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Related Experiment Videos

A negative regulatory element required for light-dependent pinopsin gene expression.

Yoko Takanaka1, Toshiyuki Okano, Kazuyuki Yamamoto

  • 1Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Japan.

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
|June 1, 2002
PubMed
Summary

Researchers identified an 18 bp DNA sequence in the chick pinopsin gene promoter essential for light-dependent gene expression. This element binds a ubiquitous factor, repressing transcription in the dark and enabling light-induced activation in pinealocytes.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Neuroscience

Background:

  • Light-stimulated gene expression in vertebrates occurs in the retina, pineal gland, and suprachiasmatic nucleus.
  • The specific cis-elements controlling light-dependent transcription remain largely uncharacterized.

Purpose of the Study:

  • To identify cis-elements regulating light-dependent transcription of the chick pinopsin gene.
  • To elucidate the mechanism of transcriptional regulation by these elements in pinealocytes.

Main Methods:

  • Luciferase reporter gene assays with varying lengths of the pinopsin 5' upstream region.
  • Site-directed mutagenesis of the identified 18 bp sequence.
  • Electrophoretic mobility-shift assays (EMSA) to detect nuclear factor binding.
  • Reporter gene assays using the SV40 promoter with the identified element in different cell types.

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Main Results:

  • Light-dependent enhancer activity was localized to the -1156 to +31 region of the pinopsin gene promoter.
  • Mutations in an 18 bp sequence (-1103 to -1086), containing an E-box, abolished light dependency by increasing dark transcription.
  • A pineal nuclear factor specifically binds to this 18 bp element.
  • This 18 bp element mediates transcriptional repression, particularly in non-photosensory cells, suggesting a ubiquitous repressor factor.

Conclusions:

  • The 18 bp element in the pinopsin promoter acts as a binding site for a ubiquitous repressor factor.
  • This repression is crucial, though not sufficient, for the light-dependent expression of the pinopsin gene in chick pinealocytes.