Functional genomics — a new addition to Chinese Medicine Research & Development

1. Functional genomics —a new addition to Chinese Medicine Research & Development (CM2006, November 24, Hong Kong) Yuhui Hu Max-Planck-Institute for Molecular Genetics Berlin, Germany Dept. Vertebrate Genomics (Prof. Hans Lehrach)

2. Accomplishment in human genome sequencing ~ 3 billion nucleotides ~ contain 20–25,000 protein-coding genes Nature (2001) Science (2001) Nature (2004)

3. New focus in post-genomic era: Functional investigation of DNA sequences Genome Phenotype Environment

4. Genome …… Phenotype Environment DNA Transcript (mRNA) Protein Functional Genomics any approach that converts the information encoded in DNA sequences into an understanding of gene functions and effects.

5. Nano-well PCR & qPCR (TaqMan) 200nl reaction (Group Nyarsik) Gene expression patterns—mouse embryo High-throughput in situ hybridization~17,000 clones & probes (EUREXPRESS consortium) (Group Yaspo) Our functional genomic tools Genomics:DNA sequence; SNP; Duplications 1st , 2nd generation of DNA sequencers; DNA microarray Epigenomics:DNA methylation; Histone code and it’s impact on gene expression Chromatin immunoprecipitation + Genomic arrays (ChIP-Chip) Transcriptome: Gene expression profiling Proteomics: Expression— High-throughput cloning & expression strategy Structure of protein complex— LC-MALDI MS (Group Gobom) Cryo-electron microscopy (Group Mielke) Function— Protein localization; Protein-protein interaction Transfected cell microarray (Group Janitz) Mammalian & Yeast two hybridization in vitro ligand screening; Protein & Antibody array (Group Konthur) Protein expression & structure (Group Büssow)

6. Regulation networks: Genome-wide promoter analysis— High-throughput evaluation of promoter activity Transcription regulatory networks— Knockdown all human Transcription Factors (TF) Expression profiling Genome location of TF binding sites (ChIP-on-Chip) Bioinformatics: Systems biology modelling Signal transduction networks

7. …… Chinese medicine Genome Phenotype Environment DNA Transcript Protein Genetics of human diseases (neurodegenerative & cardiovascular diseases; stroke; trisomy 21; etc) Functional genomics tools

8. …… Genome Phenotype Environment DNA Transcript Protein Therapeutic mechanism Chinese medicine Diseases Functional genomics tools

9. New drugs • Organism level • (Mouse, Rat, etc.) Screening at Effective compounds • Cell level DNA Transcript Protein (Transfected Cell Microarray) Therapeutic mechanism Chinese medicine Diseases Functional genomics tools

10. Principle of Cell Microarray pEXP26-C21orf Manual or robot Spotting Reverse transfection Hek293 / HeLa Expression vectors siRNA or shRNA vectors High-throughput Functional Detections Ziauddin and Sabatini, Nature, 2001 Hu, Y.H., et al., Biochemical Society Transaction,2005

11. Apoptosis 100x fluorescence Localization BioCCD scanning green/red 1000x Confocal / fluorescence Applications of Cell Microarray Labelled cell array 25x fluorescence Hu, Y.H., et al., BMC Genomics, 2006

12. Protein co-localization with 9 organelles Localization Categories (MX1) Cytosol NOT co-ER (HLCS) Cytosol (PCBP3)Nucleus+Cytosol (TMPRSS3) ER, co-ER (HMGN1)Nucleoplasm (NNP1)Nucleolus (KCNJ6)PM (KCNE2)Lysosome The localization patterns provide the first hints to the functions. Red: Chr21ORF Blue: Nucleus (DAPI) Green: organelle stain

13. Intracellular trafficking on cell array 6xHis-Chr21 Compartments Merged gene: KCNE2 1. Golgi vesicles Golgi 2.Plasma membrane Mito 3. Plasma membrane & Lysosome/Endosome Lysosome

14. Nucleus (M phase) Nuc Cell cycle-related translocationon cell array 6xHis-Chr21 Compartments Merged gene: BACH1 Cytoplasm (interphase) Nuc BACH1: BTB and CNC homology 1 (transcription factor 1)

15. Cell deathdetection Green:CLDN-14Red: Annexin V Red:CLDN-14Green: TUNEL Alteration of cell morphology due to protein overexpression CLDN-14 ER + Nuclei Merged gene: CLDN-14 Protein localization: ER Cell retracted; Nuclear shape altered CLDN14: Claudin-14 (Component of tight junction (TJ) strands )

16. siLaminA/C Control siRNA Bax over-expression siCyclinB1 siSGTA EGFP frame Apoptosis due to gene overexpression & knockdown    Apoptosis staining: Annexin V-Alexa488 TUNEL-Alexa488 Cleaved-Caspase3 Ab-Alexa488 SGTA: small glutamine-rich tetratricopeptide repeat-containing, alpha

17. Subcellular localization siRNA-mediated gene silencing Cell phenotypes, e.g. apoptosis, proliferation Applications of Cell Microarray Cell Microrray High-throughput Functional Evaluation of cDNA, sh/siRNA High-throughput Evaluation of promotor activity Large-scale cDNA/siRNA/shRNA libraries Predicted promotor regions Protein-protein interactions Cell type-specific gene expression

18. Transfected Cell Microarray • robust • high-throughput expression & knockdown & detection For Chinese Medicine • Therapeutic compound screening: • Cell death inducer — potential anti-cancer drug • Inhibitor of disease-related protein translocation & interaction, • e.g. transcription factors (NF-B), cell surface receptors (e.g. -Amyloid) • Multiple targets of Chinese Medicine—complexity and holism

19. New drugs Screening  • Organ level Effective compounds (Organotypic slice culture) • Organism level • (Mouse, Rat, etc.) • in vivo, low-throughput at • Cell level (Cell microarray) in vitro, high-throughput Chinese medicine

20. Organotypic Brain Slice Culture — Hippocampus Brain Dissection P0-P9 hippocampus ~300 m Slicing roller-tube interface collagen Culture (for months)

21. Advantages of Brain Slice Culture • Organotypic architecture • 3-dimensional connectivity • neuron-glia interactions • tissue-specific transport, ion diffusion systems For drug development: • Easy manipulation of defined neurons and synapses • NO blood-brain barrier— better accessment • Higher throughput for screening

22. Application of Brain Slice Culture • Neural development, Neurogenesis • Brain damage models + OGD • stoke/ischemia • ––oxygen/glucose deprivation (OGD); • or excitotoxicity PI: + OGD • epilepsy PI:  • Alzheimer‘s disease • infection-related (e.g. HIV) neurotoxicity; traumatic injury; etc. • Neuroprotection, neurorepair mechanism • Neuropharmacology—protective & toxic chemicals Chinese Medicine —neuroprotection and repair

23. Functional genomics tools facilitate research & development of Chinese medicine New drug screening Organisms (Mouse, Rat, etc.) Organs (Organotypic slice culture) Phenotype Cells (Cell microarray) Chinese medicine Mechanism DNA Transcript Protein

24. Genomics Diseases Pharmacology Phytochemistry etc. Chinese Medicine

25. Acknowledgements Department director Prof. Hans Lehrach Collaborators: Leibniz Institute for Neurobiology: Klaus Reymann Ulrich Schröder Automation & Technology Wilfried Nietfeld Cell Microarray Michal Janitz Dominique Vanhecke Andrea Fiebitz Charite Medical Center: Johannes Vogt Leslie Schlüter German Resource Center for Genome Research: Florian Wagner Human Chromosome 21 Marie-Laure Yaspo Hans-Jörg Warnatz 歡迎開展中醫藥研究合作, 共同探索中醫藥發展新路。 Max-Planck-Institute for Molecular Genetics Berlin, Germany http://www.molgen.mpg.de/