2-2 Henderson

Epigenetic and genetic control of Arabidopsis meiotic recombination

Ian Henderson Department of Plant Sciences University of Cambridge

- High quality reference sequence - Extensive natural variation resources - 1,001 genomes project - Rich array of epigenomic maps - Great genetics - Huge array of mutants and genetic tools available - Fast progress, as long as biology occurs in this species

Arabidopsis is an key model species for discoveries in genome biology

Coordinates (Mb)

15 205 100

Historical cM/Mb versus DNA methylation

H2A.Z versus DNA methylation

Genes versus transposons Chromosome 3

Evolution of recombination

Eukaryotes

Archaebacteria

Eubacteria

Diploid

Haploid

Clonal

Sexual Fisher (1930) The Genetical Theory of Natural Selection Muller (1932) Am Nat

Crossover frequency in plant genomes

Wheat chromosome 3B 774 Mb : Choulet et al., (2014) Science

Recombination rate

Gene density

Arabidopsis thaliana chr1 30 Mb : Yelina et al., (2012) PLoS Genetics

Plant versus yeast versus mammalian hotspots

Mus musculus (prdm9-/-) SSDS Brick et al. (2012) Nature

S.cerevisiae SPO11-oligos Pan et al. (2011) Cell

Figure 2

a

bIntron/exon Intron/exon Intron/exonIntron/exon

H2A.Z LNDH3K4me3cM/Mb

Exon Intron

5

3

1

TSS 2 4 6 8

2.5

1.5

0.5

0.08

0.04

0

0.4

0.2

0

10 TSS 2 4 6 8 10 TSS 2 4 6 8 10TSS 2 4 6 8 10

3.5

Intron/exon

DNA methylation

0.05

0.03

0.01

TSS 2 4 6 8 10

H2A.Z LNDH3K4me3cM/Mb DNA methylation

-2000 +2000TSS

0.4

0.2

0

-2000 +2000TSS -2000 +2000TSS -2000 +2000TSS -2000 +2000TSS

2.0

1.0

0

0.2

0.1

0

0.3

0.8

0.4

0

1.2

c

0.4

0.2

0

-2000 +2000TSS -2000 +2000TSS -2000 +2000TSS -2000 +2000TSS -2000 +2000TSS

cM/M

b

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

2.0

1.0

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H2A.Z H3K4me3

0.4

0.2

0

0.2

0.1

0

0.3

0.8

0.4

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1.2LND DNA methylation

Coordinates (bp) Coordinates (bp) Coordinates (bp) Coordinates (bp) Coordinates (bp)


HotColdRandom

ALL

d e fmotif@TSShot vs cold vs random

motif vs cMMb vs H2AZ

CTT-motif

HotColdRandom

A.thaliana historical crossovers Choi et al. (2013) Nature Genetics

Arabidopsis historical crossover frequency

Cao .. Weigel (2011) Nat Genet Long .. Nordborg (2013) Nat Genet

Collaboration with Gil McVean (WTCHG, Oxford) Choi, Zhao .. Henderson (2013) Nat Genet

80 Eurasian vs 180 Swedish accessions

19.57 SNPs/kb vs 17.91 SNPs/kb

LDhat: Diallelic, >10% minimum allele frequency,

Nucleosomes, recombination and diversity at TSS cM/Mb SNPs

TSS TSS TSS

Merge

TSS TSS

Nucleosomes vs cM/Mb Nucleosomes vs SNPs

MNase digestion & sequencing

Figure 2

a



Exon Intron

5

3

1

TSS 2 4 6 8

2.5

1.5

0.5

0.08

0.04

0

0.4

0.2

0

10 TSS 2 4 6 8 10 TSS 2 4 6 8 10TSS 2 4 6 8 10

3.5

Intron/exon

DNA methylation

0.05

0.03

0.01

TSS 2 4 6 8 10


-2000 +2000TSS

0.4

0.2

0

-2000 +2000TSS -2000 +2000TSS -2000 +2000TSS -2000 +2000TSS

2.0

1.0

0

0.2

0.1

0

0.3

0.8

0.4

0

1.2

c

0.4

0.2

0


cM/M

b

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

2.0

1.0

0

H2A.Z H3K4me3

0.4

0.2

0

0.2

0.1

0

0.3

0.8

0.4

0




HotColdRandom

ALL



CTT-motif

HotColdRandom

Figure 2

a



Exon Intron

5

3

1

TSS 2 4 6 8

2.5

1.5

0.5

0.08

0.04

0

0.4

0.2

0

10 TSS 2 4 6 8 10 TSS 2 4 6 8 10TSS 2 4 6 8 10

3.5

Intron/exon

DNA methylation

0.05

0.03

0.01

TSS 2 4 6 8 10


-2000 +2000TSS

0.4

0.2

0

-2000 +2000TSS -2000 +2000TSS -2000 +2000TSS -2000 +2000TSS

2.0

1.0

0

0.2

0.1

0

0.3

0.8

0.4

0

1.2

c

0.4

0.2

0


cM/M

b

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

2.0

1.0

0

H2A.Z H3K4me3

0.4

0.2

0

0.2

0.1

0

0.3

0.8

0.4

0




HotColdRandom

ALL



CTT-motif

HotColdRandom

Crossovers and H2A.Z nucleosomes overlap at promoter hotspots

~10% of promoters are hotspots and contain the majority of crossovers H2A.Z data from Daniel Zilberman (UC Berkeley)

Choi et al., (2013) Nature Genetics Kyuha Choi

Pollen-typing: allele-specific PCR to amplify crossovers

Pollen-typing hotspots via sequencing

crossovers n=197

crossovers n=~1,500

RAC1 is a disease resistance gene & major historical crossover hotspot

A subset of R genes are the major Arabidopsis hotspots

Figure 2

a



Exon Intron

5

3

1

TSS 2 4 6 8

2.5

1.5

0.5

0.08

0.04

0

0.4

0.2

0

10 TSS 2 4 6 8 10 TSS 2 4 6 8 10TSS 2 4 6 8 10

3.5

Intron/exon

DNA methylation

0.05

0.03

0.01

TSS 2 4 6 8 10


-2000 +2000TSS

0.4

0.2

0

-2000 +2000TSS -2000 +2000TSS -2000 +2000TSS -2000 +2000TSS

2.0

1.0

0

0.2

0.1

0

0.3

0.8

0.4

0

1.2

c

0.4

0.2

0


cM/M

b

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

2.0

1.0

0

H2A.Z H3K4me3

0.4

0.2

0

0.2

0.1

0

0.3

0.8

0.4

0




HotColdRandom

ALL



CTT-motif

HotColdRandom

Figure 2

a



Exon Intron

5

3

1

TSS 2 4 6 8

2.5

1.5

0.5

0.08

0.04

0

0.4

0.2

0

10 TSS 2 4 6 8 10 TSS 2 4 6 8 10TSS 2 4 6 8 10

3.5

Intron/exon

DNA methylation

0.05

0.03

0.01

TSS 2 4 6 8 10


-2000 +2000TSS

0.4

0.2

0

-2000 +2000TSS -2000 +2000TSS -2000 +2000TSS -2000 +2000TSS

2.0

1.0

0

0.2

0.1

0

0.3

0.8

0.4

0

1.2

c

0.4

0.2

0


cM/M

b

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

0.4

0.2

0

2.0

1.0

0

H2A.Z H3K4me3

0.4

0.2

0

0.2

0.1

0

0.3

0.8

0.4

0




HotColdRandom

ALL



CTT-motif

HotColdRandom

H2A.Z deposition is required for crossover hotspot activity

SWR1 [ARP6] ATP, H2A.Z

Choi et al. (2013) Nat Genet

RNA-directed DNA methylation of hotspots

dsRNA!

siRNA!

Inverted repeat!

m m m m

RNA-directed DNA methylation of hotspots

Natasha Yelina

HAIRPIN

RNA-directed DNA methylation silences a hotspot

Epigenetic control of plant crossover frequency

Coordinates (Mb)

+1 nucleosome, H2A.Z

Coordinates (kb)

Spo11-oligo purification and sequencing

Lange .. Keeney (2011) Nature

Spo11-oligo purification from Arabidopsis

- Input=30 grams of flowers - IP and end-labelling shows ~30-40 bp species - Gel purify, G-tail and ligate Illumina adapters - Amplify and gel purify ~160 bp library

Kyuha Choi

Arabidopsis Spo11-oligos are upstream of TSSs

S.cerevisiae SPO11-oligos Pan .. Keeney (2011) Cell

Spo11-oligos @ TSSs

Spo11-oligos @ random

Spo11-oligos @ TSSs

Nucleosomes @ TSSs

Spo11-oligos vs cM/Mb

Spo11-oligos and crossovers are opposite across TSSs Spo11-oligos vs H2A.Z

Hotspot-enriched DNA motifs Choi et al., (2013) Nat Gen

A-rich

Spo11-oligos vs A-rich

CTT-repeat

Spo11-oligos vs CTT-repeat

Epigenetic control of plant crossover frequency

Coordinates (Mb)

+1 nucleosome, H2A.Z

Coordinates (kb)

Acknowledgements

Funding The Royal Society The Gatsby Foundation BBSRC The Isaac Newton Trust Rijk Zwaan Marie Curie ITN

Lab members Natasha Yelina Kyuha Choi Piotr Ziolkowski Xiaohui Zhao Christophe Lambing Heidi Serra Carsten Reinhard Patrick Diaz Charlie Underwood (CSHL) Catherine Griffin Divya Nageswaran

Collaborators Gil McVean (WTCHG, Oxford) Magnus Nordborg (GMI, Vienna) Greg Copenhaver (UNC, Chapel Hill) Chris Franklin (Birmingham) Christine Mezard (IJPB, Versailles) Rob Dirks (Rijk Zwaan)

Xiaohui Zhao Natasha Yelina Kyuha Choi

2-2 Henderson

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