A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays
- Danette D Hartzell1Email author,
- Nathan D Trinklein2Email author,
- Jacqui Mendez1,
- Nancy Murphy†1,
- Shelley F Aldred†2,
- Keith Wood1 and
- Marjeta Urh1
© Hartzell et al; licensee BioMed Central Ltd. 2009
Received: 11 June 2009
Accepted: 27 October 2009
Published: 27 October 2009
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.
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 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.
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 [22–25]. 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, 22, 26–29]. 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, 24]. Upon activation of the protein kinase A pathway or stimulation of other kinases, CREB is directly phosphorylated on several critical serines [22, 30], though phosphorylation is not required for binding to DNA . 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 [32–35]. 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 [34–39].
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, 29]. 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.
Specific binding and enrichment of CREB promoters in HaloCHIP
HaloCHIP-chip DNA oligo array design
CREB HaloCHIP-chip array data analysis and cut-offs
CREB HaloCHIP-chip positive and negative predictive values.
12 of 12
11 of 12
6 of 12
2 of 26
Comparison of CREB HaloCHIP-chip and ChIP-chip array data
Comparison of CREB HaloCHIP-chip and CREB ChIP-chip data.
Number of Promoters
Enrichment over Random
Number of Promoters
Enrichment over Random
Number of Promoters
Enrichment over Random
Comparisons were also performed to between HaloCHIP-chip and previously published CREB ChIP-chip data (Table 2) . As the CREB ChIP-chip DNA microarray covered approximately 8,000 fewer number of promoters, the Top 1% and Top 5% of the list correspond to 182 and 898 promoters , respectively, which were then used for the comparison (Table 2). A slightly lower overlap of 26% and 23.8%, respectively is observed, corresponding to a 5-fold over-representation for both categories compared to what would be predicted by chance (Table 2). This is not surprising given the differences between these experiments including; the cell lines used, method of amplification for the array, as well as the different array design and platform . Nevertheless, given these differences it was very encouraging to see a significant overlap between these independent results.
Gene Ontology analysis of CREB HaloCHIP-chip promoters.
Number of Promoters
12 of 65
20 of 261
26 of 392
26 of 395
38 of 638
Nucleic acid binding
110 of 2764
High resolution mapping of CREB binding sites relative to endogenous transcription start sites
Match of full and half CRE consensus sites in CREB HaloCHIP-chip data.
% with Full CRE site
% with Half CRE Site
CREB high-throughput reporter assay analysis
Percentage of promoters activated in various conditions in functional macroarrays.
Total CREB Set Tested (235)
HaloCHIP Subset (84)
TORC1 + FSK
Given the interesting CREB binding patterns at bidirectional promoters (Figure 4b), we looked specifically at the promoter activities of bidirectional promoters in our reporter assay dataset. There were a total of 7 bidirectional gene pairs for which we collected promoter activity data for each direction. The majority of the pairs showed very low activity in both directions in the untreated cells suggesting that CREB-bound bidirectional promoters are not transcriptionally active in an un-induced state. Two of the 7 bidirectional gene pairs, which regulate two pairs of histone genes, had constitutively high promoter activities in both directions, irrespective of stimulation conditions.
We applied here two technologies, HaloCHIP and high-throughput promoter assays, to study and more fully characterize the CREB transcription pathway than previously done. The HaloCHIP method (Figure 1) offers an alternative approach for the capture of intracellular DNA-protein complexes and was developed to address the challenges of antibodies required for the existing ChIP method. The use of a robust protein tag eliminates the need for a qualified antibody and enables researchers to study highly similar paralogs, different isoforms, or point mutants of a transcription factor that may not be distinguishable by an antibody. Also, due to rapid and covalent binding kinetics HaloTag with its ligands, protein complexes can be captured efficiently from dilute solutions without concern of loss due to diffusion off the resin, allowing for the use of a much smaller number of cells (2-4 × 105) per HaloCHIP experiment as compared to the standard ChIP experiment (~1 × 107) [4, 10]. As with the use of any protein fusion tag for ChIP or HaloCHIP experiments there are concerns as to potential alteration of DNA binding due to interference by the fusion tag or changes in expression level. The CREB HaloCHIP-chip results show that binding to DNA on a genomic-scale was specific for the CREB protein and had a significant degree of overlap with conventional CREB ChIP-chip data, suggesting the HaloTag-CREB fusion protein is binding to DNA similarly to the endogenous CREB protein.
In addition to identification of new CREB-bound promoters with these array studies, we extended our studies of the CREB pathway by measuring the functional activity of over 200 CREB-target promoters [26, 27, 29] in a high-throughput reporter assay experiment (Figure 6a). Many diverse responses are regulated through the CREB pathway and unique subsets of CREB-bound genes may be transcriptionally activated and responsive to particular stimuli. The high-throughput reporter assays of CREB-bound promoters gives the ability to stratify CREB binding events based on the transcriptional activity of the fragments of DNA to which they bind. Analysis of the CREB pathway using the functional promoter macroarrays revealed only a small percentage of promoters were responsive to FSK, correlating well with the HaloCHIP-chip data showing minimal changes in binding between untreated and FSK treated cells. Interestingly, a much larger percentage of promoters were responsive to PMA, and an even greater percentage to the TORC proteins. These functional results provide further support for the idea that co-factors are a crucial part of the CREB signaling pathway and while reporter assays lack the full chromatin context of the genome, by using extended promoters regions that are 1 kb in length, we were able to observe transcriptional effects by co-factors, which may interact with proximal sites. In order to analyze CREB-enriched sites at a much higher resolution than was previously performed, custom oligo microarrays were used. This detailed analysis provided interesting and novel insight into the localization of CREB at the promoters of genes. A simple assumption is that the experimental enrichment for CREB binding would be coincident with the location of the CRE. Indeed this was observed for the majority of unidirectional promoters (Figure 4a). However, a distinctly different pattern is observed for a large fraction of bidirectional promoters, where the peaks of highest enrichment are seen downstream of the closest TSS, often not coinciding with the location of CREs (Figure 4b). This pattern was seen consistently for many bidirectional promoters, in both HaloCHIP-chip and ChIP-chip data sets, indicating this is not a phenomenon associated with a particular method.
These particular binding results suggest a number of interesting scenarios for which additional experiments will be needed. It may be the case that there is a secondary structure of the CREB-DNA complex at bidirectional promoters where the peaks of enrichment reflect the higher order crosslinked structure rather than the true localization of the CREB protein on the linear strand of genomic DNA. An alternative explanation is that CREB may be a part of a paused transcription initiation complex. In this scenario, CREB could initially bind upstream of the TSS in the bidirectional promoters, form its known interactions with the RNA PolII complex, move after initiation with the complex, and then pause at particular sites downstream of the TSS. Recent work has shown that a paused transcriptional complex containing transcriptional regulators are more abundant than previously thought [54–56], and this explanation would produce the enrichment pattern that we observe for CREB at bidirectional promoters (Figure 4b, 6b).
Results from the promoter reporter assays for bidirectional promoters are also consistent with this scenario, since a paused transcriptional machinery would likely result in lower reporter activity as was seen for the majority of bidirectional promoters tested. Perhaps most interesting is the functional behaviour of a subset of the bidirectional promoters in the presence of TORC1. In 2 out of 7 cases tested, the activity of a bidirectional promoter was strongly induced in one direction and strongly repressed in the opposite direction in the presence of TORC1. The strongly repressed promoters show CREB binding which is downstream the TSS, while the strongly induced promoters show expected upstream promoter binding. Also, the ability of the TORC1 protein to differentially regulate promoter activity, suggests that CREB co-factors may also help to regulate the directionality of transcription. This is particularly relevant for the CREB transcription factor, since over 50% of CREB binding sites are located in bidirectional promoters as we have reported for the first time.
This broad survey of the transcriptional activity of CREB-bound promoters provides a valuable layer of functional data for the CREB protein. Future efforts to compare the activity of these promoters in many more conditions will help to further understand CREB signaling and mutational analysis of the bidirectional class of CREB-bound promoters will help to dissect the mechanism of bidirectional gene regulation. The use of the new technologies presented here however is not limited to the study of the CREB pathway, rather can be generalized to study any transcriptional pathway. The HaloCHIP method, like the standard ChIP process, can be used to study DNA binding both on a small scale, as well as genome-wide scale, however follow up studies characterizing the functional consequences of these binding events have lagged much further behind. By expanding the use of high-throughput reporter assays, we hope to advance our understanding of these functional consequences. This comprehensive comparison reveals the challenges and potential pitfalls of extrapolating binding events to transcriptional activation and shows the need for both approaches, as well as other experiments to truly characterize transcriptional activity.
Cloning, cell lines, and transfections of HaloTag vectors
Full-length human CREB1-α and -Δ cDNAs were obtained from OriGene, [NCBI:NM_134442.2 and NCBI:NM_004379.2], respectively. All CREB variants were subcloned into the pFN21A HaloTag CMV Flexi Vector (Promega) using SgfI and Pme, generating N-terminal HaloTag fusion constructs for each. HeLa cells (ATCC #CCL-2) were maintained in DMEM supplemented with 10%FBS at 37°C in an atmosphere of 5% CO2. Cells were transfected using Lipofectamine LTX transfection reagent (Invitrogen) according manufacturer's protocols.
HaloCHIP Protocol and Whole Genome Amplification
A detailed version of the HaloCHIP protocol can be found at: http://www.promega.com/tbs/tm075/tm075.html
For these experiments, HeLa cells (2-4 × 105) were plated in a single well of a standard 6-well plate. After reaching 70-80% confluency, typically 18-24 hours later, cells were transfected with the HaloTag-CREB fusion constructs (experimental sample) or left untransfected (control sample). Twenty four hours post-transfection, cells were crosslinked with formaldehyde (Sigma) at a final concentration of 0.75% for 10 minutes at 22°, quenched with 0.125 M glycine for 10 min. and processed using the HaloCHIP kit (Promega). For experiments involving Forskolin, cells were treated with 10 μM Forskolin for 45 minutes at 37°C prior to crosslinking. Isolated DNA was further purified using a PCR Clean-up kit (Qiagen), and eluted 2 × 50 μl with nuclease-free water, yielding a final volume of 100 μl. To prepare sufficient HaloCHIP DNA for downstream amplification steps required for microarrays, an entire 6-well plate was transfected and processed through the HaloCHIP method as recommended. The isolated DNA was pooled before final purification on the PCR clean-up columns and lyophilized to a final volume of 12 μl. The concentrated HaloCHIP DNA was then amplified to 2-10 μg using the Whole Genome Amplification kit (Sigma) following the recommended adaptation for ChIP samples .
HeLa cells (4 × 106) were plated in several 150 mm plates and grown at 37°C to 80-90% confluency. Cells were treated with 10 uM forskolin for 45 minutes at 37°. crosslinked with formaldehyde (Sigma) at a final concentration of 1.0% for 10 minutes at 22°, quenched with 0.125 M glycine for 10 min. and processed using the ChIP Assay Kit (USB). Chromatin was sheared by sonication using a Misonix MicroTip Probe 418, output of 5.5, with a program of 15 cycles of 5 seconds on and 25 seconds off on ice. Co-immunoprecipitation was performed using 1 μg of anti-CREB1 antibody (Millipore #06-863) for the experimental sample and 1 μg of anti-IgG antibody (Sigma) for the control sample with incubation at 4°C for 15 hours. Isolated DNA was further purified using a PCR Clean-up kit (Qiagen), processed, and amplified using WGA identical as the HaloCHIP samples.
Quantitative PCR and primers
HaloCHIP DNA was analyzed using either Plexor (Promega) or SYBR green (Applied Biosystems) qPCR according to their respective manufacturer's recommendations. Plexor primers were supplied from Biosearch Technologies and SYBR green primers were from IDT DNA. The following sequences were used for amplification: Fos forward 5'-GTCTTGGCTTCTCAGATGCTCG-3', reverse 5'-GTTGAGCCCGTGACGTTTACA-3', Jun forward 5'-GAGAAAGAAGGGCCCGACTGT-3', reverse 5'-GGAGACTCCACCCTAGAAGATTCT-3', p27 forward 5'-GGGAGGCTGACGAAGAAGAAAAT-3', reverse 5'-CAACCAATGGATCTCCTCCTCTG-3', C1 forward 5'-CTGGTCTCACCTACCTTCCTGT-3', reverse 5'-ATCCATGAACTCCAGGAGCTCA-3' C2 forward 5'-TCTGTTGCCTATTGACCAGAACATG-3', reverse 5'-AGGAGCTGTAGGCTGAGTCAC-3', C3 forward 5'-CTGCTTCTTAACAGCTTAATTCGGAAGA-3', reverse 5'-ATGAGCAAAGATAGCTCAGGGAG-3'. Primers sequences used for PPV and NPV qPCR validation along with their corresponding amplified promoter can be found in supplemental materials: http://www.switchgeargenomics.com/creb_supp_data/
Oligo array design and analysis
A custom oligo array was designed to cover a genome-wide set of human promoter regions predicted by SwitchGear Genomics (more detail can be found at http://www.switchgeargenomics.com). The oligo array composed of approximately ~385,000 50mer probes was manufactured by Roche-NimbleGen Systems. The amplified enriched samples described above were shipped to Roche-NimbleGen to be hybridized according to their standard service protocol. The raw data from the arrays were analyzed as follows; the log2 ratio (enriched-cy5/total input-cy3) was calculated for each probe and data were then smoothed by averaging across a sliding window of 3 neighbouring probes shifting 1 probe at a time, minimizing noise from single probes. The median and standard deviation were calculated from the smoothed ratios for each sample. The median was subtracted from each ratio and divided by the standard deviation to center and normalize the data from each array. To summarize the enrichment for an entire promoter, the top 4 probe values were averaged for a given promoter region to approximate the 75th percentile value. The raw data, normalized data, and collapsed data for each array are available as supplemental at the following site: http://www.switchgeargenomics.com/creb_supp_data/. All microarray probes and data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE18347 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18347.
Gene Ontology (GO) analysis
The Top 1% of the CREB HaloCHIP-chip promoters, 277 in total, were deposited and analyzed using Gene Ontology (GO) http://www.geneontology.org/ and AmiGO as the search engine. GO categorized each promoter with respect to protein function, showed the number of promoters within each category, and reported a corresponding p-value based upon the calculations.
High-throughput reporter assays and analysis
The promoter reporter assays used 235 promoter-reporter vectors (utilizing the luc2P reporter cassette from Promega) containing ~1 kb promoter fragments from known CREB-bound genes. These cloned promoters were selected from SwitchGear Genomic's genome-wide promoter clone collection (details on this panel of reporter constructs can be found at http://www.switchdb.com/pathways/id_46/). A panel of promoter controls was also used to normalize signals between plates and replicates. The 32 plate normalization controls, include ~1 kb fragments representing constitutively active human promoter fragments and random regions from the genome. The promoter reporter assay experiments were all conducted in 384-well format. A detailed protocol can be found at: http://www.switchgeargenomics.com/creb_supp_data/. Transfection complexes were formed by incubating 50 ng of each individual promoter construct with 0.3 μL of Fugene 6 transfection reagent and Opti-MEM media in a total volume of 3 μL and incubated for 30 minutes. The co-transfection of the TORC1 expression construct was set up the same as the standard transfection reaction, but with the inclusion of 25 ng of TORC1 expression plasmid per reaction (TORC1 expression construct was provided by the Montminy lab). Transfection complexes were mixed with resuspended HeLa cells such that 4,000 HeLa cells were seeded in a volume of 50 μL in each well of a 384-well white tissue culture treated plate. Fifteen replicate wells of each promoter construct were performed representing triplicate assays in 5 different conditions: 1) no treatment, 2) PMA, 3) Forskolin, 4) TORC1, and 5) TORC1 + FSK.
After seeding and transfection, cells were incubated for 24 hours before inductions. Inductions were conducted for each plate by removing the old media and replacing with new media depending on the condition. For the untreated cells, fresh media was applied to each well. For the PMA induction, fresh media with 100 nM PMA was added to each well. For the forskolin induction, fresh media with 20 μM FSK was added to each well. Cells were kept in their respective induction condition for 4 hours and then frozen overnight at -80 degrees.
To read luminescent activity plates were thawed for 45 minutes at room temperature. Then 50 μL of Steady-Glo reagent (Promega #E2520) was added and incubated for 30 minutes at room temperature. Then luminescence was read for 2 seconds per well on a 384-well compatible plate luminometer (Molecular Devices LMax384).
The raw luminescent reads from each well were normalized as follows. Each 384 well plate contained 32 control wells that were comprised of 16 positive control promoters and 16 random genomic fragments that serve as background signal controls. These plate controls were used to normalize the per well values between plates within a condition. The average of the 3 replicates was taken, and the ratio of induced/untreated was calculated from the averages of the treated values and the untreated sample. A t-test for significance was also calculated between the 3 replicates of the induced and untreated samples. The background controls were also used to measure whether the average absolute signals were above background in each condition (>3 standard deviations from the mean of the negative controls). For a given promoter to be called induced or repressed it must pass the following criteria: 1) At least a 2-fold change (+/-) 2) Pass t-test with significance of p < 0.05 3) Must have absolute signals significantly above background.
We are grateful to Marc Montminy and Pankaj Singh for generously sharing their TORC1 expression construct along with providing helpful comments on our results. We are also grateful to Martin Rosenberg, Michael Slater, Luciano Di Croce, Patrick Collins, and Mike Rose for providing helpful discussion and experimental support for the project. DDH, JM, NM, KW, and MU are all funded by Promega Corporation, and NDT and SFA are funded by SwitchGear Genomics. Both Promega Corporation and SwitchGear Genomics contributed funding to this research and the preparation of the manuscript.
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