1. Today:
Regulating Gene Expression

2. Thinking About Gene Regulation
Why is it important?
What options does a cell have to accomplish regulation?

3. Introduction to Regulating Gene Expression in Eukaryotes
In eukaryotes, gene regulation functions primarily by either enhancing or restricting RNA Polymerase’s access to promoters
Gene regulation in eukaryotes is complex, and occurs at many levels (i.e. regulation of translation, enzyme activity, etc.). Most regulation, however, is done at the level of transcription. Unlike prokaryotes, most eukaryotic genes are OFF.

4. Introduction to Regulating Gene Expression in Eukaryotes
Eukaryotes must be able to create enormous diversity in their expression patterns. Two primary mechanisms accomplish this:
promoter-proximal elements (transcription factors near the promoter, ~-100-200)- tend to affect expression of many genes
enhancers or upstream activation sequences- tend to more specifically target a smaller subset of genes

5. How could you demonstrate the role of the promoter and proximal control elements?

6. Introduction to Regulating Gene Expression in Eukaryotes
The Model: Saccharomyces cerevisiae
As with the lac operon in bacteria, the galactose genes are usually off unless galactose is present and glucose is absent. (Galactose is formed when the disaccharide lactose is split into galactose and glucose.)

7. Regulating Gene Expression in Eukaryotes: Lessons from Yeast
Key Regulator is Gal4 Protein
Gal4 regulates the expression of GAL1, GAL2, GAL7, and GAL10
Each of these genes has 2+, 17 bp Gal4 binding sites (enhancers) located upstream of its promoter

8. Gal4 Function
Experimental work with reporter genes demonstrates that two critical domains, the DNA-binding domain, and the activation domain, are both required for Gal4 function. ? ? ?
An example of lacZ as a reporter in Drosophila embryos. ?

9. Gal3 only binds to Gal80 when it is bound to galactose!!
Gal4 Regulation
A second set of proteins regulates Gal4. Gal80 normally blocks Gal4 function, unless Gal3 inactivates it. When would you expect Gal 3 to inactivate Gal80??
The Gal pathway is well-conserved and can activate proteins in insects, humans, and other eukaryotes.
Gal3 only binds to Gal80 when it is bound to galactose!!

10. Now What? Gal4 Activity
Gal4, like many activators, can both attract other proteins involved in initiating transcription and recruit proteins to modify chromatin structure.

11. 2nd Mode of Action: Chromatin Remodeling
Promoters wound in nucleosomes are not accessible to the RNA polymerase!
Our understanding of this process comes from the SWI-SNF complex in yeast. Yeasts defective in this complex show a reduced level of Gal4 activity.

12. Let’s Watch: Chromatin Packaging

13. Chromatin Remodeling
The histone tails of histone proteins can be modified by the covalent attachment of a acetyl or methyl group.

14. Chromatin Remodeling
Acetylation can make an octomer more likely to slide along the DNA and can influence the binding of regulatory proteins. In general histone acetylation promotes gene transcription.

15. Enhancesomes
Enhancesomes are complexes of regulatory proteins that synergistically activate transcription to very high levels when all members are present.
Example: Beta-interferon

16. Pulling Together the Pieces in the β–interferon Gene

17. Combinatorial Control in Yeast: the MAT Locus
Two haploid and one diploid mating types are known in yeast. Interestingly, cells can switch mating types! Despite mating type being controlled by a single locus, each type expresses a unique set of genes!

18. Combinatorial Control in Yeast: the MAT Locus

19. Enhancer-Blocking Insulators
Prevent an enhancer from activating transcription when positioned between an enhancer and promoter

20. This causes a very unusual pattern of monoallelic inheritance!
Imprinting
Imprinting explains unusual inheritance patterns of autosomal genes in mammals
In paternal imprinting, the paternal copy is inactive; in maternal imprinting the maternal copy is inactive
Callipyge sheep 1 and 3 are descendents of Solid Gold. Sheep 2 and 4 are normal. Courtesy Sam P. Jackson
This causes a very unusual pattern of monoallelic inheritance!

21. Imprinting
Imprinting occurs because DNA is methylated in sex-specific manner during the development of the gametes.
These methylation patterns can be stably inherited.
At a molecular level, methylation can prevent the binding of insulators.

22. An Interesting Example…
These two mice are genetically identical!
How is this possible?
A lack of DNA methylation triggers the Agouti phenotype (as does maternal exposure to BPA).

23. The Genomic Neighborhood
Chromatin condensation patterns change during the course of the cell cycle
Heterochromatin contains few genes, while euchromatin is rich in genes

24. The Genomic Neighborhood
In fruit flies, chromosomal rearrangements can move an eye pigment gene to a region of heterochromatin, an example of epigenetic silencing.
When nearby, heterochromatin can spread into euchromatin, silencing genes. This is known as position-effect variation (PEV).

25. X-Inactivation
X-inactivation allows for dosage compensation of the genes on the X chromosomes in mammals.
This is accomplished through the formation of a Barr body (DNA and histones associated with Barr bodies are heavily methylated!)

26. X-Inactivation
The X-inactivation center (Xic) produces a long non-protein-coding RNA (ncRNA) called Xist
The chromosome producing Xist becomes inactivated as Xist coats the chromosome, triggering the production of heterochromatin.

27. For Thursday
Read one of the three papers for our Journal Club, and answer the questions provided!