Transcript Biology of the Transcriptome

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
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•
•
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Transcript exploration: P Kapranov
Chromatin: R Young
Nucleus biology: P Silver
Genome analysis: D Haussler