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Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function
Valentina Salsi1, Giuseppina Caretti, Mark Wasner
1Dipartimento di Biologia Animale, Università di Modena e Reggio, Via Campi 213/d, 41100 Modena, Italy.
The Journal of Biological Chemistry
|December 17, 2002
Summary
Multiple CCAAT boxes in the cyclin B2 promoter are crucial for function, with precise spacing enabling coactivator p300 to bind NF-Y transcription factors, especially at weaker sites.
Area of Science:
- Molecular Biology
- Gene Regulation
Background:
- The CCAAT box is a common eukaryotic promoter element, typically bound by the NF-Y transcription factor.
- While usually singular, some promoters feature multiple CCAAT boxes, like the cyclin B2 cell-cycle promoter.
Purpose of the Study:
- To investigate the interplay between NF-Y transcription factor and the p300 coactivator on the cyclin B2 promoter.
- To understand the functional significance of multiple CCAAT boxes and their spacing.
Main Methods:
- Chromatin immunoprecipitation (ChIP) to analyze in vivo binding of NF-Y and p300.
- Cotransfection experiments to assess coactivator function.
- In vitro binding assays to determine NF-Y complex order and affinity.
Main Results:
- NF-Y and p300 bind the cyclin B2 promoter in vivo, with binding levels correlating with cell cycle stage and promoter activity.
- p300 enhances NF-Y binding, particularly to the proximal Y3 CCAAT site, but requires precise spacing between elements.
- NF-Y binding affinities decrease from the distal Y1 to the proximal Y3 site; binding is not cooperative but stable at Y1 and Y2.
Conclusions:
- Precise spacing of multiple CCAAT boxes is critical for coactivator p300 function.
- Regulation of transient binding to weak NF-Y sites may be a key mechanism during the cell cycle for promoters with multiple CCAAT boxes.