Regulation of gene expression

by signalling pathways

F. Guesdon
GEM6410 - October 2006
Thinking about gene expression…
 In a typical animal cell, about 10,000 genes are
expressed into proteins
 Most of these are housekeeping proteins,
needed in all cell types
 Certain proteins can only be detected in specific
cell types (differentiation) or in specific
circumstances (response to stimuli, stress)
 The circumstances under which a gene is
expressed and the manner by which its
expression is regulated are important aspects of
its function
 Gene Expression is regulated at
several levels
 Control by transcription factors
Transcriptional control  Long-range control
 Epigenetic control

RNA processing control  Splicing

 Half-life
 RNA surveillance
Translation control

Protein activity control

Topics for this lecture
1. Transcription factors and promoters
2. Regulation by signalling pathways
3. Studying transcription factors
 Euchromatin:
Euchromatin:
•• Uncoiled
UncoiledDNA
DNA
••Available
Availablefor
fortranscription
transcription

Heterochromatin:
Heterochromatin: coiled
coiled (compacted)
(compacted) DNA,
DNA,
not
not available
available for
for transcription
transcription
 Three RNA Polymerases
transcribe different genes
1. RNA polymerase I most rRNA
2. RNA polymerase II mRNA
snRNA

3. RNA polymerase III tRNA

5S rRNA
miRNA
What are the elements controlling
transcription in euchromatin?
 Cis-acting elements : DNA sequence motifs
• Transcription start
• Promoter elements (close-range regulation)
• Enhancer elements (medium-range regulation)

 Trans-acting factors: Proteins

• How do they recognise genes?
• How do they know which gene needs to be
expressed and when?
Examples of cis-acting DNA motifs

Element Consensus Factor

TATA box TATAAA TFIID
CAAT box CCAAT CTF/NF1, C/EBP
GC box GGGCGG SP1
octamer ATTTGCAT Oct-1, Oct-2
TRE (TPA) GTGAGT(A/C)A AP-1 (Jun, Fos)
B GGGACTTTCC NF-B
CRE (cAMP) GTGACGT ATF
Basal transcriptional machinery
Transcription Initiation complex
 Required for expression of all protein-
coding genes
 A macromolecular assembly of approximately 50
proteins
 Rate limiting step in the transcription of many genes
Basal trancription
factors
 TFIIA
 TFIIB TATA-binding
protein +
 TFIID: associated
proteins
 TFIIE
 TFIIF
 TFIIH

Alberts 9-30 - The basal transcriptional machinery

Two common protein–DNA interactions
Arginine (R)- Guanine Asparagine (N)- Adenine
Upstream regulatory elements (UREs)
Comparison of many eukaryotic promoters identified
conserved motifs that were involved with the regulation
of gene transcription
Some UREs are common to many genes but others
were found only in genes expressed in specific cells or
as a result of specific stimuli

TATA First exon

-25 1
UREs
Upstream regulatory elements
An example of a non-specific URE
SP1 box – GGGCGG
This motif is present in a number of genes
Using the techniques of footprinting and gel retardation it
was found that a specific protein SP1 could bind this
motif and modulate gene expression
SP1 was shown to be expressed in all cell types and is an
example of a constitutive transcription factor
 Examples of binding motifs

Element Consensus Factor

TATA box TATAAA TFIID
CAAT box CCAAT CTF/NF1, C/EBP
GC box GGGCGG SP1
octamer ATTTGCAT Oct-1, Oct-2
TRE (TPA) GTGAGT(A/C)A AP-1 (Jun, Fos)
B GGGACTTTCC NF-B
CRE (cAMP) GTGACGT ATF
Two-domain structure of
Transcription Factors
 Transcription factors comprise at least two structural
domains that correspond to two molecular functions

 One domain binds DNA

 The other domain activates transcription by interacting with

other components of the transcriptional machinery

 Example: SP1

 In dimeric transcription factors, the two domains are in

two different polypeptide chains (examples: NF-B)
 Mechanism of action of
transcription factors: SP1

DNA-binding Transactivation
domain domain

RNA pol II

TBP
TAF
SP1 TATA
TAF
 Mechanism of action of
transcription factors
1. SP1 DNA-binding domain binds SP1 DNA motif via zinc fingers
2. The transactivation domain interacts with TAFs
3. This stabilises the transcription initiation complex

RNA pol II

TBP
TAF
SP1 TATA
TAF
Most transcription factors belong
to 4 major structural groups
The 4 groups are defined by the three-dimensional
structure of the DNA-binding domain”

 Homeodomain / Helix-Turn-Helix (HTH)

 Zinc Finger
 Leucine Zipper
 Helix-Loop-Helix (HLH)
DNA-binding domain 1:
Homeodomain

In the homeodomain
(helix-turn-helix),
three  helices interact
with the DNA.

Asparagine in helix
number 3 binds to
adenine in the DNA
DNA-binding domain
2: Zinc finger

The zinc-finger domain

is composed of an 
helix and a  sheet.

Each zinc-finger
domain is held together
by an atom of zinc
Examples of sequence-specific interactions
between zinc fingers and DNA
DNA-binding domain 3:
Leucine zipper
In the leucine zipper motif,
two  helices interact with
each other to form a DNA
binding domain.

The two  helices may be

from different polypeptide
chains.

Example: Fos / Jun hetero-

dimer, binds TPA response
elements.
 DNA-binding domain 4:
Helix-loop-helix dimers
The two monomers are
held together in a four-
helix bundle.

Each monomer
contributes two a helices
connected by a flexible
loop of protein ( red ).

A specific DNA sequence

is bound by the two a
helices that project from
the four-helix bundle
Enhancers
• In addition to DNA elements located proximal to the
promoter (promoter elements) it was also found that
stretches of DNA could influence gene expression at
much greater distances (up to 10 kb)
• They can be upstream or downstream of the gene or
even within the transcription unit
• They act by increasing the activity of the promoter but
lack promoter activity themselves and are thus known
as enhancers
• Enhancers contain multiple binding sites for
transcription factors
 Enhancers (2)
• Some of the cis-acting regulatory motifs found in
enhancers are the same as those found in promoters
• In some cases the transcription factors bound are the
same as those found in the proximal promoter elements
• Enhancers can act in tissue-specific manner
• It is thought that the basis of their long range action is
structural changes of the DNA brought about by
transcription factor interaction
Promoter and enhancer
Promoter
 Core promoter: position +1 to -25 (Transcription start,
TATAA box, GC box)
 Upstream elements: positions -26 to -200

Enhancer / silencer
 Discrete elements
 Located at variable distance from the promoter (>200 bp)
 Exact position and orientation is not important
 Confer responses to environmental / developmental
signals
 Transcription factors
A transcription factor is any protein that meets these 3 criteria:
 Is involved in the initiation or regulation of transcription
 Binds specifically to cis-acting elements or RNA polymerase
 And is not itself part of RNA polymerase

 Transcription factors include activators and inhibitors of

transcription

 Constitutive transcription factors are present in all cells

 Specific transcription factors regulate gene expression in

particular cell types or in response to particular signals
How do specific transcription factors
react to extracellular stimuli?
Some transcription factors are
directly regulated by hormones
Genes induced by TNF
Transcription factors Membrane proteins
c-jun IL-2 receptor (T cells)
c-fos E-selectin / ELAM (endothelial cells)
c-Myc ICAM
NF-IL-6 (C/EBP )
IFN response factor 1
Secreted proteins
IL-2 (T cells)
Metabolic enzymes IL-6, IL-8, IFN-ß, G-CSF, GM-CSF
Cytosolic phospholipase A2 NGF, PDGF A chain,
Cyclo-oxygenases I and II procollagenase
Nitric oxyde synthase prochymotrypsin
Glucose transporter plasminogen activator
acute phase proteins (liver)
Transduction is the process by which
extracellular signals that cannot cross the
plasma membrane are relayed inside the cell

1. agonist binds to a receptor - e.g. TNF- / TNF receptors

2. The is triggers a series of interactions between the
receptor and cytosolic proteins
3. The signal reaches transcription factors in the nucleus
The TNF- signalling pathway
 How do the TNF receptors activate the
expression of the target genes?

 Clue1: Most target genes are regulated by the

same transcription factors, AP-1 and / or NF-B
 Regulatory elements in IL-8
gene promoter

GATA ISRE AP-1 NF-IL-6 NF-B CRE exon 1

- 151 - 145 - 125 - 91 - 81 - 69 +1

Key regulatory motifs

 Regulation of IL-8 gene
1 - Inactive (basal) state
p65
IB
ATF-2 p50

Cytoplasm

Nucleus
p50
Jun
fos p50

AP-1 NF-IL-6 NF-B exon 1

GACTCAG GTTGCAAATC TGGAATTTCC

- 125 - 91 - 81 +1
 Regulation of IL-8 gene:
Activated state

Cytoplasm
p50
p50
Nucleus
Jun p65
fos ATF-2
p50
AP-1 NF-IL-6 NF-B exon 1
GACTCAG GTTGCAAATC TGGAATTTCC

- 125 - 91 - 81 +1
Nuclear factor B
Discovery of NF-B: Expression of
immunoglobulin B light chain gene

 Induced in activated B cells by a transcription factor

that binds the B light chain gene promoter
 The factor was called Nuclear Factor (NF) -B
Biological roles of NF-B
 Innate immunity
 Adaptive immunity
 Inflammation
 Lymphopoesis
 Viral infections
 Cancer
 Apoptosis
 Development
NF-B is a family of proteins with similar but
not identical functions
Mammalian family members
 p65 (Rel-A, c-Rel)
 p50
 v-Rel
 Rel-B
 P105 (p50 precursor)
 P100 (p52)

Invertebrate homologues
 dorsal
 dif
NF-B proteins are dimers
 The prototypical NF-B is a p50/p65 hetero-dimer
 P50 is the DNA-binding subunit - recognises
GGGACTTTCC
 P65 is the trans-activating subunit - it binds to and
activates RNA polymerase II
 Some NF-B dimers are inactive (e.g. p50/p50);
They are constitutive inhibitory factors that prevent
gene expression
 Regulation of NF-B proteins
 Latent NF-B activating dimers
(p50/p65) reside in the cytoplasm
 They contain inhibitory domains or are
associated with inhibitory proteins called
IB
 Release of active NF-B involves a
proteolytic step
 Regulation of NF-B

1 - Activation of IB kinase

IKK
p65
TNF IB
receptor Ub p50
Ub
Ub
Ub

2 - Phosphorylation of IB by IB kinase

3 - Ubiquitination of phospho-IB
 Ubiquitin
 76-amino acid protein that is ubiquitously expressed.
ubiquitin PDB 1TBE
 Highly conserved in all eukaryotes

 Ubiquitination is the covalent linkage of the terminal carboxyl of ubiquitin to the

-amino group of a lysine residue of a target protein

 Once the first ubiquitin is attached, anothers can link to it, on either residue K29,
K48 or K63. This starts the building of a poly-ubiquitin chain

 As many as 10 ubiquitin can be linked in chains to the target protein

 K29- and K45-poly-Ubiquitinations mark proteins for degradation in the

proteasome

 Ubiquitin units are split off intact during protein breakdown and recycled
 Regulation of NF-B - part 2

4 - Proteolysis of ply(Ub)-IB

IKK
p65
TNF IB
receptor Ub p50
Ub
Ub
Ub

5 - Migration of p65/p50
(active NF-B) to nucleus
 Regulation of NF-B - part 3

p65
p50

TNF
receptor

6 - Binding of p65/p50 to B motifs

7 - Activation of transcription
Techniques for investigating
Transcription factors activity
Cell-free assays:
 - DNA footprinting
 - EMSA

In vivo assays
 - Reporter genes (see tomorrow)
DNAse I Footprinting
A protein bound to a specific DNA * *
sequence will interfere with the
digestion of that region by DNase I.
An end-labelled DNA probe is
incubated with a protein extract or a * *
purified DNA-binding factor.
The unprotected DNA is then partially
digested with DNaseI such that on
average every DNA molecule is cut
once.
Digestion products are then resolved Footprint:
by electrophoresis.
Region
Comparison of the DNase I digestion protected
pattern in the presence and absence from
of protein will allow the identification cleavage
of a footprint (protected region)
Denaturing PAGE
Electrophoretic Mobility Shift Assay
(EMSA)

Gel retardation assays,

Gel Shifts, Band Shift
Incubating a purified protein, or a
complex mixture of proteins e.g.
nuclear or cell extract, with a 32P
end-labelled DNA fragment
containing the putative protein
binding site (from promoter region).
Reaction products are then
analysed on a non-denaturing
polyacrylamide gel.
Specificity controls: excess
unlabelled probe, probes with
altered sequences
Gel Retardation with Antibody Supershift:
Use to identify proteins in gel-shift complex

- +
Ab Ab

* supershifted DNA

shifted DNA

free DNA
Gel Retardation with Protein-Induced DNA Bending:
If bending is induced, circularly permuted fragments will gel shift
differently.
The closer the binding site is to the fragment center the greater the shift.

shifted DNA

free DNA
Summary
 In this lecture you will have learned the meaning of the
terms cis-acting elements, promoter, enhancer, silencer
and the difference between constitutive and specific
transcription factors
 You will know how transcription factors functions and
why they need to have a two-domain structure
 You will know the structures of SP1 and NF-B and the
mechanism of regulation of the later