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Logo of bmcgenoBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Genomics
 
BMC Genomics. 2009; 10: 497.
Published online Oct 27, 2009. doi:  10.1186/1471-2164-10-497
PMCID: PMC2774331
A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays
Danette D Hartzell,corresponding author2 Nathan D Trinklein,corresponding author2 Jacqui Mendez,1 Nancy Murphy,#1 Shelley F Aldred,#2 Keith Wood,1 and Marjeta Urh1
1Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
2SwitchGear Genomics 1455 Adams Drive, Suite 1317, Menlo Park, CA 94025, USA
corresponding authorCorresponding author.
#Contributed equally.
Danette D Hartzell: danette.hartzell/at/promega.com; Nathan D Trinklein: nathant/at/switchgeargenomics.com; Jacqui Mendez: jacqui.mendez/at/promega.com; Nancy Murphy: nancy.murphy/at/promega.com; Shelley F Aldred: shelley/at/switchgeargenomics.com; Keith Wood: keith.wood/at/promega.com; Marjeta Urh: marjeta.urh/at/promega.com
Received June 11, 2009; Accepted October 27, 2009.
Abstract
Background
Regulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells.
Results
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.
Conclusion
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.
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