1. Thank you for the midterm feedback!
Projects will be assigned this weekend.
[Bejerano Fall11/12]

2. Mid Term Feedback Feedback
Lecture 12
Mid Term Feedback Feedback
Cis-Regulation
Cellular Signaling
[Bejerano Fall11/12]

3. MidTerm Feedback Feedback
You like us!
You like your teachers and love your TAs
We like you too ;)
We also like recruiting (at all levels) from this class
Talk to us if you’re interested
[Bejerano Fall11/12]

4. Big Picture Reminder
We are surveying the functional classes of elements encoded by the human genome.
We have previously discussed:
Repetitive sequences (mobile elements & low complexity)
Protein coding genes (and pseudogenes)
Non coding RNAs (structural, micro, long, anti-sense etc.)
Today we’ll cover the last (known :) large class of functional elements, concluding the functional tour.
[Bejerano Fall11/12]

5. Gene Regulation gene (how to) control region (when & where) DNA
Protein coding
RNA gene
DNA
DNA binding
proteins
[Bejerano Fall11/12]

6. Unicellular vs. Multicellular
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7. Broad View of Transcription
Chromatin / Proteins
Extracellular signals
DNA / Proteins
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8. Vertebrate Transcription Regulation
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9. Pol II Transcription Key components: Proteins DNA sequence
DNA epigenetics
Protein components:
General Transcription factors
Activators
Co-activators
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10. Activators & Co-Activators
Protein - Protein
Protein - DNA
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11. The Core Promoter
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12. Chromatin Remodeling “off” “on”
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13. Nucleosome tail modifications
Lysine acetylations.
Histone Acetyl-Transferases (HAT) & Histone Deacetylases (HDAC).
Lysine and Argenine Metylations.
Modified by histone- metyl-transferase.
Phosphorilation.
Ubiquitination.
H2A ubiquitination affects % of this histone in most eukaryotic cells
ADP-ribosylation.

14. http://cs273a.stanford.edu [Bejerano Fall11/12]

15. CpG islands Functionally: Unmethylated
Evolutionary: CpGs are rare outside “CpG islands”
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16. Transcription Factor (TF) Binding Sites
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17. TFs in the Human Genome
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18. Synergy / non-linear additivity
Gene
DNA
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19. Combinatorial Regulatory Code
2,000 different proteins can bind specific DNA sequences.
DNA
Proteins
Protein binding site
Gene
DNA
A regulatory region encodes 3-10 such protein binding sites.
When all are bound by proteins the regulatory region turns “on”, and the nearby gene is activated to produce protein.
[Bejerano Fall11/12]

20. Distal Transcription Regulatory Elements
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21. Enhancers
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22. Enhancers: action over very large distances
Basal factors
RNAP II
promoter
Enhancer with bound protein
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23. Transient Transgenic Enhancer Assay
in situ
Conserved
Element
Minimal Promoter
Reporter Gene
Construct is injected into 1 cell embryos
Taken out at embryonic day
Assayed for reporter gene activity
transgenic
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24. Vertebrate Enhancer Combinatorics
limb
neural
tube
brain
Sall1
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25. Vertebrate Enhancer Combinatorics
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26. What are Enhancers? What do enhancers encode?
Surely a cluster of TF binding sites.
[but TFBS prediction is hard, fraught with false positives]
What else? DNA Structure related properties?
So how do we recognize enhancers?
Sequence conservation across multiple species
[weak but generic]
[Bejerano Fall11/12]

27. Most Non-Coding Elements likely work in cis…
“IRX1 is a member of the Iroquois homeobox gene family.
Members of this family appear to play multiple roles
during pattern formation of vertebrate embryos.”
gene deserts
regulatory jungles
9Mb
Every orange tick mark is roughly 100-1,000bp long, each evolves under purifying selection, and does not code for protein.
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28. Many non-coding elements tested are cis-regulatory
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29. Vertebrate Gene Regulation
gene (how to)
control region (when & where)
distal: in 106 letters
DNA
DNA binding
proteins
proximal: in 103 letters
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30. Gene Expression Domains: Independent
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31. Repressors / Silencers
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32. What are Enhancers? Repressors What do enhancers encode?
Surely a cluster of TF binding sites.
[but TFBS prediction is hard, fraught with false positives]
What else? DNA Structure related properties?
So how do we recognize enhancers?
Sequence conservation across multiple species
[weak but generic]
Verifying repressors is trickier [loss vs. gain of function].
How do you predict an enhancer from a repressor? Duh...
repressors
repressors
[Bejerano Fall11/12]

33. Insulators
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34. Cis-Regulatory Components
Low level (“atoms”):
Promoter motifs (TATA box, etc)
Transcription factor binding sites (TFBS)
Mid Level:
Promoter
Enhancers
Repressors/silencers
Insulators/boundary elements
Cis-regulatory modules (CRM)
Locus control regions (LCR)
High Level:
Epigenetic domains / signatures
Gene expression domains
Gene regulatory networks (GRN)
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35. Disease Implications: Genes
genome
protein
Limb Malformation
Over 300 genes already
implicated in limb malformations.
[Bejerano Fall11/12]

36. Disease Implications: Cis-Reg
gene
genome
NO protein
made
Limb Malformation
Growing number of cases (limb, deafness, etc).
[Bejerano Fall11/12]

37. Transcription Regulation & Human Disease
[Wang et al, 2000]
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38. Critical regulatory sequences
Lettice et al. HMG :
Single base changes
Knock out
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39. Other Positional Effects
[de Kok et al, 1996]
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40. Genomewide Association Studies point to non-coding DNA
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41. WGA Disease
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42. 9p21 Cis effects Follow up study:
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43. Cis-Regulatory Evolution: E.g., obile Elements
Gene
Gene
Gene
Gene
What settings make these “co-option” events happen?
[Yass is a small town in New South Wales, Australia.]
[Bejerano Fall11/12]

44. Britten & Davidson Hypothesis: Repeat to Rewire!
[Davidson & Erwin, 2006]
[Britten & Davidson, 1971]
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45. Modular: Most Likely to Evolve?
Chimp
Human
With the vast genomic resources available nowadays, we can isolate both genotypic and phenotypic differences between species. However, a gap still exists in our ability to associate genotype with phenotype, and an overarching goal in biology today is to start bridging that gap, to be able to say:
This inversion causes foot shape alterations
This duplication and that substitution both play a role in brain size
Or going from phenotype to genotype,
This sweat-related gene has some change in chimpanzee relative to human
The study I am talking about today tries to link genotype to phenotype for a single type of genomic rearrangement, human-specific deletions.
[Bejerano Fall11/12] 45

46. Human Accelerated Regions
Human-specific substitutions in conserved sequences
Human Chimp
HAR1 expressed in Cajal-Retzius neurons at border of marginal zone 46
[Pollard, K. et al., Nature, 2006]
[Prabhakar, S. et al., Science, 2008]
[Beniaminov, A. et al., RNA, 2008] 46

47. Signal Transduction
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48. Cell Communication
Lodish, 20-1

49. Wnt and Hedgehog signaling
Jacob & Lum Science 2007

50. Signaling Pathways Important in Developmental Biology
Wnt/Frizzled through b-catenin
Hedgehog
TGF-b family through Smads
Growth factors via JAK-STATs
Notch
Integrin
TNF

51. Giant State Machine… Chromatin / Proteins Extracellular signals
DNA / Proteins
[Bejerano Fall11/12]