GENE REGULATION

1. GENE REGULATION Key control mechanism for dictating cell phenotype and function

2. Two kinds of genes • Housekeeping genes • Needed for cell metabolism • Required for nearly all cell types • Regulated Genes • Controls cell phenotype and function

3. Housekeeping Genes • Pol I transcripts • Ribosomal RNA genes (18S, 5.8S, and 28s rRNAs) • Some Pol II transcripts • e.g. Structural Proteins such as Actin • Pol III transcripts • 5S rRNA (ribosomal RNA) • tRNAs (translation) • snRNA (splicing)

4. Regulated Genes • Pol II transcripts • For e.g. • Transcription factors • Cytokines • Kinases • Phosphatases • Ubiquitinases • etc., etc., etc. • Pol III transcripts • miRNAs (siRNA like molecules that inhibit specific gene expression)

5. Multiple Mechanisms of Gene Regulation • Transcription • Initiation • Elongation • Post-transcription • RNA level • RNA processing (alternative splicing, polyadenylation) • RNA transport • mRNA stability • Protein level • Translation initiation • Protein transport • Protein stability • Protein processing • Epigenetic

6. Transcription • Housekeeping • Pol I transcription • Ribosomal RNA genes (18S, 5.8S, and 28s rRNAs) • Pol III transcription • 5S rRNA (ribosomal RNA) • tRNAs (translation) • snRNA (splicing) • miRNAs (control of gene regulation) • Regulated • Pol II transcription • Protein encoding transcripts

7. Pol I Transcription • 18S, 5.8S, and 28S RNA genes transcribed as one 13 kb transcript. • This transcript is subsequently processed (cleaved) to give individual 18S, 5.8S and 28S rRNAs. • On chromosome, 13 kb transcription unit (along with a 27kb intergenic region) is repeated 30-40 times • Helps provide higher expression of these housekeeping gene products.

8. Pol I Transcription

9. Pol I Promoter Function

10. Pol III Transcription

11. Pol II Transcription • Three types of cis elements: • Core promoter elements (-45 to +40) • Binds basal factors required for initiation and elongation. • Proximal promoter elements (-1kb to +200) • Binds regulatory transcription factors involved in activating or suppressing basal transcription • Enhancers/Silencers (far up or downstream) • Binds regulatory transcription factors involved in activating or suppressing basal transcription

12. Pol II Transcription Core promoter function - facilitates transcription initiation and elongation - Pol II - Catalyzes RNA synthesis - TFIID - Provides scaffold for general transcription factors (TBP is at core of this complex and is associated with TAFs (TBP Associated Factors) - TFIIB - Binds TBP, selects start site and recruits Pol II - TFIIA - Stabilizes binding of TFIIB and TBP to promoter - TFIIF - Binds TFIIB and Pol II - TFIIE - Recruits TFIIH - TFIIH - Helicase and kinase (Unwinds DNA phosphorylates C-terminus of Pol II)

13. Pol II Transcription Proximal promoter function - regulates core promoter function Proximal promoter binding factors activate transcription through multiple mechanisms: - Recruit basal factors - Recruit coactivators and mediators (for example, CBP, p300, p/caf, etc) - helps recruit basal factors - contain or recruit histone acetyl-transferases - recruit histone methyl-transferases - contain or recruit ATP dependent chromatin remodeling enzymes - helps form stable complexes between multiple proximal transcription factors

14. Regulation of promoter function by proximal promoter factors and enhancer/silencers • Recruitment of basal factors* • Recruitment of histone acetylases • Recruitment of histone methylases • Recruitment of ATP dependent chromatin remodeling complexes*

15. Histone modification • Histone acetylation - generally associated with promoter activation • (histone deacetyleses (HDACs) inhibit transcription • Neutralizes basic charges on lysines and arginine residues - relaxes nucleosome • Allows direct binding of activating proteins to promoter bound histones • Histone methylation • Arginine methylation associated with promoter activation • Lysine methylation associated with promoter inactivation

16. Promoter Elements Function in Either Orientation

17. Pol II Promoters Contain Multiple Transcription Factor Binding Sites Human Insulin Promoter - red, specific for pancreatic beta cells Reasons? - Coactivator interactions stabilized by binding to multiple transcription factors at the same time - Some factors cannot carry out all steps required for promoter activation (SP1) - Some elements play a role in signaling activation by different effectors

18. Solved DNA Interaction Motifs

19. DNA Recognition

20. Regulation of Transcription factor function • Tissue specific expression of transcription factors • Translocation of transcription factor to nucleus • Post-transcriptional modification of transcription factors

21. Steroid Hormone Receptors - Zinc fingers - Bind as dimers

22. Regulation of Steroid Hormone Receptors

23. Regulation of CREB

24. NF-kB Regulation

25. Other Mechanisms for Regulating Gene Expression • Regulation of translation • Regulation of mRNA stability • Alternative promoter usage • Alternative Splicing • Alternative polyadenylation

26. Translational Control and RNA Stability Control

27. Tissue Specific Promoter Usage Dystrophin Gene C - Corical M - Muscle P - Purkinje R - Retinal CNS - Central Nervous System S - Schwann cell G - General

28. Alternative Splicing

29. Alternative Splicing - Examples