Transcription and gene regulation

sven.nelander@wlab.gu.se

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