MW 12:50-2:05pm in Beckman B302 Profs: Serafim Batzoglou & Gill Bejerano

1. CS273A Lecture 5: Transcription Regulation I MW  12:50-2:05pm in Beckman B302 Profs: Serafim Batzoglou & Gill Bejerano TAs: HarendraGuturu & PanosAchlioptas http://cs273a.stanford.edu [BejeranoFall13/14]

2. Announcements • HW1 is out. Due by 11.00 AM Friday, October 18. • Check it out. http://cs273a.stanford.edu [BejeranoFall13/14]

3. TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATTAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGACTAAATCTCATTCAGAAGAAGTGATTGTACCTGAGTTCAATTCTAGCGCAAAGGAATTACCAAGACCATTGGCCGAAAAGTGCCCGAGCATAATTAAGAAATTTATAAGCGCTTATGATGCTAAACCGGATTTTGTTGCTAGATCGCCTGGTAGAGTCAATCTAATTGGTGAACATATTGATTATTGTGACTTCTCGGTTTTACCTTTAGCTATTGATTTTGATATGCTTTGCGCCGTCAAAGTTTTGAACGATGAGATTTCAAGTCTTAAAGCTATATCAGAGGGCTAAGCATGTGTATTCTGAATCTTTAAGAGTCTTGAAGGCTGTGAAATTAATGACTACAGCGAGCTTTACTGCCGACGAAGACTTTTTCAAGCAATTTGGTGCCTTGATGAACGAGTCTCAAGCTTCTTGCGATAAACTTTACGAATGTTCTTGTCCAGAGATTGACAAAATTTGTTCCATTGCTTTGTCAAATGGATCATATGGTTCCCGTTTGACCGGAGCTGGCTGGGGTGGTTGTACTGTTCACTTGGTTCCAGGGGGCCCAAATGGCAACATAGAAAAGGTAAAAGAAGCCCTTGCCAATGAGTTCTACAAGGTCAAGTACCCTAAGATCACTGATGCTGAGCTAGAAAATGCTATCATCGTCTCTAAACCAGCATTGGGCAGCTGTCTATATGAATTAGTCAAGTATACTTCTTTTTTTTACTTTGTTCAGAACAACTTCTCATTTTTTTCTACTCATAACTTTAGCATCACAAAATACGCAATAATAACGAGTAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTTTCCTACGCATAATAAGAATAGGAGGGAATATCAAGCCAGACAATCTATCATTACATTTAAGCGGCTCTTCAAAAAGATTGAACTCTCGCCAACTTATGGAATCTTCCAATGAGACCTTTGCGCCAAATAATGTGGATTTGGAAAAAGAGTATAAGTCATCTCAGAGTAATATAACTACCGAAGTTTATGAGGCATCGAGCTTTGAAGAAAAAGTAAGCTCAGAAAAACCTCAATACAGCTCATTCTGGAAGAAAATCTATTATGAATATGTGGTCGTTGACAAATCAATCTTGGGTGTTTCTATTCTGGATTCATTTATGTACAACCAGGACTTGAAGCCCGTCGAAAAAGAAAGGCGGGTTTGGTCCTGGTACAATTATTGTTACTTCTGGCTTGCTGAATGTTTCAATATCAACACTTGGCAAATTGCAGCTACAGGTCTACAACTGGGTCTAAATTGGTGGCAGTGTTGGATAACAATTTGGATTGGGTACGGTTTCGTTGGTGCTTTTGTTGTTTTGGCCTCTAGAGTTGGATCTGCTTATCATTTGTCATTCCCTATATCATCTAGAGCATCATTCGGTATTTTCTTCTCTTTATGGCCCGTTATTAACAGAGTCGTCATGGCCATCGTTTGGTATAGTGTCCAAGCTTATATTGCGGCAACTCCCGTATCATTAATGCTGAAATCTATCTTTGGAAAAGATTTACAATGATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATAAAGTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATTAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGACTAAATCTCATTCAGAAGAAGTGATTGTACCTGAGTTCAATTCTAGCGCAAAGGAATTACCAAGACCATTGGCCGAAAAGTGCCCGAGCATAATTAAGAAATTTATAAGCGCTTATGATGCTAAACCGGATTTTGTTGCTAGATCGCCTGGTAGAGTCAATCTAATTGGTGAACATATTGATTATTGTGACTTCTCGGTTTTACCTTTAGCTATTGATTTTGATATGCTTTGCGCCGTCAAAGTTTTGAACGATGAGATTTCAAGTCTTAAAGCTATATCAGAGGGCTAAGCATGTGTATTCTGAATCTTTAAGAGTCTTGAAGGCTGTGAAATTAATGACTACAGCGAGCTTTACTGCCGACGAAGACTTTTTCAAGCAATTTGGTGCCTTGATGAACGAGTCTCAAGCTTCTTGCGATAAACTTTACGAATGTTCTTGTCCAGAGATTGACAAAATTTGTTCCATTGCTTTGTCAAATGGATCATATGGTTCCCGTTTGACCGGAGCTGGCTGGGGTGGTTGTACTGTTCACTTGGTTCCAGGGGGCCCAAATGGCAACATAGAAAAGGTAAAAGAAGCCCTTGCCAATGAGTTCTACAAGGTCAAGTACCCTAAGATCACTGATGCTGAGCTAGAAAATGCTATCATCGTCTCTAAACCAGCATTGGGCAGCTGTCTATATGAATTAGTCAAGTATACTTCTTTTTTTTACTTTGTTCAGAACAACTTCTCATTTTTTTCTACTCATAACTTTAGCATCACAAAATACGCAATAATAACGAGTAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTTTCCTACGCATAATAAGAATAGGAGGGAATATCAAGCCAGACAATCTATCATTACATTTAAGCGGCTCTTCAAAAAGATTGAACTCTCGCCAACTTATGGAATCTTCCAATGAGACCTTTGCGCCAAATAATGTGGATTTGGAAAAAGAGTATAAGTCATCTCAGAGTAATATAACTACCGAAGTTTATGAGGCATCGAGCTTTGAAGAAAAAGTAAGCTCAGAAAAACCTCAATACAGCTCATTCTGGAAGAAAATCTATTATGAATATGTGGTCGTTGACAAATCAATCTTGGGTGTTTCTATTCTGGATTCATTTATGTACAACCAGGACTTGAAGCCCGTCGAAAAAGAAAGGCGGGTTTGGTCCTGGTACAATTATTGTTACTTCTGGCTTGCTGAATGTTTCAATATCAACACTTGGCAAATTGCAGCTACAGGTCTACAACTGGGTCTAAATTGGTGGCAGTGTTGGATAACAATTTGGATTGGGTACGGTTTCGTTGGTGCTTTTGTTGTTTTGGCCTCTAGAGTTGGATCTGCTTATCATTTGTCATTCCCTATATCATCTAGAGCATCATTCGGTATTTTCTTCTCTTTATGGCCCGTTATTAACAGAGTCGTCATGGCCATCGTTTGGTATAGTGTCCAAGCTTATATTGCGGCAACTCCCGTATCATTAATGCTGAAATCTATCTTTGGAAAAGATTTACAATGATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAAGTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCACAAACTTTAAAACACAGGGACAAAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCTTGACATGATATGACTACCATTTTGTTATTGTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTCGATTATCTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATAAAG Genome Content http://cs273a.stanford.edu [BejeranoFall13/14]

4. Gene Products long non-coding RNA reverse transcription microRNA rRNA, snRNA, snoRNA

5. Gene Regulatory Switches • Gene = genomic substring that encodes HOW to make a protein (or ncRNA). • Genomic switch = genomic substring that encodes WHEN, WHERE & HOW MUCH of a protein to make. [0,1,1,1] B Gene H Gene Gene H N Gene N B [1,0,0,1] [1,1,0,0] http://cs273a.stanford.edu [BejeranoFall13/14]

6. If you only measure gene expression It’s like only seeing the values change in RAM as a program is running. http://cs273a.stanford.edu [BejeranoFall13/14]

7. Cis (=close) regulatory elements CIS REGULATION • Encode causality • Disease susceptibility • Driver sequences • Alter cell state • Key for evolution promoters, enhancers, silencers, insulators http://cs273a.stanford.edu [BejeranoFall13/14]

8. Transcription Activation http://cs273a.stanford.edu [BejeranoFall13/14]

9. RNA Polymerase • Transcription = Copying a segment of DNA into (non/coding) RNA • Gene transcription starts at the (aptly named) TSS, orgene transcription start site • Transcription is done be RNA polymerase, a complex of 10-12 subunit proteins. • There are three types of RNA polymerases in human: • RNA pol I synthesizes ribosomal RNAs • RNA pol II synthesizes pre-mRNAs and most microRNAs • RNA pol III synthesizes tRNAs, rRNAand other ssRNAs TSS RNA Polymerase http://cs273a.stanford.edu [BejeranoFall13/14]

10. RNA Polymerase is General Purpose • RNA Polymerase is the general purpose transcriptional machinery. • It generally does not recognize gene transcription start sites by itself, and requires interactions with multiple additional proteins. generalpurpose contextspecific http://cs273a.stanford.edu [BejeranoFall13/14]

11. Terminology • Transcription Factors (TF): Proteins that return to the nucleus, bind specific DNA sequences there, and affect transcription. • There are 1,200-2,000 TFs in the human genome (out of 20-25,000 genes) • Only a subset of TFs may be expressed in a given cell at a given point in time. • Transcription Factor Binding Sites: 4-20bp stretches of DNA where TFs bind. • There are millions of TF binding sites in the human genome. • In a cell at a given point in time, a site can be either occupied or unoccupied. http://cs273a.stanford.edu [BejeranoFall13/14]

12. Terminology • Promoter: The region of DNA 100-1,000bp immediately “upstream” of the TSS, which encodes binding sites for the general purpose RNA polymerase associated TFs, and at times some context specific sites. • There are as many promoters as there are TSS’s in the human genome. Many genes have more than one TSS. • Enhancer: A region of 100-1,000bp up to 1Mb or more upstream or downstream from the TSS that includes binding sites for multiple TFs. When bound by (the right) TFs an enhancer turns on/accelerates transcription. • Note how an enhancer (E) very far away in sequence can in fact get very close to the promoter (P) in space. promoter TSS gene http://cs273a.stanford.edu [BejeranoFall13/14]

13. TFBS Position Weight Matrix (PWM) Note the strong independence assumption between positions. Holds for most transcription binding profiles in the human genome. http://cs273a.stanford.edu [BejeranoFall13/14]

14. Promoters http://cs273a.stanford.edu [BejeranoFall13/14]

15. Enhancers http://cs273a.stanford.edu [BejeranoFall13/14]

16. Terminology • Gene regulatory domain: the full repertoire of enhancers that affect the expression of a (protein coding or non-coding) gene, at some cells under some condition. • Gene regulatory domains do not have to be contiguous in genome sequence. • Neither are they disjoint: One or more enhancers may well affect the expression of multiple genes (at the same or different times). TSS promoter enhancers for different contexts http://cs273a.stanford.edu [BejeranoFall13/14]

17. Imagine a giant state machine Transcription factors bind DNA, turn on or off different promoters and enhancers, which in-turn turn on or off different genes, some of which may themselves be transcription factors, which again changes the presence of TFs in the cell, the state of active promoters/enhancers etc. Proteins DNA transcription factorbinding site Gene DNA http://cs273a.stanford.edu [BejeranoFall13/14]

18. One nice hypothetical example requires active enhancers to function functions independently of enhancers http://cs273a.stanford.edu [BejeranoFall13/14]

19. The State Space Discrete, but very large. All states served by same genome(!) 1012cells 1cell http://cs273a.stanford.edu [BejeranoFall13/14]

20. Transcription Activation: Some measurements and observations http://cs273a.stanford.edu [BejeranoFall13/14]

21. Transcription Factor Binding Sites (TFBS) • An antibodyis a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria. • Antibodies can be raised that instead recognize specific transcription factors. • Chromatin Immunoprecipitation followed by deep sequencing (ChIP-seq): Take DNA (region or whole genome) bound by TFs, crosslink DNA-TFs, shear DNA, select DNA fragments bound by TF of interest using antibody, get rid of TF and antibody, sequence pool of DNA.  Obtain genomic regions bound by TF. http://cs273a.stanford.edu [BejeranoFall13/14]

22. ChIP-seq  Position Weight Matrix Computational challenge: The sequenced DNA fragments are 200-500bp. In each is one or more instance of the 6-20bp motif. Find it… http://cs273a.stanford.edu [BejeranoFall13/14]

23. Transcription Factors have Large “fan outs” • We could have had one TF regulate two TFS, each of which regulates two other TFs, etc. and each of those contributing to the regulation of a modest number of target genes (that do the real work). • Instead TFs reproducibly bind to thousands of genomic locations almost anywhere we’ve looked. • Gene regulation forms a dense network. http://cs273a.stanford.edu [BejeranoFall13/14]

24. Transfections As far as we’ve seen, enhancers work “the same” irrespective of distance (or orientation) to TSS, or identity of target gene. enhancer reporter gene minimalpromoter in cellular contextof choice • Which enhancers work in what contexts? • What if you mutate enhancer bases (disrupt or introduce binding sites) and run the experiment again? • What if you co-transfect a TF you think binds to this enhancer? • What if you instead add siRNA for that TF? http://cs273a.stanford.edu [BejeranoFall13/14]

25. Transcription factors bind synergistically,often with preferred spacing Transcription factor complexes prefer specific spacings! Sox:1 bp:Pax Sox2 Pax6 Sox2 Pax6 0 0 5 5 10 10 60 60 80 80 100 100 120 120 140 140 160 160 180 180 {+2} Fold activation Sox:3 bp:Pax Sox2 Pax6 Sox2 Pax6 Fold activation Adapted from Kamach et al., Genes Dev, 2001 http://cs273a.stanford.edu [BejeranoFall13/14]

26. Strict spacing between binding sites is important for structural interactions http://cs273a.stanford.edu [BejeranoFall13/14]

27. Complexes may leave genomic footprints • If a complex prefers TF : spacer : TF • This pattern may be abundant in the genome TAAACAGGAAGT AAAACAGGAATA ATAACAGGATGC TTAACAGGAAAG TAAACAGGATAG AAAACAGGAAAA Can we read complexes from individual predictions? http://cs273a.stanford.edu [BejeranoFall13/14]

28. Cooperative binding of complexes can be detected as the co-occurrence of individuals Each dot = different spacer http://cs273a.stanford.edu [BejeranoFall13/14]

29. Co-occurrences can be filtered for only structurally feasible patterns Fox { spacer } Ets = = Remove physically incompatible configurations compatible incompatible http://cs273a.stanford.edu [BejeranoFall13/14]

30. Complex motifs were grouped to reduce redundancy Started with: 300 transcription factor motifs Statistically Significant (p < 1×10-8) & valid motifs … Searched: (TF1 {spacer} TF2) 6,548,947 motif spacing combinations Fox { spacer } Ets Found: 6,180 significant motif spacing combinations Grouping 422 unique complex motifs http://cs273a.stanford.edu [BejeranoFall13/14]

31. Transgenics enhancer reporter gene minimalpromoter Observe enhancer behavior in vivo. Qualitative (not quantitative) assay. Can section and stain to obtain more specific cell-type information. http://cs273a.stanford.edu [BejeranoFall13/14]

32. BAC transgenics: necessity vs sufficiency You can take 100-200kb segments out of the genome, insert a reporter gene in place of gene X, and measure regulatory domain expression. You can then continue to delete or mutate individual enhancers. http://cs273a.stanford.edu [BejeranoFall13/14]

33. Gene Regulation: Enhancers are modular and additive brain limb neural tube Sall1 Temporal gene expression pattern “equals” sum of promoter and enhancers expression patterns. http://cs273a.stanford.edu [BejeranoFall13/14]

34. Genome Engineering • Technologies are in fact constantly improving that allow us to edit the nuclear genome itself. • Edit the genome of an embryonic stem cell, breed homozygous modified animals. http://cs273a.stanford.edu [BejeranoFall13/14]

35. Chromosome conformation capture (3C) • People are also developing methods to detect when two genomic regions far in sequence are in fact interacting in space. • Ultimately this will allow to determine experimentally the regulatory domain of each gene (likely condition dependent). http://cs273a.stanford.edu [BejeranoFall13/14]

36. 4C example result (in a single context) TSS probe Irreproducible peaks http://cs273a.stanford.edu [BejeranoFall13/14]

37. Gene Regulation is HOT • Despite its complexity gene regulation is currently one of the hottest topics in the study of the human genome. • Large projects are pouring tons of money to generate huge descriptive datasets. • The challenge now is to glean logic from these piles. To be continued… http://cs273a.stanford.edu [BejeranoFall13/14]