Evolutionary conservation and divergence of cis-regulatory interactions in Drosophila eye...

EVOLUTIONARY CONSERVATION AND DIVERGENCE OF CIS-REGULATORY INTERACTIONS IN DROSOPHILA EYE

DEVELOPMENT

MARINA NAVAL SÁNCHEZ

Cell Differentiation in Drosophila eye-antennal imaginal disc

Differentiated cellsUnDifferentiated cells

Introduction

Using cross-species information

Introduction

Comparative motif discovery and comparative transcriptomicsEye-antennal imaginal

disc

Wing imaginal disc

Results

Last 3‘NlaIII site

Tag-seq

Naval-Sánchez et al. Genome Research 2013

Identification of eye-enriched genes across species

Results

GO enrichment

Who regulates who?

TG3TG3TG3TG3

TG4TG4TG4TG4

TG2TG2TG2TG2

TG1TG1TG1TG1

TF1TF1TF1TF1

TF2TF2TF2TF2

RegulonsSet of genes

OUTPUTINPUTB. TARGET SELECTION

• Leading edgeA. MOTIF DETECTION

• 3731 candidate motifs• HMM motif clustering•12 Drosophilaver genomes• whole-genome rankings• Receiver Operating Characteristic

Aerts et al. PLoS Bioll. 2010

245 eye-enriched genes

Results

Motif Discovery Output

Novel regulatory interactions in photoreceptor development

I: Computational Predictions

Results

Novel regulatory interactions in photoreceptor development

II: Experimental Validations

Results

Experimental Validations of the Glass targetome

Results

chp

wild-type

glass mutant

Can we predict functional enhancers?

enhancer-GFP gl

80% success rateusing conservation

Results

Can we detect functional divergent regulatory interactions across species?

Strong motif

Weak motif‣ Homotypic clustering of TF binding sites‣ TF motif score enhancer across 12 genomes‣ Hidden Markov Model : score for a cluster of TF binding sites

Results


Strong motif

Weak motif‣ TF binding intensity ‣ Chromatin accessibility

Results


Strong motif

Weak motif‣ TF binding intensity ‣ Chromatin accessibility‣ Gene expression

How to model changes across phylogeny?

Quantitative traits: Ornstein-Uhlenbeck model

Results

Scoring TF binding site turnover

Random drift

= white noise

= intensity of random fluctuations

Results

Strong motif

Weak motif‣ Homotypic clustering of TF binding sites‣ TF motif score enhancer across 12 genomes‣ Hidden Markov Model : score for a cluster of TF binding sites


Random drift Stabilizing selection

= quantitative trait

= strength of selection

= optimum = white noise


Evolutionary Framework

Results

01020304050

LR,AIC,BIC

Do conserved Glass targets present lineage-specific regulatory interactions?

Previously validated Glass targets

scrt,chp, retn, dpr10, CG6329, Lim3, dmrt99B

Results

Gene Location Selective regime p-val

Lim3 chr2L: 19100674-19102387M1: melanogaster

subgroup0.001706

CG6329 chr2R:9718524-979132 M5: obscura group 0.000413

scrt chr3L: 3980929-398100 M10: D.virilis 0.004121

scrt chr3L: 3982641-3983568M1: melanogaster

subgroup0.002996

Validation of OU model as a divergent motif discovery method

scrt

Divergent enhancer p.val= 0.00412

Results

D.virilis

cloned enhancer-GFP gl Elav cloned enhancer-GFP

D.melanogaster

cloned enhancer-GFP gl Elav cloned enhancer-GFP

OU model can lead to the discovery of a de novo enhancer !

Public Cross-species data

‣ TF binding intensity ‣ Twist binding (He et al. 2011)‣ Kr, Gt, Hb, BCD (Paris et al. 2013) ‣ Enhancer-reporter assays‣ STARR-seq (Arnold et al. 2014)

Lineage-specific motif evolution

Lineage-specific functional traits

Significant Overlap

Twist binding

Evo-cisTarget

Results

Evo-cisTarget6272 PWMs

Twist BCD GT HB KR GATA VFL

Results

Validation on ChiP-seq public data

First motif

Evo-cisTarget6272 PWMs

Detecting cis-regulatory changes causing chromatin accessibility alterations

Results

D.pseudoobscura

D.virilis

D.melanogaster


Results

Conclusions

Conservation

Divergence

‣ OU model in motif discovery can lead to the discovery of functional divergent

enhancers; scrt;

‣ Evo-cisTarget identifies lineage-specific ChIP-changes

‣ Species as replicates improves the discovery of genes involved in conserved

developmental processes

‣ 80% success rate when testing enhancers

• Stein Aerts

• Delphine Potier

• Valerie Christiaens

• Gert Hulselmans

• Kristofer Davie

• Jelle Jacobs

• Zeynep Kalender

• Mark Fiers

• Hana Imrichova

• Dmitry Slivelinsky

•Thank you

Question! How many models should we compare?







Results

01020304050

LR,AIC,BIC








Results

01020304050

LR,AIC,BIC

In-house Cross-species data‣ Open-chromatin (FAIRE-seq)‣ 3 species


Significant Overlap

Can we find correlations between lineage-specific CRM changes and other lineage-specific traits?

Results

Transcriptional Regulation

Pluripotentstem cell

Genome Control

Introduction

ConservationI: Comparative motif discovery combined with comparative transcriptomics yield accurate targetome and enhancer predictions

DivergenceII: Ornstein-Uhlenbeck models identify lineage-specific cis-regulatory changes associated to altered transcription binding, chromatin activity and gene expression across Drosophila species

Aims

Development of new methods for the identification of enhancers in Drosophila eye

development using multiple species

Aims

Comparative motif discovery combined with comparative transcriptomics yields accurate targetome and enhancer predictions

Genome Research 2013Naval-Sánchez M, Potier D, Haagen L, Sánchez M,

Munck S, Van de Sande B, Casares F, Christianes V, Aerts S

Aims




Aims

DivergenceII: Ornstein-Uhlenbeck models identify lineage-specific cis-regulatory changes associated to altered transcription binding, chromatin activity and gene expression across Drosophila species

Aims




Aims


Introduction

GTGTGCAGGTGGTGCGCAGGGATGAGAAGGCAGAGATGAGAGGCGCGAGAAGCCTACAACTGGGGTTCATGAGGGATGAGAGGAGGAGGGTGTGGGATGGTGGAGGGGTTTGAGAAGGCAGCAGGTGAGCAGGTGGCGCGACTGGGGTTCATGAGGAGAAGCCTACAAAAGGGCAGGTGAGGGGGAGAAGCCTACAAGGATGTGGGATGGATGGTGGAGGGATGAGAGGCTGCAGAGAAGCCTACAACTCTGGGCTAGGGAAAGCTGGGATGTCTCTAAAGGTTGGAATGAATGGCCTAGAATCCGACCCAATAAGCCAAAGCCACTTCCACCAACGTTAGAAGGCAGGTGCCTTGGCCCCCAGAGAGCCAATTTCACGGACCTGCGTGCCCACCTGCTAACACCTGAGCACCTGGGTCCACCTGTGCCCACCTGCTGATTACTCCACCTGCCTTGTCACCTGGGGGCACCTGGCTCAGGCACCTGCATGGCACACCTGGGGCTGGGAGCACCTGTACCAGCAGGCACTCAAGCCAGGTGGGCTTAAGTGTTCCATGACAGACTGGTATGAAGGTGGCCACAATTCAGAAAGAAAAAAGAAGAGCACCATCTCCTTCCAGTGAGGACAGGTGAGCGGGACCACCACAGGTGCCCAGCGTGAAGCCTACAAGTGCTCCATCTTTTCTGGCTGGGGAGAGGCCTTCATCTGCTGTAAAGGGTCCTCCAGCACAAGCTGTCTTAATTGACCCTAGTTCCCAGGGCAGCCTCGTTCTGCCTTGGATTACTGCCAGGAAGCCTACAACCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACACAGGTGTAAAAACCCTCAGGTGTGTGTGCAGGGGGCTCAGGCATGGCA

Cis-regulatory modules

Transcription Factor Binding Sites- 6-20 nt- degenerated- clusters

OU on gene expression

Brain Eye-antenna Wing Brain Eye-antenna Wing

Results

In-house Cross-species data

‣ Gene Expression (RNA-seq)‣ 8 species


Significant Overlap

Results

Evo-cisTarget on divergent D.pseudoobscura gene expression

top 500

Collaborations

Tissue overgrowth induced by coexpression of the progenitor genes hth/MEIS1 and ts/TSHZ results from an imbalance in the estrogen

response pathway in DrosophilaMarta Neto and Fernando Casares

CABD - Centro Andaluz de Biología del Desarrollo ; Universidad Pablo de Olavide - CSIC

RNA-seq analysis Motif DiscoveryOpen-chromatin

Differential expression analysis

- wt- HTH-TSH

-TSH-HTH

Differential analysis- wt

- HTH-TSH-HTH

i-cisTargetHTH ChIP-seq

- wt- HTH-TSH

-HTH

Collaborations

RNA-seq analysis Motif Discovery

TDP-43 loss-of-function causes neuronal loss due to defective steorid receptor-mediated gene program switching in Drosophila

Lies Vanden Broeck, Bart Dermaut, Patrick CallaertsLaboratory of Behavioral and Developmental Genetics

VIB Center for the Biology of DiseaseINSERM U7444, Institut Pasteur de Lille, Université de Lille Nord de France

Differential expression analysis

-dTDP-43 upregulation and dTDP-43 downregulation

- wt

Upregulation of Map205

Decoding Smad5-mediated BMP functions in early amnion development by mRNA-seq analysis

Mariya Dobreva and An ZwijsenLaboratory for Developmental Signaling

VIB Center for Human Genetics

RNA-seq analysis Motif DiscoveryOpen-chromatin


lots of conservation at first sight

Results

D.melanogaster

D.pseudoobscura

D.virilis

Alignment to species genome

Amendments on genome annotation

Comparison of Differential expression methods

TAG-seq Analysis

Can we determine the regulatory interactions driving photoreceptor development?

Identification of conserved regulatory

interactions

Down-regulation in mutant context

II:Experimental Validation

Gene Level Enhancer Level

Enhancer-reporter assays


Cross-species Motif

Discovery (cisTargetX)

Identification of eye-enriched genes across

Drosophila species

Eye-antennal imaginal disc

Cross-species Motif Discovery (cisTargetX)





Identification of eye-enriched genes across Drosophila

species

TA

G-se

q A

naly

sis


Identification of conserved regulatory interactions

Motif D

iscovery

Cross-species Motif Discovery (cisTargetX)






species

TA

G-se

q A

naly

sis


Identification of conserved regulatory interactions

Motif D

iscovery



Gene Le

vel

En

hance

r Le

vel Enhancer-reporter assays

Cro

ss-s

peci

es

Moti

f D

isco

very

(ci

sTarg

etX

)






species

TAG-seq Analysis



interactions

Motif Discovery



Gene Level Enhancer Level

Enhancer-reporter assays

TAG-seq Analysis: Alignment to non-model organisms

478 bp

Genome Annotation Amendments



TAG-seq Analysis


interactions

Cross-species Motif



Drosophila species

I: Experimental Validations


Presence of motif

Absence of motif

TF binding site


Presence of motif

Absence of motif‣ TF binding intensity ‣ Chromatin accessibility‣ Gene expression


Presence of motif

Absence of motif‣ TF binding intensity ‣ Chromatin accessibility


Presence of motif

Absence of motif

TF binding site

‣ TF binding intensity ‣ Chromatin accessibility‣ Gene expression


Random drift

= white noise


- log (p.value)

GO term

p.adj

Genes

response to stimulus

3.39E-06

Fas2, jing, msps, slgA, mask, Mrtf, mam, Camta, St4, Fmrf, plexA, Spn27A, knk, l(3)L1231, Spec2, RhoGAP19D, ab, Tfb1, sno, Rh2, aop, shot, eIF-2beta, trr, Itp-r83A, Gbeta76C, Wwox, InR, wdb, rg, E(Pc), wit, pk, ninaC, Gap1, Graf, FK506-bp2, SPR, beat-Ib, stan

developmental process

1.42E-05

Klp61F, Fas2, exd, jing, msps, mask, Mrtf, mam, kel, ctrip, plexA, didum, Spn27A, knk, Cpr65Au, Thiolase, RhoGAP19D, ab, tud, Apc10, sno, aop, Roe1, shot, Kdm2, eIF-2beta, trr, Itp-r83A, Cpr72Ec, gpp, sano, InR, wdb, rg, Cpr31A, E(Pc), zuc, wit, Trn-SR, pk, Cpr78E, Gap1, fd68A, beat-Ib, Afti, stan

cell surface receptor signaling pathway

1.43E-05Fas2, aop, trr, mask, Gbeta76C, mam, Camta, InR, Fmrf, pk, ninaC, plexA, Spn27A, Gap1, knk, SPR, stan, Rh2, sno

cellular response to stimulus

3.05E-05Fas2, aop, trr, mask, Gbeta76C, Mrtf, mam, Camta, InR, wdb, St4, Fmrf, wit, pk, ninaC, plexA, Spn27A, Gap1, knk, Graf, SPR, FK506-bp2, Spec2, RhoGAP19D, stan, Tfb1, sno, Rh2

response to external stimulus

4.56E-05shot, eIF-2beta, jing, msps, slgA, mask, Gbeta76C, Camta, Wwox, InR, E(Pc), ninaC, plexA, Spn27A, l(3)L1231, beat-Ib, ab, stan, Rh2

signal transduction

6.91E-05Fas2, aop, trr, mask, Gbeta76C, mam, Camta, InR, wdb, Fmrf, wit, pk, ninaC, plexA, Spn27A, Gap1, knk, Graf, SPR, Spec2, RhoGAP19D, stan, Rh2, sno

Increase in stregth of the drift

Text

- Scanning for DNA words- Scanning for Clusters- Classification of Enhancers- clusters

Computational predictions of Enhancers

Sp1

Sp2

- Regulator binding

- Chromatin accessibility

- Histone modifications

- Transcription Factors (TFs)- Chromatin regulation

Experimental predictions of Enhancers

CAGGTG

Detection of Regulatory Elements in gene signatures

Sp1

Sp2

Sp3

Gene Regulatory Evolution

FlexibleRigid


DNA GTGTGGTGCGCAGGGATGAGAAGGCAGAGGCGCGACTGGGGTTCATGAGGAAGGGCAGGAGGAGGGTGTGGGATGGTGGAGGGGTTTGAGAAGGCAGAGGCGCGACTGGGGTTCATGAGGAAAGGGAGGGGGAGGATGTGGGATGGTGGAGGGGCTGCAGACTCTGGGCTAGGGAAAGCTGGGATGTCTCTAAAGGTTGGAATGAATGGCCTAGAATCCGACCCAATAAGCCAAAGCCACTTCCACCAACGTTAGAAGGCCTTGGCCCCCAGAGAGCCAATTTCACAATCCAGAGTCCCCGTGCCCTAAAGGGTCTGCCCTGATTACTCCTGGCTCCTTGTGTGCAGGGGGCTCAGGCATGGCAGGGCTGGGAGTACCAGCAGGCACTCAAGCGGCTTAAGTGTTCCATGACAGACTGGTATGAAGGTGGCCACAATTCAGAAAGAAAAAAGAAGAGCACCATCTCCTTCCAGTGAGGAAGCGGGACCACCACCCAGCGTGTGCTCCATCTTTTCTGGCTGGGGAGAGGCCTTCATCTGCTGTAAAGGGTCCTCCAGCACAAGCTGTCTTAATTGACCCTAGTTCCCAGGGCAGCCTCGTTCTGCCTTGGATTACTGCCAGCCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCTTGTGTGCAGGGGGCTCAGGCATGGCAGGGCTGGGAGAATAATAAATACTTGACCACCTGTAGTACATAAAAACCTATATATTGC



Genetic RegionCorepromoter

Proximal Promoter

Enhancer




Proximal Promoter




Proximal Promoter

Enhancer




Proximal Promoter

Enhancer

Computational

Experimental




Proximal Promoter

EnhancerEnhancer

Nucleosome

Cell differentitation




Significant Overlap

Can we find correlations between lineage-specific CRM changes and other lineage-specific

traits?




Significant Overlap

Can we find correlations between lineage-specific CRM changes and other lineage-specific

traits?

Eye Eye

I: Comparative motif discovery combined with comparative transcriptomics yield accurate targetome and enhancer predictions

• Whole-genome expression data (TAG-seq)

• Three Drosophila species

• Two tissues

• Eye-antennal imaginal disc

• wing imaginal disc

Can we determine the regulatory interactions driving photoreceptor

development?

Eye-antennal imaginal disc Wing imaginal disc





TAG-seq Analysis


interactions

Cross-species Motif



Drosophila species

OUTLINE

‣ Introduction

‣ Transcriptional regulation

‣ Drosophila Eye Development

‣ Cross-species information

‣ Aims

‣ Results

‣ Cross-species conservation

‣ Cross-species divergence

‣ Collaborations

‣ Discussion

Comparative motif discovery together with comparative transcriptomics

• Whole-genome expression data (TAG-seq)

• Three Drosophila species

• Two tissues

• Eye-antennal imaginal disc

• wing imaginal disc

Can we determine the regulatory interactions driving photoreceptor

development?

Eye-antennal imaginal disc

Wing imaginal disc


II: Experimental Validations

Results

TAG-seq Analysis: Alignment to species genome

eye counts: 122

wing counts: 4

Last 3‘NlaII site

Results

scarlet (st) is decreased in the melanogaster group

chr3L:16,489,708-16,494,743st

* p.adj = 0.036

Dmel_eyeDsim_eyeDyak_eyeDana_eyeDpse_eyeDwil_eyeDmoj_eyeDvir_eye

CS_eye

Collaborations

Cross-species gene expression analysis in the Drosophila Auditory OrganRyan Kavlie and Joerg Albert

UCL Ear Institute; University College of London

RNA-seq analysis Motif Discovery6 Drosophila

speciesD.melanogaster

D.simulansD.yakuba

D.pseudoobscuraD.persimilis

Hr39 known to influence courtship behavior

OU modelIdentification of

genes with change in

optimum across species

i-cisTarget

Change in trans

Gene expression divergence

Brain Eye-antenna Wing Brain Eye-antenna Wing

Results



Proximal Promoter

Enhancer

Introduction


Comparative motif discovery and comparative transcriptomicsEye-antennal imaginal

disc

Wing imaginal disc

Results

Last 3‘NlaII site

Tag-seq

Experimental Validations of the Glass targetome

Wild-type(Canton-S)

gl[60j]

Results

RNA-seq (TruSeq, Illumina HiSeq2000)

TFTF

=

∆ expressed genes

TGTG TGTGTGTG

Regulon discovery

TFTF=

Imported Author Yesterday, 10:59 motif that is important to bring this co-expression about

predict targets for which this motif is most likely to play a role

Imported Author Yesterday, 10:59 motif that is important to bring this co-expression about

predict targets for which this motif is most likely to play a role

245 eye-enriched genes across species

TAG-seq Analysis: Genome Annotation Amendments

Can we determine eye-specific genes across species?

Results

OUTLINE

‣ Introduction

‣ Transcriptional regulation

‣ Drosophila eye development

‣ Cross-species information

‣ Aims

‣ Results

‣ Cross-species Tag-seq and Glass conserved targetome

‣ Evolutionary model across species at the motif, chromatin and expression level

‣ Conclusions

Consequences of Animal Domestication of Genes and

Genomes

•Marina Naval Sánchez

•OCE Postdoctoral Fellowship

•CSIRO Agriculture Flagship

•Supervisor Dr. James Kijas

Evolutionary conservation and divergence of cis-regulatory interactions in Drosophila eye...

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