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<p>DNA replication is closely associated with transcriptional activity, chromatin state, and higher-order genome organization, but how transcriptional activation influences replication remains incompletely understood. Here, we established a doxycycline-inducible transcription system by inserting a short tetO-EGFP reporter cassette into a large, gene-poor, late-replicating region on chromosome 2 in mouse embryonic stem cells. Dox treatment induced robust EGFP expression and was associated with a reproducible, localized increase in early S-phase replication signal around the knock-in site. However, the resulting replication timing shift was modest, and the targeted region remained later-replicating than endogenous constitutively early-replicating domains. Time-course Repli-seq analysis indicated that local replication activity changes could be detected following Dox induction and persisted over prolonged induction. The induced replication profile showed a subtle asymmetry relative to the insertion, however, inversion of the reporter cassette did not obviously reverse the asymmetry, suggesting that, at the resolution of our assay, the spatial pattern of the induced shift is not simply determined by transcriptional orientation. Together, our results show that Dox-induced activation of an inserted reporter cassette in a late-replicating gene desert is associated with a localized increasing of replication activity. This gene-desert system minimizes confounding effects from nearby endogenous genes and regulatory elements and provides a useful platform for future studies dissecting the interplay among transcription, chromatin state, three-dimensional genome organization, and DNA replication timing.</p>

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Inducing transcription in a gene desert is associated with locally increased replication activity

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月1日

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  • Fanli Zhou,
  • Chaozhang Feng,
  • Ruxin Zhang,
  • Haizhen Long,
  • Zengqi Wen

摘要

DNA replication is closely associated with transcriptional activity, chromatin state, and higher-order genome organization, but how transcriptional activation influences replication remains incompletely understood. Here, we established a doxycycline-inducible transcription system by inserting a short tetO-EGFP reporter cassette into a large, gene-poor, late-replicating region on chromosome 2 in mouse embryonic stem cells. Dox treatment induced robust EGFP expression and was associated with a reproducible, localized increase in early S-phase replication signal around the knock-in site. However, the resulting replication timing shift was modest, and the targeted region remained later-replicating than endogenous constitutively early-replicating domains. Time-course Repli-seq analysis indicated that local replication activity changes could be detected following Dox induction and persisted over prolonged induction. The induced replication profile showed a subtle asymmetry relative to the insertion, however, inversion of the reporter cassette did not obviously reverse the asymmetry, suggesting that, at the resolution of our assay, the spatial pattern of the induced shift is not simply determined by transcriptional orientation. Together, our results show that Dox-induced activation of an inserted reporter cassette in a late-replicating gene desert is associated with a localized increasing of replication activity. This gene-desert system minimizes confounding effects from nearby endogenous genes and regulatory elements and provides a useful platform for future studies dissecting the interplay among transcription, chromatin state, three-dimensional genome organization, and DNA replication timing.