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Clustered cis-regulatory elements underlie adaptive divergence in sticklebacks Felicity Jones Friedrich Miescher Laboratory of Max Planck Society Tbingen, Germany Male threespine stickleback Molecular mechanisms of adaptation &

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Clustered cis-regulatory elements underlie adaptive divergence in sticklebacks

Felicity Jones

Friedrich Miescher Laboratory

  • f Max Planck Society

Tübingen, Germany

Male threespine stickleback

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SLIDE 2

3

Molecular mechanisms of adaptation & speciation

2 1 1 The regulatory control of adaptive divergence in gene expression

Functional dissection of adaptive regulatory elements The evolution & role of recombination during adaptive divergence

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Threespine sticklebacks have undergone a recent adaptive radiation

Extensive morphological, physiological, behavioural diversity

Marine Marine Freshwater eshwater

Bell & Foster (1994)

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SLIDE 4

Marine Marine Freshwater eshwater

Repeated & independent evolution provides biological replicates of the evolutionary process

Limnetic Limnetic Benthic Benthic

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SLIDE 5

Jones et al (2012) Nature

High resolution map of 81 parallel marine-freshwater adaptive loci (FDR 0.02)

Whole genome sequencing of marine–freshwater species pairs revealed parallel adaptive divergence at ~81 predominantly intergenic genomic loci

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SLIDE 6

Jones et al (2012) Nature

Suggests two molecular mechanisms play a role in parallel adaptive divergence of sticklebacks: Regulatory mechanisms

1 1

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SLIDE 7

Jones et al (2012) Nature

Regulatory mechanisms

1 1

How is adaptive divergence in gene expression regulated?

cis

mRNA

What are the mutations & functional elements?

Suggests two molecular mechanisms play a role in parallel adaptive divergence of sticklebacks:

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SLIDE 8

0.5cM/Mb

Adaptive loci (marine-freshwater divergence)

The genomic recombination landscape

shapes the loci of adaptation

2

Jones et al (2012) Nature

Suggests two molecular mechanisms play a role in parallel adaptive divergence of sticklebacks:

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SLIDE 9

How is adaptive divergence in gene expression regulated?

Identifying adaptive gene expression differences using comparative transcriptomics

Marine Freshwater eshwater Dr Jukka-Pekka Verta Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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SLIDE 10

How is adaptive divergence in gene expression regulated?

Identifying adaptive gene expression differences using comparative transcriptomics

Marine Freshwater eshwater Genes Genes Marine Marine Expr Expression ession (RPKM) (RPKM)

RNAseq RNAseq of

  • f Gill

Gill Tissue Tissue

Freshwater eshwater Expr Expression ession (RPKM) (RPKM) Dr Jukka-Pekka Verta Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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SLIDE 11

How is adaptive divergence in gene expression regulated?

Identifying adaptive gene expression differences using comparative transcriptomics

Marine Freshwater eshwater Genes Genes Marine Marine Expr Expression ession (RPKM) (RPKM)

RNAseq RNAseq of

  • f Gill

Gill Tissue Tissue

Freshwater eshwater Expr Expression ession (RPKM) (RPKM) Composite PC Composite PC Freshwater eshwater-lik

  • like

e Marine-lik Marine-like e

Adaptive gene expression differences (parallel divergence in gene expression)

Dr Jukka-Pekka Verta Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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SLIDE 12

Molecular signatures of natural selection acting on genes with parallel gene expression divergence

control loci loci with parallel expression divergence control loci control loci loci with parallel expression divergence loci with parallel expression divergence

Elevated genetic divergence (FST MAR v FW) around TSS Reduced nucleotide diversity (pi) around TSS Marine Freshwater

Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932 Marine-Freshwater

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SLIDE 13

cis

mRNA

trans

How are adaptive expression differences controlled? trans vs cis-regulation

Marine Freshwater eshwater F1 Hybrids F1 Hybrids

Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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SLIDE 14

Allele-Specific Expression assays: RNAseq of F1 hybrids to determine cis- vs trans-regulation

Parental Expression Divergence (log freshwater/marine) Allele specific expression in F1 Hybrid (log freshwater allele /marine allele) Genes regulated in trans

Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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SLIDE 15

Cis-regulation is the predominant mechanism underlying gene expression divergence

Genes regulated in cis Genes regulated in trans

Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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Tyne yne Forss

  • rss

Shiel Shiel Lit Little C tle Campbell ampbell

cis cis tr trans ans cis+tr cis+trans ans cis cis-tr

  • trans

ans compensatory compensatory cis cis tr trans ans cis+tr cis+trans ans cis cis-tr

  • trans

ans compensatory compensatory cis cis tr trans ans cis+tr cis+trans ans cis cis-tr

  • trans

ans compensatory compensatory cis cis tr trans ans cis+tr cis+trans ans cis cis-tr

  • trans

ans compensatory compensatory

0.25 0.20 0.15 0.10 0.05 0.00

Proportion of Transcripts

Marine Freshwater eshwater

Cis-regulation is the predominant in multiple mar-fw pairs (and is associated with the degree of genome-wide genetic divergence)

1.62M 1.54M 2.27M 2.75M

Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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SLIDE 17

Gene regulatory mechanisms and their magnitude evolve in parallel across populations

Degree of Parallel Divergence in Expression

Genes upregulated in freshwater relative to marine Genes with no difference in expression

Correlation in cis-regulation among populations (Pearson’s r)

Verta & Jones (in review) BioRxiv doi: https://doi.org/10.1101/412932

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SLIDE 18

Degree of Parallel Divergence in Expression

Genes upregulated in freshwater relative to marine Genes with no difference in expression

Correlation in cis-regulation among populations (Pearson’s r)

Genes downregulated in freshwater relative to marine

The degree of parallelism in gene regulation appears to be associated with regulatory effect size

Verta & Jones (in review) BioRxiv doi: https://doi.org/ 10.1101/412932

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SLIDE 19

Cis-regulation is more additive than other forms of gene expression regulation – more visible to natural selection

Additive Dominant Dominant Dominance : Additivity Verta & Jones (in review) BioRxiv doi: https://doi.org/ 10.1101/412932

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SLIDE 20

Cis-regulation is more additive than other forms of gene expression regulation – more visible to natural selection

Additive Dominant Dominant Dominance : Additivity

And stable under different environmental conditions

Siblings raised under different environmental conditions show strong correlation in cis-regulation Correlation in allele-specific expression

Verta & Jones (in review) BioRxiv doi: https://doi.org/ 10.1101/412932

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SLIDE 21

Cis-regulation is more additive than other forms of gene expression regulation – more visible to natural selection

Additive Dominant Dominant Dominance : Additivity

And stable under different environmental conditions

Siblings raised under different environmental conditions show strong correlation in cis-regulation Correlation in allele-specific expression Correlation in gene expression

Verta & Jones (in review) BioRxiv doi: https://doi.org/ 10.1101/412932

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SLIDE 22

Jones et al (2012) Nature

Molecular mechanisms playing a role in parallel adaptive divergence of sticklebacks: Regulatory mechanisms

1 1

How is adaptive divergence in gene expression regulated?

cis

mRNA

What are the mutations & functional elements?

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SLIDE 23

Ra Raw Data

Marine Marine Fr Freshwater

CSS MAR - FW

<5kb 20kb 40kb

Jones et al (2012) Nature

EDA

CSS Marine – Freshwater Divergence

Genes

High resolution signals, good for functional follow-up … but ancient haplotypes carry many candidate mutations

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Comparative functional annotations of adaptive loci using epigenomics ChipSeq, ATACseq, RNAseq & CaptureC

Mapping poised & active enhancers, open chromatin differential expression and regulatory interactions in marine and freshwater genomes

Identifying regulatory elements underlying divergent adaptation

Dr Stanley Neufeld

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Open chromatin and epigenetic modifications to histones are predictive of functional and active regulatory elements

Modified from image from ENCODE - NHGRI & EBI

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HMM HMM epigenet epigenetic states ic states H3K27ac ChIPseq H3K4me1 ChIPseq H3K4me3 ChIPseq Open chr Open chromat

  • matin

in ATACseq Histone Histone mod modificat ifications: ions: Gene expr Gene expression ession RNAseq

Marine males Marine females Freshwater males Freshwater females F1 Hybrids Outgroups

Liver Kidney Gill Transcript ranscript annotat annotations ions

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SLIDE 27

HMM HMM epigenet epigenetic states ic states H3K27ac ChIPseq H3K4me1 ChIPseq H3K4me3 ChIPseq Open chr Open chromat

  • matin

in ATACseq Histone Histone mod modificat ifications: ions: Gene expr Gene expression ession RNAseq

Marine males Marine females Freshwater males Freshwater females F1 Hybrids Outgroups

Liver Kidney Gill Marine v Fr Marine v Fresh esh diver ivergent gene gent gene expr expression ession Marine v Fr Marine v Fresh esh diver ivergent gent enhancers enhancers Transcript ranscript annotat annotations ions

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Enhancer marks are enriched around Transcription Start Sites

Marine Chromatin State

  • pen chromatin

H3K27ac H3K27ac & H3K4me1 H3K4me1 no mark Freshwater

  • pen chromatin

H3K27ac H3K27ac & H3K4me1 H3K4me1 no mark Fold enrichment

0 5 10 15

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SLIDE 29

Open chromatin marks are enriched for CTCF binding sites p=1x10-1679

Marine Freshwater +125bp

  • 125bp

+125bp

  • 125bp

CTCF: 11zinc finger DNA binding protein

  • 3D chromatin structure
  • Transcriptional regulation
  • Insulation
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SLIDE 30

StickleCODE reveals high resolution signals of differential regulatory marks in marine & freshwater genomes

Marine Marine Fresh esh

Genes

Marine Marine Fresh esh

Open chromatin ATACseq ChIPseq (H3K27ac)

Freshwater enhancer eshwater enhancer absent in marine absent in marine

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SLIDE 31

marine fresh active enhancers

(H3K27ac ChIPseq)

  • pen

chromatin

(ATACseq)

500bp marine fresh GFP reporter

xkr9 tram1 eya1

Δ

b

Δ

eya1-xkr9 Genomic Region a Liver

500bp GFP reporter constructs

tram1

fresh 500bp + 84bp marine indel

Δ

GFP

b marine 500bp

GFP

marine 500bp + 84bp fresh indel

Δ

GFP

fresh 500bp

GFP

Functional tests of divergent regulatory elements Divergent chromatin states to cis-controlled enhancers

  • f divergent liver expression
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marine fresh active enhancers

(H3K27ac ChIPseq)

  • pen

chromatin

(ATACseq)

500bp marine fresh GFP reporter

xkr9 tram1 eya1

Δ

b

Δ

eya1-xkr9 Genomic Region a Liver

An 84bp insertion is An 84bp insertion is necessary and suf necessary and sufficient for ficient for fr freshwater liver expr eshwater liver expression ession

500bp GFP reporter constructs

tram1

fresh 500bp + 84bp marine indel

Δ

GFP

b marine 500bp

GFP

marine 500bp + 84bp fresh indel

Δ

GFP

fresh 500bp

GFP

Functional tests of divergent regulatory elements Divergent chromatin states to cis-controlled enhancers

  • f divergent liver expression
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The liver is particularly enriched for divergent regulatory marks between ecotypes

Tissue Tissue Total peaks

  • tal peaks

Diver Divergent peaks gent peaks (FDR 1%) (FDR 1%)

% %

Liver 23394 2148 2148 9.1 9.1 Kidney 27638 458 1.7 Gill 17006 178 1.0

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Genes proximal to marine-freshwater divergent liver enhancers are enriched for lipid metabolism

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The livers of marine and freshwater sticklebacks differ considerably in size and lipid content

Fr Freshwater Marine Marine

Lipid vacuoles

Litc MAR

Fr Freshwater Marine Marine Fr Freshwater Lipid levels ( Lipid levels (pmol pmol) )

250 250 200 200 150 150 100 100 50 50

Cholesterol Esters Diacylglycerol Phosphatidate Phosphatidyl- choline Phosphatidyl- ethanolamine Phosphatidyl- glycerol Phosphatidyl- inositol Phosphatidyl- serine Shingomyelin Triacylglycerol

Under common lab conditions Under common lab conditions fr freshwater sticklebacks have eshwater sticklebacks have >30x mor >30x more e triacylglycerides triacylglycerides in their livers than marine in their livers than marine sticklebacks sticklebacks

Major form of energy storage Blood glucose adipose

Orbitr Orbitrap ap Mass spectr ass spectrome

  • metr

try

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Divergent enhancers around genes that play key roles in obesity, fatty liver and insulin resistance

Overexpression in mice blocks high-fat-induced insulin resistance Knock-out improves glucose metabolism in obese mice

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Divergent regulatory marks

  • ften occur in tissue-specific clusters

Kidney Gill Liver H3K27ac ChIPseq

Divergent regulatory elements often appear in clusters across the genome in a tissue-specific manner

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MAR MAR FW FW K27ac ATAC MAR FW Empty MAR FW Empty Genes Cis controlled

An inversion on chromosome XXI contains multiple freshwater enhancers driving expression in the liver

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Genome-wide, a small number of genomic windows contain many divergent regulatory marks

  • 1000

2000 3000 4000 2 4 6 8 10

Liver Number of differential regulatory marks in 200 kb windows

Windows ranked by #divergent marks

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SLIDE 40
  • 1000

2000 3000 4000 2 4 6 8 10

Liver Number of differential regulatory marks in 200 kb windows

Windows ranked by #divergent marks

Genome-wide, a small number of genomic windows contain many divergent regulatory marks (and are “old friends” in the lab)

chrXXI inversion chrIV Wnt7b chrXI inversion

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Adaptive genomic regions identified in previous study are enriched in divergent enhancers

Liver Kidney Gill 1 2 3 4 5 6 Tissue Enrichment of Divergent H3K27ac Peaks in Adaptive Regions

***

7.9 x 10 - 7 0.28

Kidney Gill Liver . Enrichment of Divergent H3K27ac Peaks in Adaptive Regions

(ns) ***

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SLIDE 42
  • 1000

2000 3000 4000 2 4 6 8 10

Number of differential regulatory marks in 200 kb windows chrXXI inversion chrIV Wnt7b chrXI inversion

Clusters of divergent enhancers drive adaptive evolution in sticklebacks These marine-freshwater adaptive loci are enriched for clusters of divergent regulatory marks

(Fisher’s exact test: p<1x10-51) Liver

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Molecular mechanisms of adaptation & speciation

2 1 1 The regulatory control of adaptive divergence in gene expression

cis-regulation is predominant large effect size, additive, stable…

Comparative epigenomics reveals clusters of enhancer elements

in adaptive loci.

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SLIDE 44

Allele-specific ATACseq to identify cis-control of open chromatin

Dr Stanley Neufeld

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SLIDE 45

Allele-specific ATAC reveals that marine-freshwater divergence in chromatin state is mostly due to cis-regulatory elements

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Molecular mechanisms of adaptation & speciation

2 1 1 The regulatory control of adaptive divergence in gene expression

  • cis-regulation is predominant

large effect size, additive, stable…

Comparative epigenomics reveals clusters of enhancer elements

in adaptive loci.

  • divergence in chromatin state is regulated in cis

Why should we care about cis-regulation in adaptation?

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SLIDE 47

Gene flow between divergently adapted ecotypes produces maladapted recombinants

X

Deleterious recombination

Charlesworth & Charlesworth (1979) Genetics 91:581-589 Kirkpatrick & Barton (2006) Genetics 173:419-434

Shuffling might be good…. Or Bad…. Helps adaptation Reduces background selection

Beneficial recombination

Hill & Robertson (1966) Genetical Res. 8:269-294 Felsenstein (1974) Genetics 78:737-756

Recombination may be beneficial …. or deleterious and its modifiers subject to selection

Fitness

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ReMIX: A novel pipeline to build high-resolution individualized cross over maps from gamete linked-read sequencing

  • Dr. Andreea Dreau

Dreau et al (in review) BioRxiv https://doi.org/10.1101/489989

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Proof of principle: Can ReMIX detect known hotspots in mice? (hlx, a well known hotspot on mouse chr1)

Fig S3. From Paigen et al (2008) PLoS Genetics

A strong male-specific recombination hotspot on mouse chr 1

Number of Recombinants 52 recombinant offspring out of 1547 total (3.3%)

Dreau et al (in review) BioRxiv https://doi.org/10.1101/489989

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184500000 184550000 184600000 184650000 184700000 50 100 150

Molecules in Mouse somatic

Variants Molecules

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  • Haplotype 1

molecules Haplotype 2 molecules 184.50 1.8455 184.60 1.8465 184.70 chr1 (Mb)

a) Somatic tissue

184500000 184550000 184600000 184650000 184700000 50 100 150

Molecules in Mouse sperm

Variants Molecules

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  • 184.50 1.8455 184.60 1.8465 184.70

chr1 (Mb) recombinant molecules (1.6%)

b) Gametes ReMIX successfully detects known recombination hotspots in mice

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ReMIX: a fast and cost effective method to build fine-scale contemporary maps of recombination rate

1 male 1 male 1547 of 1547 offspring fspring

Dreau et al (in review) BioRxiv https://doi.org/10.1101/489989

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SLIDE 52

X

Deleterious recombination

Marine Freshwater

What is the molecular basis of recombination variation? Is it subject to selection?

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SLIDE 53

What is the genomic basis of adaptation and speciation?

  • Whole genome sequencing of nuclear families
  • High resolution individualized maps of

recombination crossovers

  • Females have 2x more recombination
  • Male cross overs are enriched with a specific motif
  • Fitness consequences for recombinants

Ongoing work:

  • Genetic mapping of recombination modifiers
  • Comparative ChIPseq

What molecular mechanisms underlie adaptation and speciation?

Cis-regulation plays an important role in adaptive gene expression divergence

Dr Jukka-Pekka Verta Dr Stanley Neufeld

Clusters of divergent enhancers drive adaptive divergence in sticklebacks

  • Dr. Andreea Dreau

Linked-read sequencing recombination hotspots and de novo diploid assemblies

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SLIDE 54

Jones Lab, FML Jones Lab, FML Max Planck, Max Planck, Tuebingen uebingen

Molecular mechanisms of adaptive divergence and speciation

Frank Chan