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The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
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Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry

Yoko Hayashi-Takanaka1, Yuichiro Hayashi2, Yasuhiro Hirano1

  • 1Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita 565-0871, Japan.

Nucleic Acids Research
|November 24, 2021
PubMed

Insights

Cell cycle progression relies on minichromosome maintenance (MCM) helicase loading. A single MCM hexamer state acts as a cell cycle halting step, requiring conversion to a double hexamer for proliferation.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Epigenetics

Background:

  • DNA replication initiation is crucial for cell proliferation.
  • Loading of minichromosome maintenance (MCM) helicase hexamers onto DNA replication origins during the G1 phase is essential for initiating DNA replication.
  • Understanding MCM hexamer dynamics and regulation is key to cell cycle control.

Purpose of the Study:

  • To investigate the states of MCM hexamers during the cell cycle in human cells.
  • To explore the relationship between MCM hexamer states and histone modifications, specifically H4K20 methylation.
  • To determine the role of MCM hexamer states in cell cycle progression and proliferation.

Main Methods:

  • Multicolor immunofluorescence-based, single-cell plot analysis in human hTERT-RPE1 cells.
  • Biochemical size fractionation to analyze MCM hexamer complexes.
  • Cell-cycle arrest and release experiments to observe MCM hexamer dynamics.
  • Analysis of histone H4 lysine 20 (H4K20) methylation levels in correlation with MCM states.

Main Results:

  • A double MCM hexamer is formed from a single hexamer during G1 phase progression.
  • A single MCM hexamer is recruited to chromatin in early G1, with a second hexamer recruited in late G1 to form the double hexamer.
  • The double MCM hexamer state correlates with di/trimethyl-H4K20 (H4K20me2/3).
  • Inhibition of H4K20me1 to H4K20me2/3 conversion and non-proliferative states (confluent or Cdk4/6 inhibitor-treated cells) result in cells remaining in the single MCM hexamer state.

Conclusions:

  • The single MCM hexamer state serves as a halting step in cell cycle progression.
  • Histone H4K20 methylation dynamics are linked to MCM hexamer formation and cell cycle progression.
  • Regulation of MCM hexamer assembly is critical for initiating DNA replication and cell proliferation.

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