African Trypanosomes

1
Regulation of Antigenic Variation
African Trypanosomes
2
Antigenic Variation
stochastic
in vitro in vivo
Immune
destruction
by host
Proliferation
Variant Surface Glycoprotein
1000 diff variants
Only one expressed
on the surface
20 nm thick
3
Regulation of Antigenic Variation
Immune
destruction
by host
Proliferation
Stochastic
No influenced by immune system
1,000 VSG genes
VSG
VSG genes
Telomere
VSG expression site
4
Regulation of Antigenic Variation
Immune
destruction
by host
Proliferation
1,000 VSG genes
VSG
VSG genes
Telomere
VSG expression site
5
Regulation of Antigenic Variation
Immune
destruction
by host
Proliferation
1,000 VSG genes
VSG
VSG genes
Telomere
VSG expression site
6
Antigenic variation systems use subtelomeric
localization
Telomeric repeat
Subtelomere
First chromosome-
internal gene
TTAGGGTTAGGGTTAGGG..
Protect chromosomes
Degradation
End-end fusions
Benefits of subtelomeric localization
Recognition by DNA repair
Reversible gene silencing
Loss of info by replication
Telomere position effect (TPE)
Allow rapid switching and exclusive expression
Ectopic recombination
Increase gene family diversification
Rapid gene switching
Why telomeric location ???
7
Subtelomeric localization of VSG gene in T. brucei
VSG
X
(19)
VSG
Benefits of subtelomeric localization
Need to regulate both
Reversible gene silencing
Telomere position effect (TPE)
Allow rapid VSG switching and exclusive expression
Ectopic recombination
Increase gene family diversification
Rapid VSG gene switching
8
Antigenic Variation
7
6
1
2
VSG
ESAGs
VSG genes
70 bp
50 bp
telomeric
1,000
Repetitive DNA sequences
9
Antigenic Variation
7
6
1
2
(1)
VSG
active
VSG genes
X
7
6
1
2
(19)
VSG
inactive
VSG switching
1. Recombination
mechanisms
duplicative transposition
telomeric exchange
2. In situ switch
10
Antigenic Variation
X
7
6
1
2
(19)
VSG
inactive
VSG genes
7
6
(1)
1
2
active
VSG
VSG switching
1. Recombination
mechanisms
duplicative transposition
telomeric exchange
2. In situ switch
How to follow switch events ??
11
Chromosome mapping of switch events
Telomere exchange
Duplicative transposition
WT Null
WT Null
WT Null
221 1.3
221 1.3
221 1.12

SLOT
221 ES
221 ES
Probe
1.12
221
1.3
Laura Cliffe
12
Why multiple Expression Sites?
6
4
2
7
7
6
1
2
(1)
70 bp
50 bp
ESAGs
VSG genes
X
7
6
1
2
(19)
(200)
177 bp
1. Recombination
Dominant mechanism
duplicative transposition
telomeric exchange
2. In situ switch
13
Transferrin receptor hetero-dimer
Expressed on surface flagella pocket
binds iron
14
Transferrin receptor in the flagellar pocket
Tf-R
ESAGs
VSG
7
6
5
4
8
3
2
1
VSG surface coat
flagellum
mitochondrion
VSG
Tf-R
nucleus
flagellar pocket
kinetoplast
Membrane of the flagellar pocket
Allows the uptake of iron
15
Why Multiple Expression Sites ?
2
4
6
7
Cow
Each ESAG 6 and 7 in ESs are Slightly different
X
7
2
4
6
Dog
Resulting in receptors with differing affinities
for transferrin from different hosts
X
7
6
2
4
Sheep
X
6
7
2
4
Human
Trypanosomes growing in Cow serum-----------put
into dog serum ????
16
Why Multiple Expression Sites ?
ESAG 6 and 7
X
6
2
4
7
Cow
Hetero-dimer transferrin receptor
7
6
2
4
Transferrin is an iron containing molecule
essential for growth
Dog
X
7
6
2
4
Each ESAG 6 and 7 are slightly different
Sheep
X
6
7
2
4
Differences reflected in differing affinities for
transferrin
Human
Experiment
Trypanosomes growing in Cow serum-----------put
into dog serum ????
Select for trypanosomes that have switched
17
Why Multiple Expression sites ?
X
6
2
4
7
Cow
7
6
2
4
Dog
X
7
6
2
4
Sheep
X
6
7
2
4
Human
Allowed host range expansion
What is the consequence of choice during
infection ?
18
Host range of Trypanosoma brucei
Buffalo
Spotted hyena
Waterbuck
Wild dog
Cokes Hartebeest
Buffalo
Spotted hyena
Wild dog
Cokes Hartebeest
Waterbuck
Lion
Zebra
Giraffe
Reedbuck
Eland
Zebra
Giraffe
Reedbuck
Lion
Eland
Hippo
Warthog
Bushbuck
Impala
Cattle
Hippo
Warthog
Bushbuck
Impala
Cattle
19
Host range of Trypanosoma brucei
Buffalo
Spotted hyena
Waterbuck
Wild dog
Cokes Hartebeest
Buffalo
Spotted hyena
Wild dog
Cokes Hartebeest
Waterbuck
Lion
Zebra
Giraffe
Reedbuck
Eland
Zebra
Giraffe
Reedbuck
Lion
Eland
Hippo
Warthog
Bushbuck
Impala
Cattle
Hippo
Warthog
Bushbuck
Impala
Cattle
20
Multiple ESs leads to multiple Mechanisms
6
4
2
7
(1)
70 bp
50 bp
ESAGs
VSG genes
1,000
X
7
6
4
2
(19)
(gt100)
177 bp
1. Recombination
Dominant mechanism
duplicative transposition
telomeric exchange
2. In situ switch
Early infection
21
Mechanisms of VSG Switching
Monomorphic
Pleomorphic
667, 927
427
Strain
Lab adapted
Lifecycle
Experimental line of choice (?)
10-2 - 10-5
10-6 - 10-7
Switch rate
slow
rapid
Recombination
Recombination
Switch Mechanism
In situ switch
In situ switch
22
Regulation of Antigenic Variation
(1)
VSG
active
VSG genes
1,000
X
(19)
VSG
inactive
Why is only one expression site active ?
ESB localization
How are the inactive ESs stably repressed ?
Lack of Pol I ?
23
Regulation of Antigenic Variation
(1)
VSG
active
VSG genes
1,000
X
(19)
VSG
inactive
Why is only one expression site active ?
ESB localization
How are the inactive ESs stably repressed ?
Lack of Pol I ?
Active repression mechanisms
chromatin structure ?
Regulation of telomeric VSG DNA recombination ?
DNA modification chromatin structure
24
(No Transcript)
25
(No Transcript)
26
Histone methytransferase
Dot1
H3 lysine 76
27
DNA methylation and histone modification
MBP
MBP
5MeC DNA binding proteins


Recruit Histone methylase
3HC
5-methylcytidine
28
Histone Code
Dot1
K79
29
(No Transcript)
30
Chromatin structure
X
7
6
1
2
(19)
VSG
inactive
7
6
(1)
1
2
active
VSG
ISWI (ATPase/helicase)
molecular motor
Slight de-repression of ES transcription
31
Chromatin structure
X
7
6
1
2
(19)
VSG
inactive
7
6
(1)
1
2
active
VSG
ISWI (ATPase/helicase)
Slight de-repression of ES transcription
32
Dot1 Histone H3K76 methylase
X
7
6
1
2
(18)
VSG
inactive
7
6
(1)
1
2
active
VSG
X
7
6
1
2
VSG
inactive
Dot1 KO (disruption of telomeric silencing)
Slow rate of silencing previously active ES
33
Dot1 Histone H3K76 methylase
X
7
6
1
2
(18)
VSG
inactive
7
6
1
2
active
VSG
Slow silencing
7
6
1
2
active
VSG
Dot1 KO (disruption of telomeric silencing)
Slow rate of silencing previously active ES
34
How are the inactive ESs stably repressed?
PvuII
PstI
PvuII
(1)
VSG
active
1984
PvuII
PstI
PvuII
X
(19)
inactive
Blocked Restriction Sites Within Silent ESs
DNA Modification?
35
The Novel Base J
J-DNA
Base J
Glucose
Thymine
1993 J.H. Gommers-Ampt
36
Consequence of glycosylated DNA ????
J
70 bp
VSG
50 bp
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
X
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
X
37
Knock-Out J-Synthesis Pathway in T. brucei
1993
structure
(1)
VSG
active
X
1999
(19)
localization
X
2007
X
Base J function
Loss of VSG gene regulation ??
38
Regulation of J-Biosynthesis
Thymidine- hydroxylase
Glucosyl- transferase
(TH)
(GT)
HOMedU
b-D-glucosyl-HOMedU
dT
PC
BS
-

HMU
Why do procyclics lack J ?
100 ng
Lack thymine hydroxylase (?)
50
25
DNA titration
12
6
3
1.5
Anti-J dot blot
39
Regulation of J-Biosynthesis
Thymidine- hydroxylase
Glucosyl- transferase
(TH)
(GT)
HOMedU
b-D-glucosyl-HOMedU
dT
PC
BS
-

HMU
Why do procyclics lack J ?
100 ng
Lack thymine hydroxylase (?)
50
25
DNA titration
12
JBP1
J-DNA binding
TH
6
JBP2
TH
SWI2/SNF2
3
1.5
Thymine-hydroxylase motif
Anti-J dot blot
40
JBP1 Binds J-DNA and Stimulates J Synthesis
JBP1 KO
ACCCTAACCCTAACCCTAAC TGGGATTGGGATTGGGATTG
Tel-T

Tel-OH
Tel-J
Tel-T
JBP1
JBP -/-
WT
-OH
ACCCTAACCCTAACCCTAAC TGGGATTGGGATTGGGATTG
Tel-OH
Bound

DNA titration
Glc
-O-
ACCCTAACCCTAACCCTAAC TGGGATTGGGATTGGGATTG
Free
Tel-J

Kd 90 nM
JBP1
J-DNA binding
L
L
L
H
D
H
R
406
411
431
189
191
239
255
How does it bind J-DNA ??
41
JBP2 Induces De-Novo J-Synthesis
Tubulin
Tubulin
Phleo
JBP
GFP
array
array
Anti-GFP
JBP1
Stimulate J synthesis
J-DNA binding
JBP2
SWI2/SNF2
Stimulates de-novo J
ATP-dependent
34 identity
47 similarity
DNA helicase
42
JBP2 Induces De-Novo J-Synthesis
Tubulin
Tubulin
Phleo
JBP
GFP
array
array
Anti-GFP
BS
PC
PC
J1
J2
WT
WT
J1KO
J2
J2/J1
JBP1
Propagates J synthesis
J-DNA binding
TH
JBP2
SWI2/SNF2
TH
Stimulates de-novo J
Thymine-hydroxylase motif
AlkB-like hydroxylase domain
43
Thymidine hydroxylase domain of JBP2 / JBP1 ?
HO
JBP1 JBP2
Thymine
Hydroxymethyluracil
O2
CO2


2-oxoglutarate
Succinate
HCHO
OH
Fe2
AlkB
Thymine
3-methylthymine
44
Iron and 2-Oxoglutarate Hydroxylase Family
Conserved structural fold eight ?-strands
Iron and Oxoglutarate binding
H1-X-D-Xn-H2-Xn-R motif
Essential residues
Strategy
Mutate JBP1 and JB2
J-DNA binding
TH
TH
SWI2/SNF2
H-X-D-Xn-H-Xn-R
H189A
D191A
H239A
Stimulate J-biosynthesis?
R255A
V258A
Zhong Yu et. al., NAR 2007 Laura Cliffe et.
al., NAR 2009
45
JBP2 Stimulates HOMedU Formation in E. coli
Anti-HOMedU IP assay
E. coli expression
E. coli DNA
- IPTG
7
8
190
7
6
120
6
5
5
4
85
CPM x 104
CPM x 104
4
3
3
60
2
2
50
1
1
0
1 mM
5 mM
HMU
?-JBP2
IPTG
Fe2
DNA oligos
pHOMedU
OH
JBP2
12
..ATTGCT..
..ATTGCT..
pA
10
8
CPM x 104
pT
6
4
pG
2
Unmod
HOMedU
Anti-HOMedU IP
Robbie Southern
46
Regulation of J-Synthesis by Two Thymidine
Hydroxylases
O2
CO2


Succinate
2-oxoglutarate
Glc
O
HO
Fe2
GT
JBP1 JBP2
J
Thymine
Hydroxymethyluracil
Why are two thymidine hydroxylases needed ??
47
Deletion of JBP2 and JBP1 Ablates J-Biosynthesis
X
T
T-OH
T-O-Glc
TH
GT
JBP2/ JBP1
J2 J1 ddKO
JBP1 dKO
JBP2 dKO
WT BF
HMU
WT PC
Glucosyl-transferase still present
JBP2 and JBP1 rescue
JBP2/JBP1 Thymidine hydroxylases
?-J dot blot
Rudo Kieft and Laura Cliffe
48
Lack of Base J and Regulation of VSG Expression
221
6
7
2009
(1)
Base J function
7
6
X
J Null cell line
(19)
6
7
X
1. De-regulation of expression sites
2. Increased VSG switching rate
3. Repeat stability
49
Does J play a role in expression site silencing ?
WT cells
VSG
VSG
6
7
Active
J J J J J J J J
X
VSG
VSG
6
7
Silent
X
J J J J J J J J
VSG
VSG
6
7
Silent
90 6/7 mRNA from 221ES
RT-PCR analysis
VSG
7
6
Cross-reactive primers
50
J plays no role in Transcriptional silencing
J null cells
VSG
6
7
Active
X
transcripts from 221 ES
VSG
6
7
Silent
X
VSG
6
7
Silent
J null
WT
Laura Cliffe
51
J null cells show enhanced switching in vitro
J null 100 VSG 221
WT cells show stable expression of 221 VSG
52
WT
Pre switch
In situ is dominant switch mechanism in WT cells
VSG
6
7
X
VSG
6
7
Post
X
VSG
6
7
VSG
6
7
53
WT
Pre switch
VSG
6
7
X
VSG
6
7
Post
VSG
6
7
100 switch by in situ switching
VSG
6
7
VSG
6
7
54
WT
J Null
Pre switch
VSG
VSG
6
7
6
7
X
X
VSG
VSG
6
7
6
7
Post
VSG
VSG
6
7
6
7
VSG
VSG
6
7
6
7
VSG
VSG
6
7
6
7
55
J Null
100 switch by Recombination
VSG
6
7
X
VSG
6
7
VSG
ES
221
1.3
VSG
6
7
1.6
221
221
1.8
221
1.11
221
VSG
1.12
6
7
Unch
221
Unch
221
Unch
221
VSG
6
7
Laura Cliffe
56
Nature of Homologous recombination?
Telomere exchange
Duplicative transposition
VSG
VSG
6
7
6
7
VSG
X
VSG
6
7
VSG
VSG
6
7
6
7
57
Chromosome mapping of switch events
Telomere exchange
Duplicative transposition
WT Null
WT Null
WT Null
221 1.3
221 1.3
221 1.12

SLOT
221 ES
221 ES
Probe
1.12
221
1.3
Laura Cliffe
58
Increased recombination at 70bp repeats ?
Telomere exchange
Duplicative transposition
VSG
VSG
6
7
6
7
VSG
X
VSG
6
7
RAD51 foci ??
dsDNA breaks
59
Increased RAD51 Foci in the J-Null
DIC
DAPI
Anti-RAD51
WT
J-Null
Anti-RAD51 Richard McCulloch, Univ. of Glasgow
2007/2008 Biology of Parasitism course
60
Base J stabilizes repetitive DNA
Telomere exchange
Duplicative transposition
VSG
VSG
6
7
6
7
VSG
X
VSG
6
7
70 bp repeats
Regulates homologous recombination
61
Consequence of glycosylated DNA ????
J
70 bp
VSG
50 bp
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
X
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
X
62
Global increase in homologous recombination????
Increased transfection efficiency in the J null
transfectants / 106 cells
Tubulin
177 bp repeats
Targeted region
63
Acknowledgments
Robert Sabatini
University of Georgia, Athens
Biochemistry and Molecular Biology
Rudo Kieft
Laura Cliffe
Shanda Birkeland
Robbie Southern
Dilrukshi Ekanayake Marion Marshall
Verena Brand
Piet Borst
Courtney DiPaolo
NKI, Amsterdam
Kate Sweeney
Mike Cross
Zhong Yu
Paul-Andre Genest
Saara Vainio