February 1, 2006 (Vol. 26, No. 3)

Using Transcription Factor Probes to Discover Unknown Signal Pathways

Marligen Biosciences (www. marligen.com) has developed assays that allow researchers to study the activation of up to 30 transcription factors (TFs) in a single sample. Profiling the activation of multiple TFs in response to different stimuli aids in the analysis of numerous signal transduction pathways and leads to the discovery of previously unknown mechanisms controlling global cellular processes. In addition, it is expected to improve the drug discovery and development process.

Figure 1: Protocol for Marligen’s multiplex transcription factor assay

Controlling Cellular Processes

Biological differences among various cell and tissue types are largely the result of differences in gene expression. Regulation of gene expression is effected through TFs. By binding to specific DNA sequences in gene promoter regions, TFs can control which genes are turned on or off in each cell, thereby modulating protein expression.

Most TFs are present in the cell in an inactive state. When cells receive an external stimulus in the form of small molecule ligands, such as hormones, peptides, vitamins, or fatty acids, these signals trigger the activation of defined sets of TFs, which then modulate the expression of target genes in response to the stimulus.

The process is both staggeringly complex and elegantly simple in design, using less than 2,000 proteins to mediate an infinite variety of finely controlled processes within cells.

Current Methods

The traditional methods for measuring TFs today are gel shift assays, reporter gene assays, and immunoassays. All of these approaches are limited to the analysis of a single TF in each sample and therefore provide limited information. In addition, they are labor-intensive and time-consuming, making them unsuitable for the high-throughput, parallel processing approaches that have been so powerful in genomics and proteomics research.

Multiplexed Transcription Factor Probes

Each TF probe contains a double-stranded protein-binding region to which a biotin label is attached. Multiple TF probes, each with a different protein-binding sequence, are mixed with a nuclear extract, and the TFs are allowed to bind to the DNA. Once binding has occurred, a proprietary reagent is added that removes the biotin from the binding sites that have not been protected by bound TFs from the nuclear extract. A summary of the protocol is shown in Figure 1.

Next, the probes are hybridized to a set of spectrally distinguishable microspheres containing covalently attached single-stranded DNA oligonucleotides that are complementary to corresponding capture sequences included in each TF probe. Biotin molecules remaining on probes containing bound, activated TF complexes are detected with a streptavidin-phycoerythrin conjugate, and the signals are measured in a Luminex 100 instrument (www.luminexcorp.com).

Anna Lokshin, Ph.D., director of the Luminex Core Facility at the University of Pittsburgh, has been using Marligens multiplex TF assay for some time. One of Dr. Lokshins ongoing projects has been a study of the mechanism and pathways involved in the stimulation of dendritic cells, which are involved in the presentation of antigens to nave T cells in the primary immune response.

The importance of antigen presentation in the generation of specific immune response has been confirmed in vivo by the demonstration that blocking antigen presentation down-regulates both humoral and cell-mediated immune responses, including antitumor immunity. Dendritic cell function is regulated by maturation state, and appropriate differentiation and maturation is a crucial factor for a fully functional immune system.

In Dr. Lokshins studies, human dendritic cells were generated from CD14+ PBMC in cultures supplemented with GM-CSF and IL-4 for 7 days. To induce activation of NF-kB, dendritic cells were treated with TNF-a (50 ng/mL) for 15 and 30 minutes. Non-treated dendritic cells served as a control.

Nuclear extracts were prepared and tested with Marligens 10-plex TF profiling assay. The data shown (see Figure 2), provides a dynamic picture of TF activation in TNF-a-treated human dendritic cells. A peak of NF-kB activity was observed at 15 minutes, in agreement with previously published data. More importantly, however, a strong time-dependent activation of EGR, NF1, and PPAR was also observed in TNF-a-treated dendritic cells. The involvement of these TFs in dendritic cells activation by TNF-a has not been previously described.

These findings allow the exploration of the role of these TFs in dendritic cell maturation, activation and function, demonstrating the utility of multiplexed TF screening assays in biological research.

Figure 2: Simultaneous detection of multiple transcription factor activities in dendritic cells stimulated with TNF alpha

Broad-based Applications

A unique feature of this multiplexed assay is its ability to simultaneously evaluate and compare activation of different factors at exactly the same time and in the same sample. In addition, Marligens profiling assays can benefit researchers using gene expression microarrays, enabling them to pinpoint the biological processes that are causing the observed changes in gene expression patterns.

Correlating TF profiles with gene expression patterns facilitates the identification of molecular mechanisms by which cassettes of genes are co-regulated and provides a useful approach for making connections among cellular perturbations, gene expression patterns, and biological responses. Profiling TFs also offers a new tool for drug discovery. More than 15% of the currently approved drugs directly target TFs (e.g., Tamoxifen, Evista, Avandia), and many leading pharmaceutical companies including Roche and GlaxoSmithKline have initiated programs to discover new TF drug targets. Others, including Johnson & Johnson, Sangamo, and Ariad have research programs devoted to developing synthetic TFs for use as therapeutics.

In summary, multiplex TF products provide a rapid, comprehensive profile of the signals controlling gene expression and are powerful tools for discovering previously unknown signaling pathways.

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