPromoter Regions, GeneticAnimalsChromatin ImmunoprecipitationHumansHistone CodeEnhancer Elements, GeneticJournal Article

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Lauren Patel, Yuwei Cao, Tianyao Xu, Eduardo Modolo, Tamar Dishon, Lingzhi Zhang, Eric Mendenhall, Sven Heinz, Itamar Simon, Christopher Benner, Alon Goren. 2026-08-25. Improved spike-in normalization clarifies the relationship between active histone modifications and transcription.. https://doi.org/10.1038/s41588-026-02728-2

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