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12-1
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
12-2
Hypotheses of Enhancer Action
Change in topology
Sliding
Looping
Facilitated tracking
12-5
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
12-6
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
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..
12-9
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.
• Short DNA acts as rigid rod
• Long DNA behaves more like a string and can be easily manipulate
12-11
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
• 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
12-13
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
12-14
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.
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?
12-17
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
12-18
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.
12-19
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
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
• 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
12-23
Model of Multiple Insulator Action
Canceling of insulator activity
12-24
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
12-25
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
Activator
Coactivator
12-27
12.6 Regulation of Transcription Factors
• Phosphorylation of activators can allow them to interact with coactivators that in turn stimulate transcription
12-28
Model for the Activation of a Nuclear Receptor-Activated Gene
12-29
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
12-30
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
12-32
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
12-33
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
12-34
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
12-35
Three pathways that use CBP/p300 to mediate transcription activation
12-36
Ras and Raf Signal Transduction
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
• Novel member of cell division cycle-associated gene family
• Myc and E2F target gene with peak expression at G1-S
• Frequently overexpressed in human tumors
• 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
• 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 changes in Myc
• Early response gene induced by growth factors, levels peak at G0-G1
14-3-3
14-3-3
P
P P
PPIP2
PIP3
rictor
TORTOR
PDK1
P
P
P
P
14-3-3
14-3-3
Transcription Pro-apoptotic Genes ?
P
P
14-3-3
14-3-3
14-3-3
14-3-3
14-3-3
14-3-3
CDCA7CDCA7
14-3-3
14-3-3
CDCA7CDCA7
CDCA7CDCA7
Growth Factors
ReceptorTyrosineKinase
P
P
Cytoplasm
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
humCDCA7
1 371
361261zinc finger
AKT consensus site R X R X X T/S F/LCDCA7 T163 R P R R R T F
T163
AKT kinase 0.005%
24 49
>90% conservedhuman monkeydogmousechickenfrogzebrafish
69 78 112 261 363190
CDCA7 | Conservation
CDCA7 is phosphorylated at t163
WT
T163A
CD
CA
7
CD
CA
7+
CIP
T16
3A
T16
3A +
CIP
Vec
tor
-FLAG
-P-T163
• Custom made antibody against phospho-T163
• Many ways to prove phosphorylation
Radioactivity and mutational analysis
Phosphotase
-FLAG
-P-T163
Ve
cto
r0 5 15 45 120 360 PDGF (min)
Merge
Ratio P-T163/Total CDCA7
1.0 2.2 3.6 4.7 4.0 3.9
T= 0’ T= 20’ T= 30’
T= 40’ T= 50’ T= 60’
CDCA7 is phosphorylated at t163
Treatmentsw/ growth factors
Immunohistochemistry
Vec
tor CDCA7
Akt inh VIII
IP: -FLAGBlot: -P-T163
IP: -FLAGBlot: -FLAG
CDCA7 is phosphorylated at t163 by AKT
Inhibitors
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
T163A CDCA7
CDCA7
-Flag DAPI
Where is cdca7 found?
humCDCA7
1 371
361261zinc finger
T163
24 49
>90% conservedhuman monkeydogmousechickenfrogzebrafish
69 78 112
NLS?
NLS?
261 363190
157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR
CDCA7 | Conservation
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
CDCA7 contains an NLS
157-167 CDCA7
167-188 CDCA7 157-188 (T163A)
157-188 CDCA7
SV40 SV40 KE 157-188 R171E
R176E
R184ER176/184E
R171/176E
157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188
• Passive diffusion into nucleus <45 KDa
Phosphorylation alters localization-CDCA7 Nuclei Merge
Unstimulated
PDGF
PDGF + LY
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
humCDCA7
1 371
361261zinc finger
14-3-3 consensus binding site R-[S/F/Y]-X-pS/T -X -P
cdcA7 T163 R R R T F PMekk2 T283 G R K T F P
T163
24 49
>90% conservedhuman monkeydogmousechickenfrogzebrafish
69 78 112
NLS?
NLS?
261 363190
157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR
CDCA7 | Conservation
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 phospho-serine/threonine motif RSXpS/TXP
• 14-3-3 regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling
• Over 200 known ligands
14-3-3 | Modes of influence
• 14-3-3 exists as a dimer and offers two binding sites for phospho-S/T motifs• Can function as adaptor protein for:a) two proteins that would otherwise not associateb) one protein with two 14-3-3 motifs = high affinity
• Affects change by:• Alteration of enzymatic activity – maintains RAF1 in
inactive state• Alteration of DNA-binding activity – increases p53
DNA-binding after DNA damage• Sequestration - BAD, FKHRL1, HDAC5 and CDC25C
are localized to cytoplasm• Altering protein-protein interactions - reduced affinity
of CDC25A to CDC2• Adaptor protein functions – bridging of RAF1 to BCR
• Sequestration • Altering protein-protein interactions
Adapted from Hermeking, 2005
CDCA7 binds14-3-3 and is phospho dependent
-FLAG
-P-T163
-14-3-3
Vec
tor
P16
5A
F16
4AT
163A
R16
2A
R16
1A
R16
0A
P15
9A
R15
8A
Wild
type
Blot:
14-3-3 consensus site - - R X pT X PS/F
/Y
Western blots
14-3-3 alters CDCA7 localization
R161A CDCA7
T163A CDCA7
R161A/T163A CDCA7
CDCA7
-Flag DAPI
Is 14-3-3 masking the NLS within the T163 region?
CDCA7 | What is known
• Novel member of cell division cycle-associated gene family
• Myc and E2F target gene with peak expression at G1-S
• Frequently overexpressed in human tumors
• 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
WTT163A
(112-137)
(1-146)
(1-172)(1-202)
(1-234)
(260-370)
(230-370)
(170-370)
(153-370)
++++
+++-
---
CDCA7
His-Myc PulldownBlot: -FLAG
InputBlot: -FLAG
WT
CD
CA
7
T16
3A C
DC
A7
(11
2-13
7) C
DC
A7
(1-
146)
CD
CA
7(
1-17
2) C
DC
A7
(1-
202)
CD
CA
7(
1-23
4) C
DC
A7
(26
0-37
0) C
DC
A7
(23
0-37
0) C
DC
A7
(17
0-37
0) C
DC
A7
(15
3-37
0) C
DC
A7
CDCA7 binds the transcription factor Myc
Co-immunoprecipitation
So how does cdca7 affect phenotype?
Apoptosis
proliferation
14-3-3/CDCA7 binding influence Myc-induced transformation
Colony formation assay
Rat1
Myc-Rat1
Sh1-Myc-Rat1
14-3-3/CDCA7 binding influence Myc-induced apoptosis
Trypan blue exclusion
14-3-3
14-3-3
P
P P
PPIP2
PIP3
rictor
TORTOR
PDK1
P
P
P
P
14-3-3
14-3-3
Transcription Pro-apoptotic Genes ?
P
P
14-3-3
14-3-3
14-3-3
14-3-3
14-3-3
14-3-3
CDCA7CDCA7
14-3-3
14-3-3
CDCA7CDCA7
CDCA7CDCA7
Growth Factors
ReceptorTyrosineKinase
P
P
Cytoplasm
Nucleus
summary• CDCA7 is a novel target of AKT required for Myc-
dependent apoptosis• 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