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Related Experiment Videos

The Pole3 bidirectional unit is regulated by MYC and E2Fs.

Fabrizio Bolognese1, Claudia Forni, Giuseppina Caretti

  • 1Dipartimento di Scienze Biomolecolari e Biotecnologie, U. di Milano, Via Celoria 26, 20133 Milano, Italy.

Gene
|January 13, 2006
PubMed
Summary

The Pol3 gene, a DNA polymerase subunit, is cell-cycle regulated, peaking during S phase entry. Its promoter is controlled by E2F transcription factors and MYC, linking histone-like gene regulation to DNA synthesis.

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Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Gene Expression

Background:

  • Pole3 (DPB4/YBL1/CHRAC17) is a subunit of DNA polymerase epsilon, featuring a histone-like domain crucial for heterodimerization with Pole4 (DPB3).
  • Pole3 also interacts with YCL1/CHRAC15 and the ACF1/SNF2H chromatin remodelling complex, suggesting roles beyond DNA replication.

Purpose of the Study:

  • To investigate the cell-cycle regulation of the Pol3 gene in NIH3T3 fibroblasts.
  • To characterize the promoter region of the Pol3 gene and identify regulatory elements involved in its expression.
  • To elucidate the interplay between transcription factors and the bidirectional Pol3 promoter during the cell cycle.

Main Methods:

  • Serum induction of starved NIH3T3 fibroblasts to study gene regulation.

Related Experiment Videos

  • Promoter analysis including sequence characterization (absence of TATA/CCAAT boxes, presence of E box and E2F sites) and mutagenesis.
  • Generation of stable cell clones to confirm cell-cycle regulation.
  • Chromatin immunoprecipitation (ChIP) assays to identify protein-DNA interactions at the promoter.

    • Main Results:

    • The Pol3 gene expression peaks at the G1/S phase transition in response to serum stimulation.
    • A minimal promoter region, containing an E box and a direct repeat, is essential for Pol3 gene activation.
    • An additional E2F site contributes to cell-cycle regulation, which was validated in stable clones.
    • E2F1/4 and MYC transcription factors bind to the Pol3 promoter in a cell-cycle-dependent manner.

    Conclusions:

    • The Pol3 gene is coregulated with core histones during DNA synthesis, highlighting a coordinated mechanism for cell proliferation.
    • This study provides the first detailed analysis of transcription factor binding and regulation on a bidirectional promoter involving a histone-like gene and cell-cycle regulators.