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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
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Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops.

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Hypoxia in tumors causes R-loop accumulation via reactive oxygen species, inhibiting ribosomal RNA synthesis and contributing to cancer progression. Restoring R-loop levels rescues this effect.

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Area of Science:

  • Molecular Biology
  • Cancer Research
  • Cellular Stress Response

Background:

  • Hypoxia is prevalent in solid tumors, correlating with poor prognosis, therapy resistance, and metastasis.
  • Physiological hypoxia (<0.1% O2) triggers replication stress, DNA damage response, and unfolded protein response.
  • Hypoxia induces robust accumulation of R-loops, structures of DNA-RNA hybrids.

Purpose of the Study:

  • To elucidate the mechanism and consequences of hypoxia-induced R-loop accumulation.
  • To investigate the role of reactive oxygen species (ROS) in R-loop formation under hypoxia.
  • To understand how R-loops contribute to the transcriptional stress response in hypoxic tumors.

Main Methods:

  • Investigated R-loop accumulation in hypoxic conditions.
  • Assessed the role of non-DNA damaging reactive oxygen species (ROS) in R-loop formation.
  • Monitored ribosomal RNA (rRNA) synthesis and nucleolin translocation.
  • Analyzed heterochromatin marker H3K9me2 deposition on rDNA.
  • Depleted R-loops to observe rescue effects.

Main Results:

  • Hypoxia-induced R-loop accumulation is dependent on ROS.
  • Hypoxia-induced R-loops repress rRNA synthesis and cause nucleolin translocation.
  • R-loops accumulate on rDNA, promoting H3K9me2 deposition and inhibiting Pol I transcription.
  • R-loop depletion rescues rRNA transcription and nucleolin translocation in hypoxia.

Conclusions:

  • A novel ROS-R-loop-H3K9me2 axis mediates hypoxia-induced transcriptional stress.
  • Hypoxia-induced R-loops contribute significantly to tumorigenesis through transcriptional dysregulation.