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Updated: Aug 18, 2026
Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
Published on: May 27, 2016
Cloning and transcriptional analysis of the mouse receptor activity modifying protein-1 gene promoter
1Department of Cellular and Molecular Medicine, St. George's Hospital Medical School, Cranmer Terrace, London, SW17 ORE, UK. mpondel@sghms.ac.uk
Background:
Receptor activity modifying protein-1 (RAMP-1) is a single transmembrane-domain protein required for the functional expression of calcitonin gene-related peptide (CGRP) receptors. To date, little is known about the molecular mechanism(s) that activate/inhibit RAMP-1 gene expression. Such mechanism(s) are likely to play a major role in modulating the responsiveness of tissues to CGRP.
Results:
To initiate studies on the transcriptional regulation of the mouse RAMP-1 gene, RAMP-1 transcriptional initiation sites were mapped in a variety of tissues. Analysis of RAMP-1 expression in C2C12 myoblasts demonstrated that RAMP-1 mRNA is expressed at greatest levels in confluent myoblasts verses non-confluent and fused myoblasts. Transfection of confluent C2C12 myoblasts and NIH 3T3 cells with RAMP-1 promoter/luciferase deletion constructs revealed that 4.7 kb of RAMP-1 5' flanking region demonstrated optimal promoter activity while 343 bp of 5' flanking region was defined as a minimal RAMP-1 promoter. In non-RAMP-1 expressing HEK293 cells, constructs containing 4.7 kb to 782 bp of RAMP-1 5' flanking region were transcriptionally inactive. However, deletion of sequences -782 to -343 activated RAMP-1 promoter activity.
Conclusion:
These findings suggest that tissue specificity of RAMP-1 gene expression is mediated by a negative acting transcription factor that represses RAMP-1 gene expression in non-RAMP-1 expressing tissues. This transcription factor is therefore likely to play an important role in modulating the responsiveness of tissues to CGRP.
Insights
Receptor activity modifying protein-1 (RAMP-1) gene expression is tissue-specific. A negative transcription factor represses RAMP-1 in non-expressing tissues, impacting CGRP receptor responsiveness.
Area of Science:
- Molecular Biology
- Gene Regulation
- Neuroscience
Background:
- Receptor activity modifying protein-1 (RAMP-1) is crucial for functional calcitonin gene-related peptide (CGRP) receptors.
- Mechanisms regulating RAMP-1 gene expression are largely unknown.
- Understanding RAMP-1 regulation is key to modulating tissue responsiveness to CGRP.
Purpose of the Study:
- To investigate the transcriptional regulation of the mouse RAMP-1 gene.
- To identify regulatory elements controlling RAMP-1 gene expression.
- To elucidate the role of RAMP-1 in tissue-specific CGRP receptor function.
Main Methods:
- Mapping RAMP-1 transcriptional initiation sites across various tissues.
- Analyzing RAMP-1 mRNA expression levels in C2C12 myoblasts under different conditions.
- Utilizing RAMP-1 promoter/luciferase deletion constructs in transfection assays (C2C12, NIH 3T3, HEK293 cells).
Main Results:
- RAMP-1 mRNA expression is highest in confluent C2C12 myoblasts.
- A 4.7 kb 5' flanking region shows optimal RAMP-1 promoter activity; a 343 bp region defines the minimal promoter.
- Sequences between -782 and -343 bp in HEK293 cells repress RAMP-1 promoter activity, suggesting a tissue-specific repressor element.
Conclusions:
- Tissue-specific RAMP-1 gene expression is mediated by a negative-acting transcription factor.
- This repressor element likely controls RAMP-1 expression in non-RAMP-1-expressing tissues.
- The identified transcription factor plays a significant role in regulating tissue sensitivity to CGRP.
