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
] with DNA microarrays analysis (ChIP-chip) [4
] or ultra high-throughput sequencing (ChIP-Seq) [11
]. While genome-wide maps of DNA-protein interactions are crucial to understanding global transcriptional networks, understanding the functional consequences of these binding events is equally important. To expand existing approaches to study DNA-protein interactions in living cells, we present two complementary technologies: HaloCHIP, an antibody-free alternative approach to ChIP, for mapping protein binding sites on DNA, and high-throughput reporter assays to measure the promoter activity associated with binding events.
The success of ChIP relies heavily on the success of the immunoprecipitation step in the process, creating a need for alternative approaches when the antibody against the DNA binding protein is either not functional or available for the ChIP assay [17
]. Such alternative approaches are derived from the standard ChIP method and include the initial formaldehyde crosslinking of protein:DNA complexes, yet typically differ in the use of protein fusion tags, which allow for complexes to be isolated using either an antibody against the tag [17
] or direct capture on a resin that interacts with the fusion tag [18
]. The latter is the basis for the HaloCHIP method, which utilizes the HaloTag protein [19
], a 33 kDa protein fusion tag, that can be cloned N- or C-terminally to a DNA binding protein of interest[19
] (Figure ). In the HaloCHIP method, the HaloTag fusion protein is expressed either transiently or stably in mammalian cells and crosslinked complexes can be directly captured from a cellular lysate via covalent binding to a HaloTag-specific resin, termed HaloLink [19
] (Figure ). The complete covalent linkage established at this point allows for extensive washing to remove non-specific protein and DNA, followed by standard reversal of the crosslinks to release the DNA fragments which were bound to the DNA binding protein (Figure ). Several controls for the HaloCHIP method are possible to show that capture is specific in this process and provide an excellent estimate of background (Figure ).
Figure 1 Schematic of the HaloCHIP process. The HaloCHIP process is initiated by cloning a desired DNA binding protein-of-interest, i.e. a transcription factor (TF) into a HaloTag (HT) fusion mammalian expression vector. For the experimental HaloCHIP sample, the (more ...)
Both the HaloCHIP and ChIP method yield information about the location and timing of binding events on DNA, but do not provide information as to the cellular response or consequence of the given binding event. Currently, mRNA and protein levels are measured to determine whether or not a gene has been activated or repressed, but a more direct measure of the transcription potential or function of bound DNA sequences would be ideal. To complement these approaches and also increase sensitivity, high-throughput reporter assays can be used (Cooper et al. 2006). High-throughput reporter assays utilize a 384-well format that enable the functional measure of thousands of endogenous human promoters. Each individual promoter is fused to a luciferase reporter gene and transiently delivered to living cells. Upon protein binding to the promoter region, the luciferase reporter gene is activated and the degree of this activation can be quantitatively determined before and after a stimulus by measuring the light output. This allows real-time monitoring of transcriptional activation or repression from the promoter-reporter construct after stimulus of a pathway or response to other cellular conditions.
To demonstrate the use of these approaches to further understanding of DNA-protein interactions in living cells, we chose to study the CREB transcription factor [22
]. The model system of the CREB signaling pathway has been elegantly studied and its binding targets have been described previously at the level of individual promoters as well as a genome-wide scale [12
]. CREB belongs to a family of transcription factors including activating transcription factor 1 (ATF1) and the cAMP response element modulator (CREM), which regulate gene expression in response to changes in cAMP and other cellular signals [23
]. Upon activation of the protein kinase A pathway or stimulation of other kinases, CREB is directly phosphorylated on several critical serines [22
], though phosphorylation is not required for binding to DNA [31
]. The phosphorylation events instead allow subsequent recruitment and binding of transcriptional co-activators CREB binding protein (CBP)/p300 as well as transducers of regulated CREB (TORCs) to the promoter region [32
]. Previous studies have shown that CREB co-factors are often necessary for transcription activation and that CREB binding to DNA, even in its phosphorylated form, is not usually sufficient to activate transcription [34
In this paper, CREB binding is mapped at a much higher resolution than previous studies and covers all known and predicted human promoters using the HaloCHIP method in conjunction with DNA microarrays, ("HaloCHIP-chip"). As this is a new approach for studying genome-wide protein:DNA interactions, these data were compared to the standard CREB ChIP-chip process using an antibody against the endogenous CREB protein, revealing a high degree of overlap between the methods and also to previously published data [26
]. To further correlate DNA binding events to potential transcription activation or repression, a subset of CREB-bound promoters were analyzed using high-throughput reporter assays in the presence or absence of protein kinase A pathway activators as well as the CREB transcriptional co-activator, TORC1. All together these data reveal new CREB-bound promoters and binding preferences on DNA, interesting functional activities provided by the high-throughput reporter assays, and new insights into CREB-mediated transcription regulation.