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The chromosome pairing locus in wheat. Graham Moore. Prophase I. Pachytene. Diplotene. Diakinesis. Zygotene. Prophase II. Metaphase I. Metaphase II. Leptotene. Telophase II. Anaphase I. Anaphase II. Telophase I. Meiosis. Homologous chromosomes must recognise each other, - PowerPoint PPT Presentation
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Graham Moore
The chromosome pairing locus in wheat
Meiosis
1 Diploid cell
4 Haploid cells
Telophase II
Prophase I
DiploteneDiakinesis
PachyteneZygotene
LeptoteneMetaphase
I
Anaphase I
Telophase IProphase IIMetaphase
IIAnaphase
II
Paired homologues align
on plate
Homologues are separated
Sister chromatids are separated
Homologous chromosomes must recognise each other,pair correctly and recombine
Incorrect pairing leads to unbalanced gametes and infertility
Hexaploid wheatTriticum aestivum
2n = 6x =42
1 2 3 4 5 6 7
A
B
D
abcd
abcd
abcd
homologues
homoeologues
Ph1 locus
Prevents pairing of homoeologous chromosomes and unbalanced gametes
To be fertile, true homologues must pair at meiosis
Ph1’s effect-is importantagronomically Wheat-rye hybrid
Ph1+
Ph1-
Ph1 locus suppresses pairingbetween related chromosomes (homoeologous pairing)
If Ph1 locus is deleted, pairing is induced between related chromosomes
**Strategic Goal**Can we from understanding Ph1 locus, switch Ph1 on and off in elite wheat varieties crossed with wild species?
Wild species of wheat carry important traits fordisease resistance and salt, cold and drought tolerance
• No natural variation in Ph1 phenotype-Can’t create segregating populations, the starting point of all previous positional cloning projects
• EMS treatments don’t yield mutants • But X-Ray and fast neutron irradiation do
-A single deletion (ph1b) of the locus = 70Mb in size
• “Ph1 locus” arose on polyploidisation • The wheat genome is very large
What is Ph1 ?Cloning - the issues
Rice
Brachypodium
Wheat
DeletionsDeletionsDeletions
Defining the Ph1 locus
Griffiths et al 2006Al-Kaff et al 2008
Rice
Wheat
Cluster of Cyclin dependent kinase-like (Cdks) genes
= Ph1 locus
All defective genes
Large segment ofHeterochromatin inserted on polyploidisation
Hypothesis- the defective 5B Cdk copies are suppressing the activity of the related Cdks elsewhere in the genome.But how to take the study further in wheat?
Ph1 Cdk-like gene shows similarity to Cdk2
Cdk2 Ph1-cdk gene
Yousafzai and Al-kaff, 2010
Cdk2 in mammals affects histone H1 phosphorylation
Ph1 cdk+cyclinA compared to Cdk2+cyclinA
So as a defective locus, does Ph1 suppress Cdk activity, hence Histone H1 phosphorylation??
Protein modelling
Human Histone H1 phosphorylation sites
Is wheat histone H1 phosphorylated at Cdk2 consensus sites and is their phosphorylation altered by Ph1 ???
_
Does Ph1 affect histone HI phosphorylation? 1 80 H11_HUMAN (1) MSETVPPAPAAS--AAPEKPLAGKKAKKPAKAAAASKKKPAGPSVSELIVQAASSSKERGGVSLAALKKALAAAGYDVEK H1T_HUMAN (1) MSETVPAASASAGVAAMEKLPTKKRGRKPAGLISAS-RKVPNLSVSKLITEALSVSQERVGMSLVALKKALAAAGYDVEK H15_HUMAN (1) MSETAPAETATP--APVEKSPAKKKATKKAAGAGAAKRKATGPPVSELITKAVAASKERNGLSLAALKKALAAGGYDVEK H12_HUMAN (1) MSETAPAAPAAA--PPAEKAPVKKKAAKKAGGTP---RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK H13_HUMAN (1) MSETAPLAPTIP--APAEKTPVKKKAKKAGATAGK--RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK H14_HUMAN (1) MSETAPAAPAAP--APAEKTPVKKKARKSAGAAK---RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK Consensus (1) MSETAPAAPAAP APAEKTPVKKKAKK AGAAGAS RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK 81 160 H11_HUMAN (79) NNSRIKLGIKSLVSKGTLVQTKGTGASGSFKLNKKASSVETKPGASKVATKT--KATGASKKLKKATGASK---KSVKTP H1T_HUMAN (80) NNSRIKLSLKSLVNKGILVQTRGTGASGSFKLSKKVIPKSTRSKAKKSVSAKTKKLVLSR-----DSKSPK----TAKTN H15_HUMAN (79) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAT--PKKAKKAAGAKKAVKKTP H12_HUMAN (76) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKVKKAGGTKPKKPVGAAKKPKKAAGGATPKKSAKKTP H13_HUMAN (77) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEGKPKAKKAGAAKPRKPAGAAKKPKKVAGAATPKKSIKKTP H14_HUMAN (76) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTP Consensus (81) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAK KKPAGAAKKPKKATGAATPKKSAKKTP 161 231 H11_HUMAN (154) KKAKKPAATRKSSKNP---KKPKTVK-PKKVAKSPAKAKAVKPKAAKARVTKPKTAKPKKAAPKKK----- H1T_HUMAN (151) KRAKKPRATTPKTVRS--GRKAKGAK-GKQQQKSPVKARASK-----SKLTQHHEVNVRKATSKK------ H15_HUMAN (157) KKAKKPAAAGVKKVAK-SPKKAKAAAKPKKATKSPAKPKAVKPKAAKPKAAKPKAAKPKAAKAKKAAAKKK H12_HUMAN (156) KKAKKPAAATVTKKVAKSPKKAKVAK-PKKAAKSAAKAVKP-------KAAKPKVVKPKKAAPKKK----- H13_HUMAN (157) KKVKKPATAAGTKKVAKSAKKVKTPQ-PKKAAKSPAKAKAPKPKAAKPKSGKPKVTKAKKAAPKKK----- H14_HUMAN (156) KKAKKPAAAAGAKKAK-SPKKAKAAK-PKKAPKSPAKAKAVKPKAAKPKTAKPKAAKPKKAAAKKK----- Consensus (161) KKAKKPAAAA TKK A SPKKAKAAK PKKAAKSPAKAKAVKPKAAKPKAAKPK AKPKKAAPKKK
TPKKTPKK
TPVK
SPAKSPKK
Cdk2 phosphorylates human histone H1 at:
Consensus motifs (S/T) –P-X-K
Wheat Histone H1 phosphorylated at Cdk2-type consensus (S/T) –P-X-K sites
Cdk2-type phosphorylation on histone H1 is increased when Ph1 locus deleted
Progenesis
WT
PH
0.00
0.02
0.04
0.06
0.08
Azahara Martinez, Ali Pendle, Alex Jones, Isabelle Colas
2 Ph1copies
6 Ph1copies
Metaphase I pairing
Bivalents
0 Ph1copies
Mutivalents
ReducedHomologouspairing-univalents
Homologouspairing
Reduced homologousPairing-univalentsHomoeologousPairing-
Homologouspairing
increasedCdk activity
reducedCdk activity
Reduced homologousPairing-univalents
Reduced homologous pairing-univalents
John Doonan Moshe Feldman1966
Mutate or over-expressArabidopsis Cdk
Greer et al2012
So key question
Can we mimic the effect of deleting Ph1 by increasing histone H1 phosphorylation and henceInduce pairing between related chromosomes??
Deleting Ph1 increases Cdk activity- which increases histone H1 phosphorylation, And we have pairing between related chromosomes
Does increased Cdk-type activity induce pairing between related
chromosomes?
Detached tiller method
• Okadaic acid inhibits phosphatases
• Okadaic acid increases Histone H1 kinase activity
• Does Okadaic acid induce pairing between related chromosomes?
Okadaic acid induces pairing of related chromosomes in a wheat x rye hybrid
Homoeologous pairing
Wheat X Rye – Ph1 deleted
No okadaic acid – mostly univalents
Okadaic acid - bivalents & other chromosomes associations
So, okadaic acid treatment produces a similar effect on chromosome pairing of related chromosomes as deleting Ph1
Knight et al., 2010
Does Okadaic acidtreatment affect the same Cdk2 consensus site asPh1??? yes
The “Ph1” Cdk2-type consensus site shows increased phosphorylation with Okadaic acid treatment
Progenesis
WT
PH
0.00
0.02
0.04
0.06
0.08
Untreat
ed
OA_100
OA_200
0.000
0.005
0.010
0.015
0.020
0.025
rati
o p
ho
sph
o /
no
n-p
ho
sph
o
Increased histone H1 phosphorylation leads to more “open” /decondensed chromatin? How does this affect pairing /recombination?
Our detailed understanding of the genes involved in recombination has come from models: yeast, C elegans, Drosophila –Little is known about homologue recognition
42 chromosomes
6 chromosomesincorrectly paired during zygotene
6 chromosomes associating incorrectly resolved at pachytene
21 correct pairsat metaphase I
42 chromosomes 14 incorrectly paired during zygotene
14 chromosomes associating incorrectly not resolved at pachytene
14 chromosomesas univalents ormultivalents
14 correct pairs
Ph1 effect in wheat-don’t want incorrect associations here
Homologue recognition altered by Ph1
Ph1+
Ph1-
Resolution ofincorrect associations altered by Ph1
Holm, 1986, 1988
Homologuespresent
DiploteneDiakinesis
PachyteneZygotene
Leptotene Metaphase I
At both these stages condensation changes occur which would be affected by histone H1 phosphorylation
Ph1 effect in wheat hybridswant incorrect associations here
No increase in incorrect associations in absence of Ph1
Ph1+
Ph1-
Resolution ofincorrect associations altered by Ph1
Wheat-rye28 chromosomes
30% pairingonly atzygotene
1 pair at metaphase I
Wheat-rye28 chromosomes 7 pairs at
metaphase I
60% pairingat pachytene
Wang 1988, Wang and Holm 1988
No homologues
30% pairing
Little change at pachytene
30% pairingonly at zygotene
<20% pairing
>50% pairing
65% pairing
>90% pairing
Wheat-kotschyi35 chromosomes
Wheat-kotschyi35 chromosomes
increase in incorrect associations in absence of Ph1
Gillies 1987
1 pair at metaphase I
14 pairs at metaphase I
Effect on recognition??
Wheat Lack of Ph1 induces more incorrect pairing between homoeologues
Wheat-rye Lack of Ph1 does not induce more incorrect pairing between the homoeologues in the absence of homologues??
What is happening??
Wheat-kotschyi Lack of Ph1 induces more incorrect pairing between homoeologues in the absence of homologues??
But
telomeres
homologoussegments
What happens at the homologue recognition stage in wheat?
The identical chromosomes zip up from their telomere regions
Pilar Prieto et al 2004 Nat Cell Biol
Rye
segment
homologues
telomeres
“Decondensation‘Elongation” ofchromatin
This conformation change now reported in C elegans
Telomeres
Ph1+ Ph1+Ph1+
Ph1-
Ph1+
In wheat- chromosomes remodel in both the presence and absence of Ph1
but there is asynchronous chromatin remodelling in the absence of Ph1 correlating with more incorrect associations at homologue recognition stage
Interstitial segments- 15% of the wheat chromosome
Pilar Prieto et al 2004 Nat Cell Biol
Decondensation of chromosome segments linked to presence of homologous chromosomes and affects pairing
• Identical segments
Segments elongated Synchronously before clustering
100% pairing
• Similar segments
Segments elongated but
Not Synchronously
50% pairing
• Distinct segments
Reduced/Delayed
15% pairing
Isabelle Colas et al 2008 PNAS
No Pairing
Ph1+
Some Pairing
Ph1-
Pairing
Ph1-
Diploid-homologues
hybrid- Ph1-homoeologues
In wheat-rye hybrids-without Ph1 homoeologous wheat-rye chromosomes only trigger a partial conformation change hybrid- Ph1+
homoeologues
heterochromatin
telomeres
Partial changenot sufficient to induce more incorrect associations
Ph1+
Ph1-
Resolution ofincorrect associations altered by Ph1
Wheat-rye28 chromosomes
30% pairingonly atzygotene
1 pair at metaphase I
Wheat-rye28 chromosomes 7 pairs at
metaphase I
At pachytenePairing increasesto 60%
Wang 1988, Wang and Holm 1988
30% pairing
Little change at pachytene
30% pairingonly at zygotene
<20% pairing
>50% pairing
65% pairing
>90% pairing
Wheat-kotschyi35 chromosomes
Wheat-kotschyi35 chromosomes
Gillies 1987
1 pair at metaphase I
14 pairs at metaphase I
The processing of incorrect associations –ie associations between related chromosomes at pachytene
The maintenance of incorrect associations without Ph1 correlates with significant increase in pairing (synapsis)
Diverged (related) chromosomes pair via pegging process Homologueswith divergentsegments
telomeres
Chromosome segmentsremodel
Chromosome segmentspeg together forming a circular structure
Colas et al., PNAS 2008
Synaptic adjustmentNon-homologous pairingwithout Ph1at pachytene/diplotene
recombination
The altered chromatin with Ph1 affects Condensation/Decondensationchange atPachytene/Diplotene
Little Synaptic adjustmentwith Ph1
No recombinationChromosomes resolve
Synaptic adjustment
enhances correct chromosome segregation through regulating centromere pairing
Finally
Metaphase I Anaphase I
Meiosis I
Cohesion
Spindle force
Chiasmata
Separase
Cohesionlost in arms
Chromosome segregation via the centromeres
enhances correct chromosome segregation through regulating centromere pairing
Finally
Centromeres in many plants (wheat, Arabidopsis, brachypodium) pair independently from telomeres and the rest of the chromosomes
Synapsis can be initiated at centromeres independently from the telomeres
Ph1 increases the stringency of centromere pairing- thus the fidelity ofsegregation
Martinez-Perez et al., 2001 Nature
telomeres
Centromeres
(Phosphorylation (Histone) hI alters chromatin and hencehomologue recognition, synaptic adjustment andchromosome segregationduring meiosis
Conclusions
Thanks to… Genomics- Comparative-BAC library-mutantsTracie Draeger (Foote), Michael Roberts, Lijia Qu, Terry Miller, Steve Reader, Simon Griffiths, Sebastien Allouis, Rebecca Sharp, Kath Mortimer, Emilie Knight, Nadia Al-Kaff, Vera Thole, Ruoyu Wen
Ph1 into other species-Brachypodium/ArabidopsisRuoyu Wen, Vera Thole, Philippe Vain, John Doonan,Peter Shaw
Protein modelling-Faridoon Yousafzai, Nadia Al-Kaff
Phosphoproteomics- advanced mass specAzahara Martinez-Raminez, Ali Pendle, Isabelle Colas, Alex Jones, Peter Shaw
Cell biology – 3-D visualisation- Luis Aragon, Fadri Martinez, Pilar Prieto, Mike Wanous, Isabelle Colas, Emma Greer, Azahara Martinez-Raminez, Peter Shaw
W h e a tc h r 5 B
P h 1 reg ion in B rac h yp o d iu m (to sca le )
B rac h yp o d iu m re g io n 1 ge n es
B rac h yp o d iu m re g io n 2 ge n es
B rac h yp o d iu m B A C s
W h e a tc h r 5 A
W h e a tc h r 5 D
D ele tio nb rea kp o in ts
D ele tio nb rea kp o in ts
clk1
(cl
v)
stk1
vmp
1
hyp
4 (n
ew)
rdr1
zip
1 (z
inc)
hsp
20-1
raf1
(ra
8)
wd
b1
(wrk
y)
ps1
v
h51
l (h
ap5)
at1(
AtH
ypIII
)
hsp
90
gtl
1 (c
hic
k)
sbp
1 (s
elen
)
cmt1
(cc
om
)
ug
g1
(ud
p)
hyp
6 (A
tHyp
2)
fim
2
slp
1 (s
ub
)
hyp
3 (O
sHyp
II)
bn
a
12
36H
11 R
ev
a
12
36H
11 R
ev
b
16
77L
4 C
33
14
17M
11 3
50
Os
Hy
pI
91
6G3
Re
v
13
90E
11 U
ni
pep
1
fim
1
mic
1 (A
tHyp
7)
hyp
5 (m
arck
s)
R ice c hr 8 g e n es
P h 1 re gio n in ric e c h r 9 (to s ca le)
R ice c hr 9 g e n es
rlk2
hyp
scf
OsH
ypI CS
379
3
CS
977
raf
wd
b
ps1
v
h5l
1
atat el
p
gtl
cmt
hyp
clk
scf
hsp
90
raf
wd
b
h5l
gtl
cmt
pd
p
gtl
2
cmt2
fim
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hyp
slp
fim
2
C84
6C
S1
002
& 4
55
-9-1
CS
10
10
pfk
hyp
hyp
plp
1
1 2 F 1 6
1 7 8 3 A 2 2
1 3 9 0 E 1 1
7 1 9 O 7
6 0 6 L 2
1 0 5 1 I3
1 7 7 2 H 1 2
1 6 7 7 L 4
5 3 2 G 11
1 6 5 D 1 2
1 0 5 P 17
1 6 5 3 J 2
1 2 1 7 B 2
9 1 6 G 3
4 3 2 L 9
1 4 1 7 M 11
1 5 6 8 O 9
1 6 6 4 J 5
9 9 3 J2
2 8 2 J8
4 7 0 M 7
1 3 2 0 C 1 4
1 4 0 5 B 1 0
3 0 4 F 6
1 5 0 C 1 4
clk
1
rlk
1
cy
cd1
raf1
wd
b1
mic
1p
s1v
h5
l1
at1
rlp
1
AtH
yp
5
gtl
1sb
p1
cmt1
AtH
yp
2
fim
1
slp
1
Ne
w H
yp
cdc
2
pfk
1
Ma
rcks
plp
1
grp
1
KH
1
KH
4
KH
2
KH
5
KH
3
KH
6K
H7
1 2 0 1 K 2 3
hyp
2 (K
H2)
hyp
3 (K
H3)
hyp
4 (K
H4)
S ub te lom e ric in se rtion from ch r 3A
1 1 6 2 B 5
2 5 4 I1 0
1 8 9 8 B 1 9
6 7 H 7
2 2 I2
5 6 3 C 24
1 2 2 3 B 6
9 6 2 M 8
1 2 3 6 H 11
Te lo m e reC e n tr o m e r e
11 9 5 H 2 2
8 4 6 A 11
hsp
90cn
bsh
aw
aklb
p
fas
9 2 5 M 1 5
3 4 2 F 1 0
2 1 8 J 1 3
8 9 7 P 24
c d c 2h o m o lo g u e s
cdc2
-4
3 0 N 2
2 8 C 1 3
3 1 8 G 1
O u tsid e d e letio n re g io n
7 7 9 O 1 2
O u tsid e d e letio n re g io n
1 5 6 7 N 6
1 8 2 1 A 3
1 5 9 A 2
1 4 9 7 M 6
3 1 B 11
1 2 7 5 L 1 5
7 0 3 L 1 6
1 2 4 P 2 2
11 4 6 J 1 3
4 3 1 E 7
1 6 2 5 E 2 1
7 2 6 N 4
1 2 6 8 O 8
11 8 3 B 1 0
1 4 6 2 G 1 5
7 4 9 H 1 5 ( A /D )
3 8 7 C 1 9
7 8 0 H 1 6
1 8 4 0 B 1 7
8 7 0 L1 4
21419
100K b