Norman H. Lee Research Interests [email protected] June 15, 2007 1. Cancer Genomics – Reconstructing Gene Networks (NCI funded) ¾ Tools: Gene expression profiling, chromatin immunoprecipitation (ChIP), loss-of- function screening using RNA interference, Bayesian statistics, site-directed mutagenesis ¾ Collaborators: H Lee Moffitt Cancer Center, NCI, NIDCR, Duke University, MD Anderson Cancer Center, UC Santa Cruz, Venter Institute, GWU 2. Cardiovascular Physiogenomics – Linking Genes to Disease (NHLBI funded) ¾ Tools: Gene expression profiling, congenic and chromosome substituted rat strains, quantitative trait locus analysis ¾ Collaborators: Medical College of Wisconsin, University of Toledo 3. Behavioral Genomics – Linking Genes to Behavior & Drugs of Abuse (NIDA) ¾ Tools: Gene expression profiling, behavior genetics in inbred mouse strains, RNAi knock-downs via lenti virus, quantitative trait locus analysis ¾ Collaborators: University of Maryland, Tel Aviv University, GWU 1 Gene Expression Fingerprint in Human Colon Cancers transcription factor/RNA processing propionyl-CoA carboxylase unknown function no database match ubiquitin/proteasome pathway IGFBP 3 hemoglobin alpha chaperonins/heat shock proteins bumetanide-sensitive Na-K-Cl signal transduction nucleosome assembly protein Normal transversum cell growth and proliferation nucleosome assembly protein tumor protein D52 cytoskeletal organization/trafficking transcription factor Sp3 lysosomal proteins no database match cytochrome b5 cytochrome b5 Adenoma Duke’s B2 Duke’s C2 Duke’s D Metastasis M1 Src531 M3 Src531 V-Src A V-Src B M14 M18 c-Src 8 c-Src 9 c-Src 12 M3 + Inhibitor RACK7 heat shock protien 40 RACK7 proteasome R-DELTA subunit IGFBP 2 chemokine CX3C proteasome RCI subunit cytosolic acetoacetyl-coenzyme A thiolase lysyl hydroxylase cytosolic acetoacetyl-coenzyme A thiolase p190-B, rho GAP family Duke’s A cyclin G metallothionein-I gene transcription activa vesicle-associated membrane protein 7 no database match 5 yr survival 85-90% YY1-associated factor 2 H. sapiens hypothetical protein KIAA0462 neuronal cell death related gene in neuron-7 proteasome C8 subunit beta-1,4-galactosyltransferase H. sapiens hypothetical protein KIAA0011 glucose-6-phosphate isomerase; neuroleukin proteasome activator PA28 beta subunit p59 immunophilin thymidine kinase MAP kinase kinase dystroglycan 1 splicing factor, arginine/serine (SC-35) microfibril-associated glycoprotein APP-binding protein 1 no database match pyrroline 5-carboxylate reductase 1 Myc-associated zinc-finger protein snRNP core Sm protein homolog Sm-X5 translational activator GCN1 (KIAA0219) zinc finger transcription factor homolog CPG20 ubiquintin c-terminal hydrolase related tousled-like kinase (KIAA0137) dynein heavy chain Duke’s D H. sapiens hypothetical protein KIAA0459 RAC protein kinase alpha Munc18-1 mannose 6-P/IGFR II 5 yr survival <5% myosin-IXb splice variant endolyn myosin-IXb splice variant endolyn no database match ubiquitin conjugating enzyme 12 glycogen phosphorylase P311 ganglioside m2 activator protein mRNA cleavage factor I, 25 kDa subunit LIM and SH3 domain protein-1 (Lasp-1) catecholamine-O-methyltransferase arylsulfatase A cyclin D1 H. sapiens hypothetical KIAA1125 cyclin D1 HGF receptor substrate Hrs cyclin D1 thymidine kinase E2I ubiquitin conjugating enzyme, Ube2i >4 1:1 >4 heat shock protein 90 Metastasis annexin V bifunctional purine biosynthesis protein p Malek et al., Oncogene 2002 E2 ubiquitin conjugating enzyme, Ubc13 Teramoto et al., Oncogene 2003 E2I ubiquitin conjugating enzyme, Ube2i heat shock protein 90 Teramoto et al., Oncogene 2005 hnRNP-E1 protein 2 Empirical Approach to Define Bayesian Strategy for Cancer Invasion Network Cancer Invasion Network SRC Single Input Motif 1st tier genes Feed Forward Loops CCR9 ? ? Multi Input Motif ADAM21 CD53 ? 2nd tier genes DEAD Regulatory Chain ? ? GLUT TFDP ? ? ? KER RPL32 ? Irby et al., Cancer Res 2005 Lee, Pharmacogenomics 2005 Goutias and Lee, Current Pharm Des 2007 3 Cancer Genomics: Mutations in Sodium Channels SCN11A (R Æ Q) P segments SCN10A SCN10A SCN1A (T Æ A) (T Æ S) (A Æ P) SCN7A SCN7A (R Æ G) (R Æ H) 4 Experimental and Computational Approaches to Associate Genes with Phenotypes (g) Lentivirus delivery of shRNA (a) Animal model phenotyping – drug addiction (f) Congenic, consomic, KO animals anxiety cardiovascular (e) Candidate genes (c) Bioinformatics (b) DNA microarrays Malek et al., Nature Genetics 2006 (d) QTL linkages Letwin et al., J Neurosci 2006 Reiner et al., Bioinformatics 2007 Lee, Exp Physiol 2007 5 Searching for Genes Involved in Anxiety • Time in open quadrants of an elevated zero-maze is a Resistance commonly used measure of anxiety state • Computational analysis identified 14 positively and 21 negatively correlated genes in the periaqueductal gray • A number of these genes map to anxiety-associated QTLs (Rims3, Cdk2, AW554615, AI853937) • Egr-1 (Axtrb4 QTL) ↑ fear conditioning; the anxiolytic drug Diazepam blocks this effect Susceptibility 6 RNAi Mediated Gene Knockdown & Anxiety Anxiogenic gene knockdown: Exploratory behavior Scrambled control: Resistance genes Resistance Susceptibility genes Susceptibility 7.