Analysis of the interaction of a non-canonical twin half-site of Cyclic AMP-Response Element (CRE) with CRE-binding protein

• Published in: Biochimie 2023-08, Vol.211, p.25-34
• Main Authors: Mukherjee, Srimoyee, Sarkar, Aditya Kumar, Lahiri, Ansuman, Sengupta (Bandyopadhyay), Sumita
• Format: Article
• Language: English
• Subjects: Base Sequence; CRE mutation; CREB dimer; Cyclic AMP - metabolism; Cyclic AMP Response Element-Binding Protein - genetics; Cyclic AMP Response Element-Binding Protein - metabolism; DNA-Protein interaction energy; Half-CRE; Response Elements; Transcription, Genetic
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2023.02.011

Differential regulation of a gene having either canonical or non-canonical cyclic AMP response element (CRE) in its promoter is primarily accomplished by its interactions with CREB (cAMP-response element binding protein). The present study aims to delineate the mechanism of the CREB-CRE interactions at the Oncostatin-M (osm) promoter by in vitro and in silico approaches. The non-canonical CREosm consists of two half-CREs separated by a short intervening sequence of 9 base pairs. In this study, in vitro binding assays revealed that out of the two CRE half-sites, the right half-CRE was indispensable for binding of CREB, while the left sequence showed weaker binding ability and specificity. Genome-wide modeling and high throughput free energy calculations for the energy-minimized models containing CREB-CREosm revealed that there was no difference in the binding of CREB to the right half of CREosm site when compared to the entire CREosm. These results were in accordance with the in vitro studies, confirming the indispensable role of the right half-CREosm site in stable complex formation with the CREB protein. Additionally, conversion of the right half-CREosm site to a canonical CRE palindrome showed stronger CREB binding, irrespective of the presence or absence of the left CRE sequence. Thus, the present study establishes an interesting insight into the interaction of CREB with a CRE variant located at the far end of a TATA-less promoter of a cytokine-encoding gene, which in turn could be involved in the regulation of transcription under specific conditions. •The non-canonical CREosm consists of two half CREs separated by a short intervening sequence of 9 base pairs.•In vitro binding of CREB to the 5' CRE site is indispensable while to the 3' CRE site is weak with low specificity.•Modeling and free energy calculations for interaction of CREB with CREosm and its mutants validated the in vitro results.•Association of CREB with the canonical CRE was more stable than that with the half-CREs or non-canonical CREs.