1. Doug Brutlag 2011 Genomics, Bioinformatics & Medicine http://biochem158.stanford.edu/ http://biochem158.stanford.edu/ Doug Brutlag Professor Emeritus of Biochemistry & Medicine Stanford University School of Medicine Gene Expression http://biochem158.stanford.edu/Gene%20Expression.html

2. Doug Brutlag 2011 Leveraging Genomic Information Understanding Metabolism Cell Signaling Differentiation Disease Novel Diagnostics DNA Microchips & Microarrays Gene Expression - RNA Proteomics - Protein

3. Doug Brutlag 2011 Gene Regulatory Mechanisms Transcriptional Mechanisms –Type of promoters & RNA polymerase –Control of Transcription –Transcription Factors and TFBSTranscription Factors and TFBS RNA processing –Capping –Splicing and Alternative Splicing –Poly-Adenylation –RNA export to cytoplasm –RNA degradation rates Micro RNAs (miRNAs) inhibit translation and degrade mRNA Silencer RNAs (siRNAs or RNAi) degrading mRNA Epigenetic Mechanisms –DNA methylation –Histone modifications Acetylation Methylation Phosphorylation, etc. –Chromatin remodeling

4. Doug Brutlag 2011 Eukaryotic Gene Structure miRNA Target

5. Doug Brutlag 2011 Multiple Enhancer Sequences © Garland Publishing

6. Doug Brutlag 2011 Enhanceosome: Exploring the Structure http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/download/Enhanceosome.pdf http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/download/Enhanceosome.pdf ATF-2/cJun IRF NF Kappa-B

7. Doug Brutlag 2011 Enhanceosome: Integrating the Signal http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb122_2.html http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb122_2.html HAT = Histone Acetyl Transferase

8. Doug Brutlag 2011 Oct3/4-Sox2 bound to Enhancer http://www.pdb.org/pdb/explore/explore.do?structureId=1GT0 http://www.pdb.org/pdb/explore/explore.do?structureId=1GT0

9. Doug Brutlag 2011 Repressor Bound to Silencer Sites

10. Doug Brutlag 2011 Gene Expression Regulatory Network © Current Opinions in Genetics & Development

11. Doug Brutlag 2011 Hematopoiesis http://www.biocarta.com/pathfiles/h_stemPathway.asp http://www.biocarta.com/pathfiles/h_stemPathway.asp

12. Doug Brutlag 2011 IL7 Regulatory Pathway http://www.biocarta.com/pathfiles/h_il7Pathway.asp/ http://www.biocarta.com/pathfiles/h_il7Pathway.asp/ © BioCarta

13. Doug Brutlag 2011 Growth Hormone Receptor Pathway http://www.biocarta.com/pathfiles/h_ghPathway.asp/ http://www.biocarta.com/pathfiles/h_ghPathway.asp/ © BioCarta

14. Doug Brutlag 2011 DNA Microarrays & DNA Chips Accelerate Gene ExpressionAnalysis Parallel Analyses –Analyze entire genomes instead of single genes –Analyze expression of entire genome –Analyze genetic polymorphisms (SNPs) Miniaturization Automation

15. Doug Brutlag 2011 Diagnosis Using DNA Arrays http://www.affymetrix.com/ http://www.affymetrix.com/ © Affymetrix Inc.

16. Doug Brutlag 2011 Affymetrix Arrays http://www.affymetrix.com/ http://www.affymetrix.com/ © Affymetrix Inc. Any collection of 25mers (1,200,000) can be synthesized in 100 steps

17. Doug Brutlag 2011 Light Directed Oligonucleotide Synthesis http://www.youtube.com/watch?v=ui4BOtwJEXs&feature=related http://www.youtube.com/watch?v=ui4BOtwJEXs&feature=related © Affymetrix Inc. Glass Light (deprotection) Mask 25-mer GeneChip Microarray Repeat

18. Doug Brutlag 2011 Automated DNA Chip Synthesis http://www.affymetrix.com/ http://www.affymetrix.com/ © Affymetrix Inc.

19. Doug Brutlag 2011 Photolithography Masks http://www.affymetrix.com/ http://www.affymetrix.com/ © Affymetrix Inc.

20. Doug Brutlag 2011 Hybridization & Detection © Affymetrix Inc. labeled probe

21. Doug Brutlag 2011 Cystic Fibrosis Array http://www.affymetrix.com/ http://www.affymetrix.com/ © Affymetrix Inc.

22. Doug Brutlag 2011 Microarrayer in Pat Brown’s Lab http://cmgm.stanford.edu/pbrown/ http://cmgm.stanford.edu/pbrown/

23. Doug Brutlag 2011 High Precision DNA Printing Arrayit Corporation

24. Doug Brutlag 2011 Mechanical Spotting Microarrays http://www.arrayit.com/ http://www.arrayit.com/ Arrayit Corporation

25. Doug Brutlag 2011 DNA Chips are used to Measure Gene Expression Searching for conserved sequence motifs regulating the expression MicroArray analysis of whole genome gene expression Clustering of genes based on their expression pattern

26. Doug Brutlag 2011 Megacluster of Yeast Gene Expression

27. Doug Brutlag 2011 T Cells Signaling DNA Damage Fibroblast Growth B Cells Signaling CMV Infection Anoxia Polio Infection Cell Signaling IL4 Growth Hormone Human Gene Expression Signatures Genes Biological Conditions

28. Doug Brutlag 2011 Finding Transcription Factor Binding Sites Upstream Regions Co-expressed Genes GATGGCTGCACCACGTGTATGC...ACGATGTCTCGC CACATCGCATCACGTGACCAGT...GACATGGACGGC GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA TCTCGTTAGGACCATCACGTGA...ACAATGAGAGCG CGCTAGCCCACGTGGATCTTGA...AGAATGACTGGC Pho 5 Pho 8 Pho 81 Pho 84 Pho … Transcription Start

29. Doug Brutlag 2011 Upstream Regions Co-expressed Genes GATGGCTGCACCACGTGTATGC...ACGATGTCTCGC CACATCGCATCACGTGACCAGT...GACATGGACGGC GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA TCTCGTTAGGACCATCACGTGA...ACAATGAGAGCG CGCTAGCCCACGTGGATCTTGT...AGAATGGCCTAT Finding Transcription Factor Binding Sites

30. Doug Brutlag 2011 Upstream Regions Co-expressed Genes ATGGCTGCACCACGTTTATGC...ACGATGTCTCGC CACATCGCATCACGTGACCAGT...GACATGGACGGC GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA TTAGGACCATCACGTGA...ACAATGAGAGCG CGCTAGCCCACGTTGATCTTGT...AGAATGGCCTAT Pho4 binding Finding Transcription Factor Binding Sites

31. Doug Brutlag 2011 Discovering Transcription Factor Binding Sites is Difficult Binding sites are short (5-15 base pairs) Not highly conserved (as little as 50%) Located in long intergenic regions (>10kb) Not always present (false positive genes)

32. Doug Brutlag 2011 Three Algorithms BioProspector –Presented in 2000 –Extends Gibb’s sampling (stochastic method) –For any cluster of sequences MDscan –Deterministic approach –Enumerative –Very fast –For sequences with some ranking information MotifCut and MotifScanMotifCut MotifScan –Graph-based –Does not use PSSMs –Novel and sensitive

33. Doug Brutlag 2011 cDNA Diagnostics Using DNA Microarrays

34. Doug Brutlag 2011 72 Breast Cancers Classified by 451 Gene Expression Assays Sorlie et al., PNAS 98, 10869-10874. 72 Patients 451 Genes

35. Doug Brutlag 2011 Breast Cancers Classified by 451 Gene Expression Assays Sorlie et al., PNAS 98, 10869-10874.

36. Doug Brutlag 2011 ERB-B2 in Signal Transduction & Oncology http://www.biocarta.com/pathfiles/h_her2Pathway.asp http://www.biocarta.com/pathfiles/h_her2Pathway.asp

37. Doug Brutlag 2011 Herceptin binds to ERB-B2 (HER-2) Blocking Cell Growth Herceptin = anti-HER2 Fab Fragment HER2 (Erb-2) Human Epidermal Growth Factor Receptor

38. Doug Brutlag 2011 Regulation of transcriptional activity by PML http://www.biocarta.com/pathfiles/h_pmlPathway.asp http://www.biocarta.com/pathfiles/h_pmlPathway.asp © BioCarta

39. Doug Brutlag 2011 Diagnosis & Gene Expression

40. Doug Brutlag 2011 Predicting Site of Origin for Cancers of Unknown Primary PathWork Diagnostics http://www.pathworkdx.com/ © Pathwork Diagnostics Inc.

41. Doug Brutlag 2011 PathWork Oncology Suite: Site of Origin 1844 tumors tested one at a time versus all 18 tissues of origin Retrospective study on well characterized patient samples Uses PathChip (functionally similar to Affymetrix HU-133A GeneChip) –604 specimens used for training –636 specimens used for test –604 specimens in reserve for final validation Reproducibility from lab to lab Performance based on sensitivity (> 70%) & accuracy (> 95%) © Pathwork Diagnostics Inc.

42. Doug Brutlag 2011  Receive microarray data file  Check file and data quality  Run model against microarray data file  Generate report file  Deliver report file back to clinical lab Production Data Flow I IINTNTEERRNENETTIINTNTEERRNENETTERTI IINTNTEERRNENETTIINTNTEERRNENETTERT Microarray data file PDF Report Sample Clinical Laboratory  Receive sample  Process specimen, mRNA extraction and microarray hybridization  Submit data file to PathWork for processing  Deliver report to oncologist

43. Doug Brutlag 2011 CONFIDENTIAL43 © Pathwork Diagnostics Inc.

44. Doug Brutlag 2011 CONFIDENTIAL44 © Pathwork Diagnostics Inc.