CREB-binding protein activates transcription through multiple domains

Area of Science:

• Molecular Biology
• Gene Regulation
• Protein Function

Background:

• CREB-binding protein (CBP) is a coactivator for various transcription factors.
• The specific regions of CBP responsible for transcriptional activation were previously unidentified.

Purpose of the Study:

• To identify and characterize the transcriptional activation domains (TADs) within CBP.
• To investigate the interaction of CBP TADs with TATA-binding protein (TBP).
• To determine the cell-type specificity of PKA-mediated regulation of CBP TADs.

Main Methods:

• Deletion analysis to identify functional domains within CBP.
• Squelching assays to assess the role of TADs in transcription.
• In vitro binding assays to examine CBP TAD-TBP interactions.
• GAL-CBP chimera constructs to study C-terminal TADs.
• Reporter gene assays in various cell lines (PC-12, F9, COS-7) to assess transcriptional activity.

Main Results:

• The N-terminal half of CBP possesses a potent TAD sufficient for CREB-mediated transcription.
• This N-terminal TAD directly binds TBP.
• Two distinct C-terminal TADs were identified adjacent to the c-Fos binding site.
• PKA enhances the transcriptional activity of the N-terminal TADs in PC-12 cells, but not in F9 or COS-7 cells, indicating cell-type specific regulation.

Conclusions:

• CBP contains multiple TADs, with the N-terminal region being crucial for CREB-mediated transcription and TBP binding.
• The regulation of CBP activity by PKA is cell-type specific.
• These findings elucidate the functional organization of CBP's transcriptional activation capabilities.