Transcription and gene regulation
RNA in a mammalian cell
Amount #genes
Protein-coding RNA 5% 20000
(mRNA)
Non-coding RNA 95% <1000
(tRNA, rRNA, miRNA)
mRNA expression levels
X axis: organ (skin, heart, brain and so on)
Y axis: mRNA expression level
Tnnt2 TROPONIN T, CARDIAC MUSCLE ISOFORMActc1 ACTIN, ALPHA CARDIAC
Srf SERUM RESPONSE FACTOR
Transcripts per cell
Num
ber
of
gen
es
Rare and abundant transcripts
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
Mack and Owens, Circ. Res. 84:852, 1999
LacZ
Angiotensin II
?
Transcriptional Regulation of SMC Genes is Dependent on Complex Combinatorial Interactions of Many Cis Elements and Trans Factors
Int C
ArG
CArG B
CA
rG A
SRF
SRF
SRF
SRF
SRF SRF
TATA
TBP
Pol IIComplex
TCE
KLF5 +
KLF4 -
+
TGF
GATA Factors
CRP2
PIAS1
EBoxes
E12/E47
MyocardinM
yocardinMyocard
in
MHOX/Prx1+
?
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
www.wlab.gu.se/lindahl/genebatteries
1 2 3 4 5 6 7 8 9 # # gene(m gene(huMybpc3 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 ITGB1BP31110028A07Rik Q8IUQ7D830019K17Rik 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
MEF2SRF
OTHERS
Two views on heart development !
Cripps RM, Olson EM. Control of cardiac development by an evolutionarily conserved transcriptional network.Dev Biol. 2002 Jun 1;246(1):14-28.
TGIF/MEIS
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