Center for Integrated Animal Genomics

1. NF-B Binding to DNA Potential Gene Consensus p65 p50 Figure 1. Binding of transcription factor to binding site helps regulates genetic expression Binding Induces Gene Expression • Research Experience in Molecular Biotechnology & Genomics • Summer 2007 Center for Integrated Animal Genomics Confirmation of Bioinformatic Prediction of NF-B Binding to Promoters of Differentially Expressed Genes During the Porcine Immune Response to Salmonella Nowlan Freeseab, Oliver Coutureb, Yanfang Wangb, Chris Tuggleb a Department of Biology, Saint Olaf College, Northfield, MN 55057, USA b Department of Animal Science and Center for Integrated Animal Genomics Iowa State University, Ames, IA 50010, USA • Introduction • Salmonella costs pig producers about $100,000,000/year • 1,400,000 cases of non-typhoid Salmonella infections/year in humans and 600 associated deaths (Wang et al. 2007) • Potential food safety issues by way of meat contamination • Response to Salmonella serovars induces inflammation • NF-B is a transcription factor important for inflammatory response • Transcription factors regulate gene expression in response to changes in the environment by binding DNA • We examined two genes which were unknown to bind with NF-B but were predicted to be part of the inflammatory response in previous work (Wang et al. 2007) • Better innate immunity in livestock species could potentially reduce the number of infections, the time of infection, and the use of antibiotics, and the cross contamination of livestock to humans EMSA Gels Figure 3. NF-B p65 antibody recognizes mouse nuclear extract proteins binding porcine sequences. Arrowheads indicate Ab-protein complex formation. Blue rectangles show loss of specific bands due to Ab binding. Figure 4. Mouse nuclear extract testing the gene probes for AIF1, UBD and TAP1 with their competitors. Blue ovals indicate loss of band due to specific competition, but no loss due to non-specific competition. Figure 2. TRANSFAC matrices for NF-B p65 and p50 are two subunits of NF-B • Materials & Methods • Prediction of NF-B Binding Sites • TFM-Explorer and TRANSFAC were used to predict binding sites on the differentially expressed genes found by Wang et al. (2007, in press) • Gene Selection • Needed porcine flanking genomic sequence (Obtained from VEGA at Sanger) • NF-B Binding Site Probe Selection • PATCH software was used to rate strength of binding sites, requiring minimum of 87.5 (Biobase) • Similar upstream location as human and/or mouse binding site • See Table 1 below for probe sequences • Selection of Cell Types • Mouse M have been shown to respond to LPS in a similar manner as S.typhimurium (Rosenberger et al. 2000) • NF-B has been shown to be activated due to LPS treated mouse M cells • Mouse models are regularly used; in Wang et al., response was shown to include M responding genes in pigs, i.e. IL-6 and IL-10 • HeLa cells have known NF-B activity • Cultured porcine cells are not yet available • Wet Lab • Mouse M cells gathered and treated with LPS by Birt lab • Standard nuclear extraction performed (Ajuwon et al. 2004) • HeLa nuclear extract ordered from Promega lot #22958701 • Cy5 fluorescent labeled probes and competitors ordered from Integrated DNA Technologies • EMSA gels made under Promega technical bulletin TB110 (50mL instead of 20mL final volume) • EMSA gels scanned by Typhoon 8600 and 9410 scanners Legend A - AIF1 U - UBD T - TAP1 S.C. - Specific Competitor N.S.C. - Nonspecific Competitor Ab - p65 Polyclonal Antibody Figure 5. Hela nuclear extract testing the gene probes AIF1, TAP1 and UBD with their competitors. Blue ovals indicate loss of band due to specific competition , but no loss due to non-specific competition. Red ovals indicate loss of specific band due to complex formation and supershift. • Summary of Results • TAP1 and UBD were both found to be strongly bound by proteins in mouse extract, while AIF1 is weakly bound • p65 antibody shows specific NF-B complexes are formed on all three probes using mouse extract • HeLa nuclear extracts showed weak binding activity with low evidence of specificity • Discussion and Conclusion • The porcine sequence of TAP1, a known direct target in human and mouse, was shown to bind and makes specific p65 complex with the human and mouse proteins • Likely direct target in the pig as well • AIF1 PATCH score for its binding site (88.8) is lower in comparison with the other probes (100) • Weaker binding of the AIF1 agrees with the lower PATCH score • Non specific bands are most likely due to other transcription factors binding to the sequences • For example the UBD probe contains a CEBP and a MyoD binding site • The UBD promoter has been predicted to contain NF-B binding sites (Canaan et al 2006), which have been verified by the formation of specific complex on the probe • Further testing with porcine extract is needed Acknowledgements Carlie LaLone and Dianne Birt for the mouse M and the LPS treatment Max Rothschild for coordination the summer REU program References Ajuwon, K.M., Jacobi, S.K., Kuske, J.L., and Spurlock, M.E. 2004. Interleukin-6 and interleukin-15 are selectively regulated by lipopolysacchride and interferon- in primay pig adipocytes. Am J Physiol Regul Intergr Comp Physiol. 286:547-553. Canaan A., Yu X., Booth C.J., Lian J., Lazar I., Gamfi S.L., Castille K., Kohya N., Nakayama Y., Liu Y.C., Eynon E., Flavell R., and S.M. Weissman. 2006. FAT10/diubiquitin-like protein-deficient mice exhibit minimal phenotypic differences. Molecular and Cellular Biology. 26;13:5180-5189 Rosenberger C.M., Scott G.M., Gold M.R., Hancock R.E.W. and B.B. Finlay. 2000. Salmonella typhimurium infection and lipopolysaccharide stimulation induce similar changes in macrophage gene expression. The Journal of Immunology. 164: 5894-5904 Wang Y., Qu, L., Uthe, J.J., Bearson, S.M.D., Khuar, D., Lunney, J.K., Couture, O.P., Nettleton, D., Dekkers, J.C.M., and Tuggle, C.K. 2007. Global transcriptional response of porcine mesenteric lymph nodes to Salmonella enterica serovar Typhimurium, Genomics. doi:10.1016/j.ygeno.2007.03.018. NF-B Binding Domain Table 1. Probe design for individual genes based on porcine genomic DNA. GC mutant was created at second conserved G to make all non-specific competitors. Yellow indicates 100% conserved nucleotides across these sites. Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371