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Transcription and gene regulation [email protected] RNA in a mammalian cell Amount #genes Protein-coding RNA (mRNA) 5% 20000 Non-coding RNA (tRNA, rRNA, miRNA) 95% <1000 mRNA expression levels X axis: organ (skin, heart, brain and so on) Y axis: mRNA expression level Number of genes Rare and abundant transcripts Tnnt2 Actc1 TROPONIN T, CARDIAC MUSCLE ISOFORM ACTIN, ALPHA CARDIAC Srf SERUM RESPONSE FACTOR Transcripts per cell Expression patterns Co-expression How are transcript levels controlled? 1. Synthesis Changes in the chromatin Transcription 2. Decay Degradation by RNases Accessible and non-accessible chromatin Quantitative chromatin profiling of the CD2 gene (Dorschner et al, Nature Methods) 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 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 Transcription initiation Gene regulation We want to define … 1. The cis regulatory elements (CREs) 2. The transcription factors that act on the CREs Studying gene regulation • Traditional experiments • Global experiments • Computational methods Example 1: a traditional approach LacZ Mack and Owens, Circ. Res. 84:852, 1999 Transcriptional Regulation of SMC Genes is Dependent on Complex Combinatorial Interactions of Many Cis Elements and Trans Factors CArG B + Angiotensin II MHOX/Prx1 SRF SRF ? SRF SRF Pol II Complex SRF CArG A Int CArG SRF TBP + TATA TGF b1 Example 2: a modern experimental approach • Which are the targets of transcription factor X? MyoD and myogenin targets in muscle development (Blais et al, Genes Dev 2005) 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 Two views on heart development ! SRF TGIF/MEIS MEF2 1 2 3 OTHERS 4 5 6 7 8 9 # # gene(m gene(hu Mybpc3 MYBPC3 Tnnt2 TNNT2 Tnni3 TNNI3 Myh6 MYH6 Adprhl1 ADPRHL1 Nppa NPPA Myl2 MYL2 Tncc TNNC1 Myoz2 MYOZ2 Actc1 ACTC Casq2 CASQ2 Fhl2 FHL2 Myl7 MYL7 Cox6a2 COX6A2 Cox7a1 COX7A1 Popdc2 POPDC2 Nkx2-5 NKX2-5 Fabp3 FABP3 Itgb1bp3 ITGB1BP3 1110028A0 7Rik D830019K1 Q8IUQ7 7Rik C9orf67 Rnf30 RNF30 Pgam2 PGAM2 Ldb3 LDB3 Ak1 AK1 Hrc HRC Adss ADSSL1 Asb2 ASB2 NM_016298 Actn2 ACTN2 Des DES Smpx SMPX Itgb1bp2 ITGB1BP2 Hspb2 HSPB2 O75155 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 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 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 Previous students • Erik Larsson • Graduate student, Wallenberg Lab / GU • Tanya Lobovkina • Graduate student, Physical chemistry / Chalmers • Nino Demetrashvili 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