Genomic Circuits

1
Genomic Circuits
2
Are Genes Hardwired?

• No, gene expression can be modified
• Cis-regulatory elements are modular offering
  sites for protein binding in repression or
  activation
• Utilization of a regulatory module in sea urchin
  development

3
Developmental biology in a eggshell

• Click here to see Quicktime movie of cleavage.
• http//occawlonline.pearsoned.com/bookbind/pubbook
  s/bc_mcampbell_genomics_1/medialib/method/UrchDev.
  html

4
Endo16

• Expression of this protein is tightly controlled
  for location, time, and amount of RNA
• Just before gastrulation, cells at the base of
  the blastula expression Endo16, and later cells
  of the archenteron express it, later becoming
  endodermal, etc.
• Protein appears to be involved in cell adhesion

5
Genetic circuitry

• Control of Endo16 during sea urchin development
  due to modules of multiple cis-acting sequences
  (A-G) (Figure 7.5)
• Use deletion series eliminating upstream
  sequences, and distinct modules independently
  fused to reporter gene

6
Dissecting cis sequences

• Can divide reporter constructs into three groups
  those that express CAT in (wt, G, B, A) or out
  (D, E, F, Bp only ) of endoderm (note 5, 4, 3, 9
  allow expression in mesoderm and ectoderm) and
  lastly, C does not allow CAT expression in any
  cell type
• What are the roles of CDEF, which promote
  expression in wrong cell type?

7
Combinatorial cis elements

• Do F-C influence transcription of G, B, A?
• GBA turn down inappropriate expression in
  combination with F-C
• Model
• GBA strongly promote appropriate expression in
  endoderm, while FE repress expression in ectoderm
  and DC repress expression in mesoderm (look at
  gray shades in Table 7.2 or look over Figure
  7.10)

8
Its all in the timing

• Blastula forms at 24 hr, gastrula at 48 hrs,
  larva at 65 hrs
• Measure CAT at different times from different
  constructs
• Although GBA all promote CAT production, they do
  so at distinct times (normalized data, also)
• A during the first 48 hrs, then drops
• B during 60-72 hrs
• G offers small quantity of CAT with peak around
  48 hrs
• When combined, observe similar profile to entire
  cis-region

9
What is G doing?

• Found transcription activation was promoter
  independent
• G acts as an amplifier
• Cranks up A, and B (if coupled to A) Figure 7.13
  b and c

10
Circuit diagram

• Link cis and trans factors into modules that form
  a regulatory circuit
• Discussion of Discovery questions 10-13
• For next Monday, DQ Chapter 7
• 3, 6
• 18
• 27-31

11
Interacting gene circuits

• http//occawlonline.pearsoned.com/bookbind/pubbook
  s/bc_mcampbell_genomics_1/medialib/method/mirror/G
  raph/u9sspp.htm

12
Protein-DNA interactions

• http//occawlonline.pearsoned.com/bookbind/pubbook
  s/bc_mcampbell_genomics_1/medialib/method/mirror/P
  etri/s_purp_w.htm

13
Switches and Engineering

• Module A serves as a bimodal toggle switch
• Its either in complete control of gene output or
  gives complete control to module B
• When B is active, A acts as a amplifier
• Can these activities be separated? - Yes

14
How do cells throw switches?

• Gene regulation is a stochastic process that must
  differentiate between signal and noise.
• Models for these stochastic processes
• Binomial model
• Normal model

15
Cell division

• 50 copies of a given proteins6 of the time one
  cell will get 19 or fewer copies and 6 of time
  it might get 31 copies
• Binomial frequency function assumes independent
  experiments
• The fate of one molecule is independent of the
  other 49

16
Whither the molecules

• 50 trials, n50
• Each trial results in success with probability of
  p, in this case p0.5 for each cell
• (n/k) pk(1-p)n-k (first feature is binomial
  coefficient)
• Probability that one cell gets 25 molecules
• (50/25) (.5)25 (1-.5)50-25 0.112

17
The effect of stochastic process and noise

• Proteins are expressed in bursts of varying
  durations with different outputs
• The total number of proteins produced from any
  one gene is not the same each time, but an
  average normal distribution

18
Stochastic effects

• Randomness means that if you measure the amount
  of protein produced by the same gene in thousands
  of identical cells the outcome will vary.
• Normal or Gaussian distribution

19
The return of lambda and the repressilator

• Pgs. 236-241

20
Ok, but how does it all work?

• Sports analogy very nice
• Cooperation through communication
• Cell sentries emit signals to organize
• Checkpoints
• DNA replication for example
• Redundancy
• Multiple ways to do anything and everything

21
How reliable is a system?

• Reliability probability of B being produced
  (arbitrarily set at 0.9)
• If B production requires two genes, R .81
• Etc. page 243