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

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.

Methods for detection of protein-protein and protein-DNA interactions using HaloTag.

HaloTag is a protein fusion tag which was genetically engineered to covalently bind a series of specific synthetic ligands. All ligands carry two groups, the reactive group and the functional/reporter group. The reactive group, the choloroalkane, is the same in all the ligands and is involved in binding to the HaloTag. The functional reporter group is variable and can carry many different moieties including fluorescent dyes, affinity handles like biotin or solid surfaces such as agarose beads. Thus, HaloTag can serve either as a labeling tag or as a protein immobilization tag depending on which ligand is bound to it. Here, we describe a procedure for immobilization of HaloTag fusion proteins and how immobilized proteins can be used to study protein-protein and protein-DNA interactions in vivo and in vitro.

HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.