Genomic Circuits

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

Developmental biology in a ‘egg’shell

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

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

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

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?

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)

It’s 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

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

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

Interacting gene circuits

• http://occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_1/medialib/method/mirror/Graph/u9sspp.htm

Protein-DNA interactions

• http://occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_1/medialib/method/mirror/Petri/s_purp_w.htm

Switches and Engineering

• Module A serves as a bimodal toggle switch

• It’s 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

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

Cell division

• 50 copies of a given proteins…6% 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

Whither the molecules…

• 50 trials, n=50• Each trial results in success with probability

of p, in this case p=0.5 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

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

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

The return of lambda and the repressilator

• Pgs. 236-241

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

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