Centers & Programs

Functional Genomics Research Program

Dr. Russell Thomas, Ph.D., Director

An important question to ask when studying gene transcript perturbations associated with chemical exposures is the cellular context in which these processes occur. Specifically, we want to know about the signaling networks and pathways that are involved in regulating the biological responses to specific compounds. The Functional Genomics Research Program at The Hamner Institutes for Health Sciences is applying a series of bioinformatics and large-scale genomics tools to investigate these processes at the molecular level and build comprehensive signaling networks that are involved in normal cellular signaling and stress-related responses. The tools for this type of research include a combination of gene expression analysis using genome-wide microarrays and large-scale, loss-of-function and gain-of-function studies using inhibitory RNA libraries and libraries of full-length genes, respectively.

For those unfamiliar with inhibitory RNAs, RNA interference (RNAi) was originally discovered in plants when two groups reported cosuppression through an overexpression of chalcone synthase (van der Krol et al., 1990; Jorgensen et al., 1996). Simply put, the researchers would sometimes create more white petunias when they were trying to create more purple ones. Further studies in the nematode Caenorhabditis elegans (Fire et al., 1998) expanded on these observations and showed that specific genes could be silenced through the addition of double-stranded RNA (dsRNA). Mechanistic studies have revealed that an enzyme in the RNAse III family called Dicer cleaves the long dsRNA into small interfering RNAs (siRNAs) of 21 to 25 nucleotides. The resulting siRNAs are guided to the complementary mRNA by the RNA-induced silencing complex (RISC), and the specific mRNA is subsequently degraded (for a review on the subject, see Hannon, 2002). The resulting technology provides a powerful tool for biological researchers by allowing a gene to be virtually knocked out inside the cell through the introduction of synthetic or plasmid-derived siRNAs. This process is known as reverse genetics and is similar to assessing the function of small numbers of genes through the production of knock-out mice.

In contrast to loss-of-function studies enabled by the siRNAs, gain-of-function studies are facilitated through more traditional means. Full-length genes are cloned into plasmids that contain viral promoters and transfected into cells. The expression of the gene is enhanced compared to its normal expression and allows an assessment for the role of the gene when overproduced. This process is similar to evaluating the function of a gene in vivo through the production of certain types of transgenic mice.

As part of the functional genomics initiative, The Hamner has purchased a set of approximately 12,000 full-length genes and is working to acquire a large collection of inhibitory RNAs. Together with the gene expression capabilities, results obtained with these tools can provide a comprehensive picture of the signaling network inside the cell. For example, cell-based screens can be constructed where luciferase or fluorescent protein reporters are introduced inside the cell and a luminescent or fluorescent signal is measured when a particular pathway is activated. In the gain-of-function studies, robotic systems individually screen thousands of full-length genes to identify which genes, when overexpressed, alter the signaling of the pathway of interest and activate or suppress the luminescent or fluorescent reporter (Michiels et al., 2002) (Figure 1). Similarly, in loss-of-function studies, the pathway is stimulated using a ligand or constitutively active mutant, and individual inhibitory RNAs are screened to identify which genes, when knocked out inside the cell, also alter the signaling of the pathway of interest (Lum et al., 2003) (Figure 1). Using the results from both screens, the data are overlaid and filtered using a set of bioinformatics tools, and a likely cellular pathway map is constructed and refined with what is known in the literature. After reconstructing the signaling pathway of interest, the downstream targets of the pathway are identified using a unique combination of microarray analysis coupled with specific gain-of-function and loss-of-function tools.

The Functional Genomics Research Program represents a significant commitment by The Hamner to a systems biology paradigm. Initially, the program will address research questions associated with existing program projects on respiratory tract responses to irritant gases and developmental responses of compounds that interfere with the male hormone testosterone during fetal development. Longer-term research goals will apply these technologies to the investigation of human susceptibility involved in gene-environment interactions and transcriptional regulation of sets of genes in liver by endocrine-active compounds.

References

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.

Hannon, G. J. (2002). RNA interference. Nature 418, 244-251.

Jorgensen, R. A., Cluster, P. D., English, J., Que, Q., and Napoli, C. A. (1996). Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol. Biol. 31, 957-973.

Lum, L., Yao, S., Mozer, B., Rovescalli, A., Von Kessler, D., Nirenberg, M., and Beachy, P. A. (2003). Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039-2045.

Michiels, F., van Es, H., van Rompaey, L., Merchiers, P., Francken, B., Pittois, K., van der Schueren, J., Brys, R., Vandersmissen, J., Beirinckx, F., Herman, S., Dokic, K., Klaassen, H., Narinx, E., Hagers, A., Laenen, W., Piest, I., Pavliska, H., Rombout, Y., Langemeijer, E., Ma, L., Schipper, C., De Raeymaeker, M., Schweicher, S., Jans, M., van Beeck, K., Tsang, I.- R., van de Stolpe, O., and Tomme, P. (2002). Arrayed adenoviral expression libraries for functional screening. Nat. Biotechnol. 20, 1154-1157.

van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N., and Stuitje, A. R. (1990). Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2, 291-299.