Transcription and gene regulation

1. Transcription and gene regulation sven.nelander@wlab.gu.se

2. RNA in a mammalian cell Amount #genes Protein-coding RNA 5% 20000 (mRNA) Non-coding RNA 95% <1000 (tRNA, rRNA, miRNA)

3. mRNA expression levels X axis: organ (skin, heart, brain and so on) Y axis: mRNA expression level

4. Rare and abundant transcripts Tnnt2 TROPONIN T, CARDIAC MUSCLE ISOFORM Actc1 ACTIN, ALPHA CARDIAC Number of genes Srf SERUM RESPONSE FACTOR Transcripts per cell

5. Expression patterns

6. Co-expression

7. How are transcript levels controlled? 1. Synthesis Changes in the chromatin Transcription 2. Decay Degradation by RNases

8. Accessible and non-accessible chromatin Quantitative chromatin profiling of the CD2 gene (Dorschner et al, Nature Methods)

9. How is a gene made (in)accessible for transcription? • 1. Chemical modifications of histones • Acetylation  open configuration • Deacetylation  closed configuration • 2. Chemical modification of the DNA • Methylation  no transcription

10. RNA polymerase II • The enzyme that makes protein-coding transcripts • RNA pol II is non-specific in its pure form: ANY DNA  RNA COPY • ~40 basal transcription factors are needed to make RNApol II promoter-specific THE DNA OF A GENE  RNA COPY • ~2000 transcription factors are needed to regulate the action of RNApol II THE DNA OF THE RIGHT GENE  RNA COPY

11. Transcription initiation

12. Gene regulation We want to define … • The cis regulatory elements (CREs) • The transcription factors that act on the CREs

13. Studying gene regulation • Traditional experiments • Global experiments • Computational methods

14. LacZ Example 1: a traditional approach Mack and Owens, Circ. Res. 84:852, 1999

15. Transcriptional Regulation of SMC Genes is Dependent on Complex Combinatorial Interactions of Many Cis Elements and Trans Factors Angiotensin II EBoxes + MHOX/Prx1 E12/E47 PIAS1 SRF SRF ? ? SRF SRF SRF SRF + TGF b1 CArG B GATA Factors Myocardin Myocardin Myocardin CRP2 CArG A Pol II Complex Int CArG KLF5 + KLF4 - TBP TCE TATA

16. Example 2: a modern experimental approach • Which are the targets of transcription factor X?

17. MyoD and myogenin targets in muscle development (Blais et al, Genes Dev 2005)

18. Example 3: a typical bioinformatic approach • Which genes are targeted by factor X? 1. Identify genomic regions likely to contain regulators 2. Identify potential sites for factor X

19. Two views on heart development ! SRF TGIF/MEIS MEF2 OTHERS Cripps RM, Olson EM. Control of cardiac development by an evolutionarily conserved transcriptional network.Dev Biol. 2002 Jun 1;246(1):14-28. www.wlab.gu.se/lindahl/genebatteries

20. Conclusions: • RNA levels are tightly regulated by transcription and degradation • Transcription is a product of • chromatin modifications • cis regulatory elements • transcription factors • There is a number of experimental and computational methods

21. Master’s projects • 1. EGFR pathway – towards combinatorial treatment of solid tumours? • 2. From stem cell to B cell • 3. Evolution of Serum Response Factor regulation

22. Previous students • Erik Larsson • Graduate student, Wallenberg Lab / GU • Tanya Lobovkina • Graduate student, Physical chemistry / Chalmers • Nino Demetrashvili

23. Reading tips • About transcriptional regulation • ’Genes and Signals’ by M Ptashne • ’Genomic regulatory systems’ by E Davidson • Some labs doing nice work • Transcript exploration: P Kapranov • Chromatin: R Young • Nucleus biology: P Silver • Genome analysis: D Haussler