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Tracking induced pluripotent stem cell differentiation with a fluorescent genetically encoded epigenetic probe.

Afanasii I Stepanov1,2, Alexandra A Shuvaeva1,2,3, Lidia V Putlyaeva1,2

  • 1Skolkovo Institute of Science and Technology, Bolshoi Blvd. 30, Bld. 1, 121205, Moscow, Russia.

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Summary

Researchers developed a fluorescent probe to visualize epigenetic landscapes in living cells. This probe tracked changes in histone modification (H3K9me3) during stem cell differentiation, revealing key reorganization events.

Keywords:
EpigeneticsFluorescent proteinsGenetically encoded sensorH3K9me3Histone modificationMachine learning

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Area of Science:

  • Molecular Biology
  • Epigenetics
  • Cell Biology

Background:

  • Epigenetic modifications, such as histone methylation and acetylation, are crucial for gene expression regulation.
  • Epigenomic changes are dynamic during biological processes like cell differentiation.
  • Existing methods for analyzing epigenetic modifications typically require fixed cells, limiting real-time observation.

Purpose of the Study:

  • To develop a genetically encoded fluorescent probe for visualizing H3K9me3, a marker of inactive chromatin, in living cells.
  • To track dynamic changes in H3K9me3 epigenetic landscapes during induced pluripotent stem cell (iPSC) differentiation into induced neurons.
  • To establish a novel method (LiveMIEL) combining epigenetic probes and machine learning for analyzing single-cell epigenetic signatures.

Main Methods:

  • Development of MPP8-Green, a genetically encoded fluorescent probe based on the MPP8 chromodomain, for detecting H3K9me3.
  • Live-cell imaging of H3K9me3 epigenetic landscapes in differentiating iPSCs.
  • Application of machine learning approaches within the LiveMIEL framework for multiparametric epigenetic signature classification.

Main Results:

  • MPP8-Green successfully visualized H3K9me3 dynamics in single living cells.
  • Two significant waves of global H3K9me3 reorganization were observed during a 4-day iPSC to induced neuron differentiation process, occurring on days 1 and 3.
  • Minimal changes in H3K9me3 landscapes were detected on days 2 and 4 of differentiation.

Conclusions:

  • The MPP8-Green probe enables real-time monitoring of H3K9me3 dynamics in living cells.
  • The LiveMIEL method offers a powerful approach for classifying single-cell epigenetic states during differentiation.
  • This technology has broad potential for studying epigenomic regulation in various biological models and disease states.