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Protein/DNA arrays identify nitric oxide-regulated cis-element and trans-factor activities some of which govern

Saravanakumar Dhakshinamoorthy1, Shiva Ranjani Sridharan, Lei Li

  • 1Cell Death and Human Diseases Group, Division of Genomics and Genetics, Institute of Molecular & Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Republic of Singapore.

Nucleic Acids Research
|August 19, 2007
PubMed

Insights

Toxic nitric oxide (NO) regulates gene expression by altering transcription factor binding in neuroblastoma cells. This leads to Bcl-2 induction, which protects a portion of cells from NO-induced apoptosis.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Neuroscience

Background:

  • Toxic nitric oxide (NO) levels are known to influence gene expression.
  • Understanding the precise mechanisms by which NO affects cellular processes, particularly gene regulation and cell death, is crucial.

Purpose of the Study:

  • To investigate how toxic nitric oxide (NO) levels impact transcription factor binding to cis-elements in neuroblastoma cells.
  • To elucidate the role of specific transcription factors and their target genes, such as Bcl-2, in NO-mediated apoptosis.

Main Methods:

  • Utilized a novel protein/DNA array to assess trans-factor binding to cis-elements under toxic NO conditions.
  • Employed electrophoretic mobility shift assays (EMSA) and reporter assays to confirm cis-element functionality.
  • Investigated the role of Bcl-2 in NO-induced apoptosis using overexpression and knockdown cell models.

Main Results:

  • Toxic NO levels altered the binding of multiple transcription factors (AP1, AP2, Brn-3a, EGR, E2F1, SP1) to cis-elements.
  • NO induced the anti-apoptotic Bcl-2 protein and its mRNA, preceding cell death in 30-60% of cells.
  • Bcl-2 overexpression conferred resistance to NO-induced apoptosis, while Bcl-2 knockdown increased sensitivity.

Conclusions:

  • NO stimulates the binding of various trans-factors, some of which regulate cell viability via target gene transcription.
  • Bcl-2 plays a significant role in counteracting NO-induced apoptosis in a subset of neuroblastoma cells.
  • A DNA/protein array approach is effective for discovering global transcription factor activities regulated by cell death molecules.

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