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<p>Developmental genes are controlled by an ensemble of <i>cis</i>-acting regulatory elements (REs), which in turn respond to multiple <i>trans</i>-acting transcription factors (TFs). Understanding how a <i>cis</i>-regulatory landscape integrates information from many dynamically expressed TFs has remained a challenge. Here we develop a combined CRISPR screening approach using endogenous RNA and RE reporters as readouts. Applied to the murine <i>Xist</i> locus, which is crucial for X-chromosome inactivation in females, this method allows us to comprehensively identify Xist-controlling TFs and map their TF–RE wiring. We find a group of transiently upregulated TFs, including ZIC3, that regulate proximal REs, driving the binary activation of Xist expression. These basal activators are more highly expressed in cells with two X chromosomes, potentially governing female-specific Xist upregulation. A second set of developmental TFs that include OTX2 is upregulated later during differentiation and targets distal REs. This regulatory axis is crucial to achieve high levels of Xist RNA, which is necessary for X-chromosome inactivation. OCT4 emerges as the strongest activator overall, regulating both proximal and distal elements. Our findings support a model for developmental gene regulation, in which factors targeting proximal REs drive binary on–off decisions, whereas factors interacting with distal REs control the transcription output.</p>

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Reporter CRISPR screens decipher cis-regulatory and trans-regulatory principles at the Xist locus

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年10月6日

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  • Till Schwämmle,
  • Gemma Noviello,
  • Eleni Kanata,
  • Jonathan J. Froehlich,
  • Melissa Bothe,
  • Alexandra Martitz,
  • Aybuge Altay,
  • Jade Scouarnec,
  • Vivi-Yun Feng,
  • Heleen Mallie,
  • Martin Vingron,
  • Edda G. Schulz

摘要

Developmental genes are controlled by an ensemble of cis-acting regulatory elements (REs), which in turn respond to multiple trans-acting transcription factors (TFs). Understanding how a cis-regulatory landscape integrates information from many dynamically expressed TFs has remained a challenge. Here we develop a combined CRISPR screening approach using endogenous RNA and RE reporters as readouts. Applied to the murine Xist locus, which is crucial for X-chromosome inactivation in females, this method allows us to comprehensively identify Xist-controlling TFs and map their TF–RE wiring. We find a group of transiently upregulated TFs, including ZIC3, that regulate proximal REs, driving the binary activation of Xist expression. These basal activators are more highly expressed in cells with two X chromosomes, potentially governing female-specific Xist upregulation. A second set of developmental TFs that include OTX2 is upregulated later during differentiation and targets distal REs. This regulatory axis is crucial to achieve high levels of Xist RNA, which is necessary for X-chromosome inactivation. OCT4 emerges as the strongest activator overall, regulating both proximal and distal elements. Our findings support a model for developmental gene regulation, in which factors targeting proximal REs drive binary on–off decisions, whereas factors interacting with distal REs control the transcription output.