1. Even-skipped gene regulation

2. Control of transcription
How is the spatial and temporal expression of genes regulated?
Enhancer elements can regulate spatial and temporal expression of genes
Different enhancers regulate the expression different stripes 1 2 3 4 5 6 7
coding=Even-skipped gene

3. How was the eve striped enhancer identified?b c d e
Randomly fragment DNA
Place DNA fragments upstream of a strong promoter.
Place a marker gene (lacZ or GFP) downstream of the promoter
Determine if cells express that marker.
These are called REPORTER ASSAYS!

4. How was the eve striped enhancer identified?b c d e
Randomly fragment DNA
Place DNA fragments upstream of a strong promoter.
Place a marker gene (lacZ or GFP) downstream of the promoter
Determine if cells express that marker.
These are called REPORTER ASSAYS!

5. How was the eve striped enhancer identified?
The even-skipped (eve) striped 2 was controlled by the DNA fragment in red.
With a reporter gene constructs, are you looking directly at the even skipped gene?
Paolo Struffi et al. Development 2011;138:

6. eve stripe 2 activators and repressors
Eve strip 2 enhancer
How can enhancers be so specific?

8. Bicoid is an example of a Morphogen
Morphogens are diffusible molecules that can specify positions in the embryo by setting up concentration gradients.
The imitation game

10. eve stripe 2 activators and repressors
Eve strip 2 enhancer
Eve2 is only expressed when the repressors Kruppel and Giant are not present!

12. eve stripe 2 activators and repressors

13. Long-range enhancers
How do enhancers acting at such a large distance “find” the promoter region
Can you come up with models that may help to to explain this?
Chromatin: Higher order chromatin structure may make long sequences much closer than if there were linear

14. Long-range enhancers Long-range enhancers
If enhancers can at such long distances, can they also activate inappropriate genes?
Yes– but insulator proteins help block this.

16. Model for how enhancer-blocking insulators might work
One proposal is that enhancer-blocking insulators create new loops that physically separate a promoter from its enhancer.
Eukaryotic enhancers can act at great distances to modulate the activity of the transcriptional apparatus. Enhancers contain binding sites for many transcription factors, which bind and interact cooperatively. These interactions result in a variety of responses, including the recruitment of additional co-activators and the remodeling of chromatin.

18. Repressors? Competition Inhibition Direct Repression
Based on Robert Tjian, "Molecular Machines that Control Genes," Scientific American.

19. Repressors?
Figure 19-22a 19

20. Repressors?
Figure 19-22b 20

21. Repressors?
Figure 19-22c 21

25. Activation of Genes in a Chromatin Environment
Enhanceosomes help recruit the transcriptional machinery
As you have seen so far… transcription of eukaryote genes has to be turned on and off during the lifetime of an organism. To understand how eukaryotes regulate genes during their lifetime, it is necessary to see how chromatin changes during transcriptional activation
The β-interferon enhanceosome. In this case, the transcription factors recruit a co-activator (CBP), which binds both to the transcription factors and to RNA polymerase II, initiating transcription.
DNA binding proteins (enhancers) bind to cis acting sequences can have a synergistic effect on transcription

26. The β-interferon enhanceosome acts to move nucleosomes by recruiting the SWI-SNF complex.
β-interferon encodes the antiviral protein interferon, and it is normally switched off except in the case of viral infection.
The key to activation of this gene is the assembly of transcription factors into an enhanceosome about 100bp upstream of the stat site.