A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays

In analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1.These data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.Control of gene expression and transcription in mammalian cells is typically achieved through a multi-layered network of protein signaling pathways containing multiple checkpoints to ensure specificity or correct transmission of external stimuli. Regulation of transcriptional activation or repression is crucial for proper development, cell growth, and routine progression through the cell cycle. There is a rapidly growing body of data describing DNA-protein interactions on a genome-wide scale, aided by availability of complete mammalian genome sequences and also the coupling of chromatin immunoprecipitation (ChIP) experiments [1-3] with DNA microarrays analysis (ChIP-chip) [4-10] or ultra high-throughput sequencing (ChIP-Seq) [11-16]. While genome-wide maps of DNA-protein interactions are crucial to understanding global transcriptional networks, understanding the f