Transcript Guest lecture 3130 2015 - Scheid Signalling Lab @ York University

Action at a Distance
• Bacterial and eukaryotic enhancers
stimulate transcription even though located
some distance from their promoters
• Four hypotheses attempt to explain the
ability of enhancers to act at a distance
– Change in topology ie. supercoiling
– Sliding
– Looping
– Facilitated tracking
12-1
Hypotheses of Enhancer Action
Change in topology
Sliding
Looping
Facilitated tracking
12-2
Complex Enhancers
• Many genes can have more than one
activator-binding site permitting them to
respond to multiple stimuli
• Each of the activators that bind at these
sites must be able to interact with the
preinitiation complex assembling at the
promoter, likely by looping out any
intervening DNA
12-5
Control Region of the Metallothionine Gene
• The metallothionine gene product helps
eukaryotes cope with heavy metal poisoning
• Turned on by several different agents
• Complex enhancers enable a gene to respond
differently to different combinations of activators
• This gives cells exquisitely fine control over their
genes in different tissues, or at different times in
a developing organism
12-6
After identifying individual regions in the 5’ UTR of the Endo16 gene that bind
nuclear proteins, the isolated binding regions were fused to a reporter cassette and
reintroduced into sea urchin.
Expression was monitored and it was determined that some regions act alone and
others in combination with each other.
Very important for the fine control of gene expression required during development..
Architectural Transcription Factors
Architectural transcription factors are those
transcription factors whose sole or main
purpose seems to be to change the shape
of a DNA control region so that other
proteins can interact successfully to
stimulate transcription.
Important when control regions are in very
close proximity to one another.
12-9
• Short DNA acts as rigid rod
• Long DNA behaves more like a string and
can be easily manipulate
Example of Architectural Transcription
Factor: Control region of the human T-cell receptor
alpha chain (TCR ) gene
• Within 112 bp upstream of the start of
transcription are 3 enhancer elements
• These elements bind to:
– Ets-1, LEF-1, CREB
– LEF-1 alone cannot activate gene. Role?
• bends DNA at the minor grove by 130deg
12-11
• Used synthetic DNA containing LEF-1
binding site in electrophoretic assay to
show DNA bending
• When the site was positioned in the middle
of the synthetic DNA, movement through a
gel was greatly retarded
DNA Bending Aids Protein Binding
• The activator LEF-1 binds to the minor
groove of its DNA target through its HMG
domain and induces strong bending of
DNA
• LEF-1 does not enhance transcription by
itself
• Bending it helps other activators bind and
interact with activators and general
transcription factors
12-13
Enhanceosome
• An enhanceosome is a nucleoprotein complex
containing a collection of activators bound to
an enhancer in such a way that stimulates
transcription
• Ex. IFN-beta contains 8 binding sites which
must all be occupied by activators. The other
end of the simple/complex enhancer spectrum
• only activated when a cell is under attack by a
virus.
12-14
Dilema:
Some enhancers act at great distances
from their promoters.
ie. Drosophila cut locus is 85kb from
promoters.
Enhancer is likely to come in proximity
to other genes, how does the cell
prevent inappropriate activation?
Insulators
• Insulators can shield genes from activation by
enhancers (enhancer blocking activity)
• Insulators can shield genes from repression by
silencers (barrier activity). Prevent condensing
• Insulators define regions between DNA
domains. ie. Between enhancers and
promoters
12-17
Mechanism of Insulator Activity
• One mechanism which can be ruled out is that insulators
induce the condensation of DNA upstream of their location.
– If a gene were placed upstream of such an insulator, it would
always be silenced
– Experiments in Drosophila show that such genes can still be active
and can be activated by their own enhancers.
12-18
Mechanism of Insulator Activity
• Sliding model
– Activator bound to an
enhancer and stimulator
slides along DNA from
enhancer to promoter
• Looping model
– Two insulators flank an
enhancer, when bound
they interact with each
other isolating enhancer
12-19
Mechanism of Insulator Activity
• Sliding model
– Activator bound to an
enhancer and stimulator
slides along DNA from
enhancer to promoter
• Looping model
– Two insulators flank an
enhancer, when bound
they interact with each
other isolating enhancer
12-20
• However…some insulators work as single copies.
• ie. The Drosophila hairy-wing insulator
- single insulator = some insulator activity
- two insulators = no insulator activity
- insulator-enhancer-insulator = increased insulator activity
Model of Multiple Insulator Action
Canceling of insulator activity
12-23
Summary
• Some insulators have both enhancer-blocking and
barrier activities, but some have only one or the
other
• Insulators may do their job by working in pairs that
bind proteins that can interact to form DNA loops
that would isolate enhancers and silencers so they
can no longer stimulate or repress promoters
• Insulators may establish boundaries between DNA
regions in a chromosome
12-24
12.6 Regulation of Transcription Factors
• Several activators do not active transcription by contacting
the basal transcription apparatus directly. Rely on:
• Coactivators = no activator function on its own, but
collaborates with one or more activators to stimulate the
expression of a set of genes
12-25
Coactivator
Activator
12.6 Regulation of Transcription Factors
• Phosphorylation of activators can allow them to interact
with coactivators that in turn stimulate transcription
12-27
Model for the Activation of a Nuclear
Receptor-Activated Gene
12-28
12.6 Regulation of Transcription Factors
• Sometimes genes are inactivated by the destruction of their
activators
• Ubiquitylation of transcription factors can mark them for
– Destruction by proteolysis
– Stimulation of activity
• Sumoylation is the attachment of the polypeptide SUMO
which can target for incorporation into compartments of the
nucleus
• Methylation and acetylation can modulate activity
12-29
Ubiquitylation
• Normal function of ubiquitylation is to mark
proteins for destruction by the proteasome.
ie. Aprx 20% of all proteins are made incorrectly
and need to be quickly disposed of
• A fine balance may exist between coactivators
and corepressors which have ubiquitylating
ability
- ie. A corepressor may mark a coactivator for
destruction, tipping the scale towards repression
12-30
Activator Sumoylation
(SUMO or small ubiquitin like modifier)
• Sumoylation is the addition of one or more
copies of the 101-amino acid polypeptide
SUMO (Small Ubiquitin-Related Modifier)
to lysine residues on a protein
• Process is similar to ubiquitylation
• Results quite different – sumoylated
activators are targeted to a specific
nuclear compartment that keeps them
stable
12-32
Activator Acetylation
• Nonhistone activators and repressors can
be acetylated by HATs
• HAT is the enzyme histone
acetyltransferase which can act on
nonhistone activators and repressors
• Such acetylation can have either positive
or negative effects
- ex. p53 acetylation by coactivator p300
results in increased DNA binding
12-33
Signal Transduction Pathways
• Signal transduction pathways begin with a
signaling molecule interacting with a
receptor on the cell surface
• This interaction sends the signal into the
cell and frequently leads to altered gene
expression
• Many signal transduction pathways rely on
protein phosphorylation to pass the signal
from one protein to another
• This leads to signal amplification at each
step
12-34
Three pathways that use CBP/p300 to
mediate transcription activation
12-35
Ras and Raf Signal Transduction
12-36
Lecture key words
-
Cell cycle
Transcription factors
Phosphorylation
Heterodimerization
Immunohistochemistry
Immunprecipitation
Growth factors
Apoptosis
Colony formation
Nuclear localization
Consensus sequences
Motifs
CDCA7 | a case study in cellular
regulation
• Cell cycle control is the endgame of cellular regulation
- critical balance between proliferation and
apoptosis  CANCER
CDCA7 | a case study in cellular
regulation
• Cell cycle control is the endgame of cellular regulation
- critical balance between proliferation and
apoptosis  CANCER
• Modes:
-phosphorylation,
-subcellular localization
- heterodimerization
CDCA7 | What is known
• Myc and E2F target gene with peak expression at
1-S member of cell division cycle-associated
• G
Novel
familyoverexpressed in human tumors
• gene
Frequently
• JPO2 binds Myc and promotes Myc dependent
transformation
• JPO2 and CDCA7 share cysteine rich C-term
which may bind DNA
• Not known if CDCA7 interacts with Myc
Myc | Just the facts
• Discovered in Burkitt’s lymphoma
• patients
Member of bHLH-LZ family of transcription
•
•
•
•
•
•
factors
Requires heterodimerization with
Max to transactivate
Regulates the expression of
~10-15% of genes
Role in development, cell division, cell growth,
metabolism, angiogenesis
Early response gene induced by growth factors,
levels peak at G0-G1
Driving force of cell cycle and
malignant transformation
Active in 70% of human cancers
• ~100,000 cancer deaths per year in the US due to
Growth
Factors
Receptor
Tyrosine
Kinase
P
P
P
P
PIP3
PI3K PIP2
PDK1
P
P
AKT
CDCA7P
P
14-33
TOR
ricto
r
14-33
P
14-33
Cytoplasm
AKT
P
14-33
14-33
CDCA7P
14-33
AKT P
CDCA7
Myc
Myc
Transcription
Pro-apoptotic
Genes ?
Nucleus
CDCA7 | a case study in cellular
regulation
• Cell cycle control is the endgame of cellular regulation
- critical balance between proliferation and
apoptosis  CANCER
• Modes:
-phosphorylation
-subcellular localization
-heterodimerization
CDCA7 | CONSERVATION
AKT consensus site
CDCA7 T163
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish
1
24
49
69 78
112
190
R X R X X T/S F/L
R P R R R T
F
261
363
AKT kinase 0.005%
371
T163
humCDCA7
zinc finger
261
361
CDCA7 is phosphorylated at t163
Phosphotase
T163A
WT
T163A + CIP
Radioactivity and mutational
analysis
Vector
CDCA7+
CIP
CDCA7
• Many ways to prove phosphorylation
• Custom made antibody against
phospho-T163
-FLAG
T163A
-P-T163
Vector
CDCA7 is phosphorylated at t163
0
5
15
45
120
360
PDGF (min)
Treatments
w/ growth factors
-P-T163
-FLAG
Merge
1.0 2.2 3.6 4.7 4.0 3.9 Ratio P-T163/
Total CDCA7
T= 0’
T= 20’
T= 30’
Immunohistochemistry
T= 40’
T= 50’
T= 60’
CDCA7 is phosphorylated at t163
by AKT
Vector
Inhibitors
CDCA7
Akt inh VIII
IP: -FLAG
Blot: -P-T163
IP: -FLAG
Blot: -FLAG
CDCA7 | a case study in cellular
regulation
• Cell cycle control is the endgame of cellular regulation
- critical balance between proliferation and
apoptosis  CANCER
• Modes:
-phosphorylation
-subcellular localization
-heterodimerization
Where is cdca7 found?
-Flag
CDCA7
T163A
CDCA7
DAPI
CDCA7 | CONSERVATION
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish
24
49
69 78
112
1
190
261
363
371
T163
humCDCA7
NLS
?
157-186
NLS
?
zinc finger
261
RRPRRRTFPGVASRRNPERRARPLTRSRSR
How do we test for a nuclear localization signal?
Isolate region in question and test its ability to target an
innocuous protein to the nucleus
361
CDCA7 contains an NLS
• Passive diffusion into nucleus <45 KDa
SV40
SV40 KE
157-188
157-188 CDCA7 157-167 CDCA7
R176E
167-188 CDCA7 157-188 (T163A)
R176/184E
R171E
R171/176E
R184E
157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188
PHOSPHORYLATION ALTERS
LOCALIZATION
-CDCA7
Unstimulated
PDGF
PDGF + LY
Nuclei
Merge
CDCA7 | a case study in cellular
regulation
• Cell cycle control is the endgame of cellular regulation
- critical balance between proliferation and
apoptosis  CANCER
• Modes:
-phosphorylation
-subcellular localization
-heterodimerization
CDCA7 | CONSERVATION
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish
24
49
69 78
112
1
190
261
363
371
T163
humCDCA7
NLS
?
zinc finger
NLS
?
157-186
261
361
RRPRRRTFPGVASRRNPERRARPLTRSRSR
14-3-3 consensus binding site
R-[S/F/Y]-X-pS/T -X
cdcA7 T163
Mekk2 T283
R
G
R
R
R
K
T
T
F
F
-P
P
P
14-3-3 | Just the facts
• Large family of highly conserved, small, acidic
polypeptides of 28-33 kDa
• Seven different isoforms in humans, 14-3-3σ directly
implicated in cancer
• Binds to protein ligands at defined phosphoserine/threonine motif RSXpS/TXP
• Over 200 known ligands
• 14-3-3 regulates process relevant to cancer biology:
cell-cycle progression, apoptosis and mitogenic
signaling
14-3-3 | Modes of influence
• 14-3-3 exists as a dimer and offers two binding sites
for phospho-S/T
motifs protein for:
• Can
function as adaptor
a) two proteins that would otherwise not associate
b) one protein with two 14-3-3 motifs = high
affinity
Adapted from Hermeking, 2005
• Affects change by:
• Alteration of enzymatic activity – maintains RAF1 in
inactive state
•
•
•
Alteration of DNA-binding activity – increases p53
Sequestration
DNA-binding
after DNA damage
Altering
protein-protein
interactions
Sequestration - BAD, FKHRL1, HDAC5 and CDC25C
CDCA7 binds14-3-3 and is
phospho dependent
P165A
X pT X P
T163A
F164A
- R
R161A
R162A
-
R158A
P159A
R160A
Vector
Wildtype
14-3-3 consensus site
S/F/Y
Western blots
Blot:
-FLAG
-P-T163
-14-3-3
14-3-3 alters CDCA7 localization
-Flag
DAPI
CDCA7
T163A
CDCA7
R161A
CDCA7
R161A/
T163A
CDCA7
Is 14-3-3 masking the NLS within
the T163 region?
CDCA7 | What is known
• Myc and E2F target gene with peak expression at
1-S member of cell division cycle-associated
• G
Novel
familyoverexpressed in human tumors
• gene
Frequently
• JPO2 binds Myc and promotes Myc dependent
transformation
• JPO2 and CDCA7 share cysteine rich C-term
which may bind DNA
• Not known if CDCA7 interacts with Myc
CDCA7 binds the transcription
factor Myc
D(170-370) CDCA7
D(153-370) CDCA7
D(230-370) CDCA7
D(260-370) CDCA7
T163A CDCA7
D(112-137) CDCA7
D(1-146) CDCA7
D(1-172) CDCA7
D(1-202) CDCA7
D(1-234) CDCA7
WT CDCA7
Co-immunoprecipitation
CDCA7
WT
T163A
D(112-137)
D(1-146)
His-Myc Pulldown
Blot: -FLAG
D(1-172)
D(1-202)
D(1-234)
D(260-370)
D(230-370)
D(170-370)
D(153-370)
Input
Blot: -FLAG
+
+
+
+
+
+
+
-
So how does cdca7 affect
phenotype?
APOPTOSIS
PROLIFERATION
14-3-3/CDCA7 binding influence Mycinduced transformation
Colony formation assay
14-3-3/CDCA7 binding influence Mycinduced apoptosis
Rat1
Trypan blue exclusion
Myc-Rat1
Sh1-Myc-Rat1
Growth
Factors
Receptor
Tyrosine
Kinase
P
P
P
P
PIP3
PI3K PIP2
PDK1
P
P
AKT
CDCA7P
P
14-33
TOR
ricto
r
14-33
P
14-33
Cytoplasm
AKT
P
14-33
14-33
CDCA7P
14-33
AKT P
CDCA7
Myc
Myc
Transcription
Pro-apoptotic
Genes ?
Nucleus
summary
• CDCA7 is a novel target of AKT required for Mycapoptosis
• dependent
Phosphorylation
of T163 inhibits CDCA7/Myc
apoptosis by:
• Promoting 14-3-3 binding
• Disruption of Myc binding
• Shuttling to the cytoplasm
• Potential for medical intervention in Myc tumors
where AKT is dysregulated