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Structural determinants for transcriptional activation by cAMP-responsive DNA elements.

P J Deutsch1, J P Hoeffler, J L Jameson

  • 1Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.

The Journal of Biological Chemistry
|December 5, 1988
PubMed
Summary
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The cAMP-responsive element (CRE) function is significantly influenced by surrounding DNA sequences. Specific mutations in CREs can abolish or, in some contexts, maintain cAMP responsiveness, highlighting the importance of sequence context.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Epigenetics

Background:

  • The cAMP-responsive enhancer element (CRE), an 8-base pair palindrome (5' TGACGTCA 3'), is crucial for regulating eukaryotic gene transcription.
  • Understanding the precise mechanisms of CRE function, including the impact of sequence context and mutations, is vital for deciphering gene expression control.

Purpose of the Study:

  • To investigate the impact of point mutations within the CRE on gene transcription.
  • To analyze the influence of flanking DNA sequences on CRE activity.
  • To determine the requirements for transcriptional synergism of tandemly repeated CREs.

Main Methods:

  • Insertion of wild-type and mutated CRE sequences into a chloramphenicol acetyltransferase reporter plasmid.
  • Analysis of transcriptional activity using reporter gene assays.

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  • Gel mobility-shift assays to assess DNA-protein interactions.

    • Main Results:

    • Point mutations at critical positions within the CRE significantly inhibited transcriptional activity.
    • The surrounding DNA sequence context profoundly affected CRE function, with some mutant CREs showing maximal activity in specific native contexts (e.g., VIP gene).
    • Tandemly repeated CREs exhibited synergistic transcriptional stimulation, but insertion of more than two repeats or intervening sequences diminished activity.

    Conclusions:

    • The transcriptional activity mediated by cAMP-responsive elements is highly dependent on the adjacent DNA sequences.
    • Sequence context can modulate the effect of mutations within the CRE, influencing cAMP responsiveness.
    • Cooperative interactions occur between tandemly repeated CREs, but optimal spacing and number are critical for synergistic effects.