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Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter
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Cha, a basic helix-loop-helix transcription factor involved in the regulation of upstream stimulatory factor activity

Clara I Rodríguez1, Núria Gironès, Manuel Fresno

  • 1Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain.

The Journal of Biological Chemistry
|August 19, 2003
PubMed

Insights

Cha is a novel basic helix-loop-helix (bHLH) transcription factor that negatively regulates upstream stimulatory factor (USF)-dependent gene transcription. It requires USF-1 for DNA binding and inhibits USF-1-activated gene expression, particularly in T cells.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Basic helix-loop-helix (bHLH) proteins are crucial transcription factors involved in various cellular processes.
  • Upstream stimulatory factor (USF)-1 is a known regulator of gene expression, including the CD2 gene.

Purpose of the Study:

  • To characterize a novel bHLH transcription factor, named Cha.
  • To elucidate the role of Cha in regulating USF-1-dependent transcription.

Main Methods:

  • Protein and mRNA expression analysis.
  • Electrophoretic mobility shift assays (EMSA) to assess DNA binding.
  • Pull-down and co-immunoprecipitation assays for protein interactions.
  • Reporter gene assays to measure transcriptional activity.
  • Chromatin immunoprecipitation (ChIP) assays to determine promoter occupancy.
  • Gene silencing and overexpression studies in T cells.

Main Results:

  • Cha exhibits characteristics of a class C bHLH transcription factor and interacts with USF-1.
  • Cha requires USF-1 for binding to E-box DNA elements and inhibits USF-1-mediated transcription.
  • Cha expression is inversely correlated with CD2 expression in T cells, with high levels in resting cells and low levels in activated cells.
  • Cha occupancy of the CD2 promoter is observed in resting T cells but diminishes upon mitogen stimulation.
  • Overexpression of Cha in T cells leads to a significant reduction in CD2 expression.

Conclusions:

  • Cha is a novel bHLH transcription factor that functions as a repressor of USF-1-dependent transcription.
  • Cha plays a role in regulating gene expression, such as CD2, in T cells, potentially by modulating USF-1 activity.
  • The findings reveal a new mechanism of transcriptional regulation involving bHLH factors and their interactions with other transcription factors like USF-1.

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