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c-Myb-binding sites mediate G(1)/S-associated repression of the plasma membrane Ca(2+)-ATPase-1 promoter
1Centre for Cardiovascular Research, Toronto General Hospital, Toronto, Ontario, Canada.
Abstract:
We demonstrate that two Myb-binding sites of the mouse plasma membrane Ca(2+)-ATPase-1 (PMCA1) promoter are required for G(1)/S cell cycle stage-associated repression of PMCA1 promoter activity. Nuclear run-on experiments revealed G(1)/S-associated repression of PMCA1 transcription. Ribonuclease protection assays revealed two transcription initiation sites between two Myb-binding sites in the PMCA1 promoter. Gel shift assays showed that c-Myb can bind to wild-type but not point mutated Myb binding sequences of the PMCA1 promoter. Transient transfection assays using cell cycle-synchronized vascular smooth muscle cells (VSMC) and PMCA1 promoter-luciferase constructs showed a 2-fold decrease in reporter activity at G(1)/S as compared with G(0). Overexpression of wild-type c-Myb severely repressed PMCA1 promoter activity at both G(0) and G(1)/S while co-transfection of a dominant negative c-Myb, or a construct encoding an anti-c-Myb neutralizing antibody, completely abolished the repression seen at G(1)/S. Single nucleotide substitutions in the first, second, or both Myb-binding sites alleviated the G(1)/S-associated repression of PMCA1 promoter activity in transformed rat VSMC and primary mouse VSMC cultures. We conclude that c-Myb mediates G(1)/S-associated transcriptional repression of the PMCA1 Ca(2+) pump in rodent VSMC by direct binding to the PMCA1 promoter.
Insights
Two Myb-binding sites in the PMCA1 promoter are essential for repressing the Ca(2+) pump during the G(1)/S cell cycle phase. The transcription factor c-Myb directly binds these sites to mediate this repression in vascular smooth muscle cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Cardiovascular Research
Background:
- The plasma membrane Ca(2+)-ATPase-1 (PMCA1) regulates intracellular calcium levels, crucial for vascular smooth muscle cell (VSMC) function.
- Cell cycle progression influences gene expression, impacting cellular processes like calcium homeostasis.
Purpose of the Study:
- To investigate the role of Myb-binding sites in the PMCA1 promoter in regulating its activity during the G(1)/S cell cycle transition.
- To determine the involvement of the transcription factor c-Myb in the cell cycle-dependent repression of PMCA1.
Main Methods:
- Nuclear run-on assays to assess PMCA1 transcription rates.
- Ribonuclease protection assays to identify transcription initiation sites.
- Gel shift assays to confirm c-Myb binding to PMCA1 promoter sequences.
- Transient transfection assays with PMCA1 promoter-luciferase constructs in synchronized VSMCs.
- Mutagenesis of Myb-binding sites and overexpression/inhibition of c-Myb.
Main Results:
- PMCA1 transcription is repressed during the G(1)/S phase.
- Two functional Myb-binding sites were identified in the PMCA1 promoter, located between two transcription initiation sites.
- c-Myb directly binds to these specific Myb-binding sites.
- PMCA1 promoter activity decreased 2-fold at G(1)/S compared to G(0) in VSMCs.
- Overexpression of c-Myb repressed PMCA1 activity, while dominant-negative c-Myb or anti-c-Myb antibodies abolished this repression.
- Mutating the Myb-binding sites eliminated the G(1)/S-associated repression.
Conclusions:
- c-Myb directly binds to two specific sites within the PMCA1 promoter.
- c-Myb mediates the G(1)/S-associated transcriptional repression of the PMCA1 Ca(2+) pump in rodent VSMCs.