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

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21419

100K b