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

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.

Specificity determinants for the abscisic acid response element.

Abscisic acid (ABA) response elements (ABREs) are a group of cis-acting DNA elements that have been identified from promoter analysis of many ABA-regulated genes in plants. We are interested in understanding the mechanism of binding specificity between ABREs and a class of bZIP transcription factors known as ABRE binding factors (ABFs). In this work, we have modeled the homodimeric structure of the bZIP domain of ABRE binding factor 1 from Arabidopsis thaliana (AtABF1) and studied its interaction with ACGT core motif-containing ABRE sequences. We have also examined the variation in the stability of the protein-DNA complex upon mutating ABRE sequences using the protein design algorithm FoldX. The high throughput free energy calculations successfully predicted the ability of ABF1 to bind to alternative core motifs like GCGT or AAGT and also rationalized the role of the flanking sequences in determining the specificity of the protein-DNA interaction.