1. Regulation of eukaryotic genes Gene silencing Enhancers Activators Functional domains of activators

2. Idea for another extra credit project Explore DNA binding domains of proteins. 1. Go to a web site with a Chime tutorial, e.g. GAL4 or Cro 2. Or use Kinemages 3. Write a roughly 2 page report on how a particular protein recognizes a DNA sequence

3. States of eukaryotic genes Inactive: –Closed chromatin –Open chromatin, but repressors or lack of activators keep frequency of initiation low. –Open chromatin, transcription has initiated, but polymerases will not elongate. Active: –Open chromatin, basal transcription: requires TATA + Inr –Open chromatin, activated transcription: requires enhancer or upstream activator sequences

4. Silent and open chromatin

5. Transcription initiation and pausing

6. Basal and activated transcription

7. Silencing Mechanism

8. Silencer Cis-acting sequences that cause a decrease in gene expression Similar to enhancer but has an opposite effect on gene expression Gene repression - inactive chromatin structure (heterochromatin) Examples –Telomeric silencing –a or  genes - silent loci of mating type switching in yeast

9. Silencer binding proteins Silencer binding protein serve as anchors for expansion of repressed chromatin Rap1 protein binds to silencer elements SIR proteins (Silent Information Regulators) Nucleates assembly of multi-protein complex –hypoacetylated N-terminal tails of histones H3 and H4 –methylated N-terminal tail of H3 (Lys 9) Experiments: Condensed chromatin –Resistant to DNaseI digestion –Delete silencer - genes are derepressed

10. Gene Silencing

11. Silencing Mechanism

12. Enhancers Cis-acting sequences that cause an increase in expression of a gene Act independently of position and orientation with respect to the gene. Can act to: –Increase the rate of initiation at a promoter –Increase the fraction of cells in which a promoter is active

13. SV40 Control region Origin of replication Promoter and upstream activator sequences for early transcription Promoter for late transcription Enhancer

14. SV40 map

15. Many regulatory DNA sequences in SV40 control region

16. Stimulation of transcription by enhancer is independent of orientation and position Early Late T-Ag + Enhancer wt Enh- pos orien T-Ag + T-Ag - SV40:

17. Enhancers also regulate cellular genes

18. Enhancer contains multiple binding sites for transcriptional activators Early Late high level Enhancer wt SV40: ACB low level deletion revertant T-Ag CB CBC high level An enhanson

19. Enhancers can occur in a variety of positions with respect to genes Transcription unitP Ex1Ex2 Enhancer Adjacent Downstream Internal Distal Upstream

20. Activator proteins

21. Modular nature of activator proteins DNA binding domain: recognition and binding to specific DNA sequences Multimerization domain: allows formation of homo- or hetero-multimers Activation domain: –Needed for increase in expression of responding gene –Targets are still under investigation General transcription factors Histone modifying enzymes Nucleosome remodeling complexes, etc

22. Modular structure of GAL4 NC 198 148 196768881 Activation DNA binding Dimerization GAL80 binding

23. Induction by galactose exposes an activation surface In the presence of galactose, GAL4 activates several genes whose products are required for galactose metabolism. GAL4 binds to a DNA sequence called UAS G. In the absence of galactose, GAL80 blocks GAL4 activation. Binding of the sugar causes GAL80 to move. This exposes the activation domain of GAL4.

24. Induction of GAL4

25. Domain swap experiments show the domains are interchangeable Fuse an DNA-binding domain (DBD) from one transcription factor to the activation domain (AD) of a different one. –DBD from LexA (E. coli) –AD from GAL4 (yeast) Now a target gene can be placed under control of the DNA binding site for the first factor –GAL1 gene with o Lex (LexA binding sites) can be activated by the fusion protein. Basis for 2-hybrid screen for any interacting proteins

26. Domain swap experiments: Diagram 1

27. Domain swap experiments: Diagram 2

28. Two Hybrid Screens (Interaction Cloning), part 1

29. Two Hybrid Screens (Interaction Cloning), part 2