Post on 05-Aug-2015
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
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
Can we detect functional divergent regulatory interactions across species?
Strong motif
Weak motif‣ TF binding intensity ‣ Chromatin accessibility
Results
Can we detect functional divergent regulatory interactions across species?
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
Scoring TF binding site turnover
Random drift Stabilizing selection
= quantitative trait
= strength of selection
= optimum = white noise
= intensity of random fluctuations
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
Detecting cis-regulatory changes causing chromatin accessibility alterations
Results
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?
Random drift Stabilizing selection
= quantitative trait
= strength of selection
= optimum = white noise
= intensity of random fluctuations
Evolutionary Framework
Results
01020304050
LR,AIC,BIC
Scoring TF binding site turnover
Random drift Stabilizing selection
= quantitative trait
= strength of selection
= optimum = white noise
= intensity of random fluctuations
Evolutionary Framework
Results
01020304050
LR,AIC,BIC
In-house Cross-species data‣ Open-chromatin (FAIRE-seq)‣ 3 species
Lineage-specific motif evolution
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
Development of new methods for the identification of enhancers in Drosophila eye
development using multiple species
ConservationI: Comparative motif discovery combined with comparative transcriptomics yield accurate targetome and enhancer predictions
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
Development of new methods for the identification of enhancers in Drosophila eye
development using multiple species
ConservationI: Comparative motif discovery combined with comparative transcriptomics yield accurate targetome and enhancer predictions
Aims
Transcriptional Regulation
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
Lineage-specific motif evolution
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
Detecting cis-regulatory changes causing chromatin accessibility alterations
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
I: Computational Predictions
Cross-species Motif
Discovery (cisTargetX)
Identification of eye-enriched genes across
Drosophila species
Eye-antennal imaginal disc
Cross-species Motif Discovery (cisTargetX)
I: Computational Predictions
Alignment to species genome
Amendments on genome annotation
Comparison of Differential expression methods
Identification of eye-enriched genes across Drosophila
species
TA
G-se
q A
naly
sis
Can we determine the regulatory interactions driving photoreceptor development?
Identification of conserved regulatory interactions
Motif D
iscovery
Cross-species Motif Discovery (cisTargetX)
I: Computational Predictions
Alignment to species genome
Amendments on genome annotation
Comparison of Differential expression methods
Identification of eye-enriched genes across Drosophila
species
TA
G-se
q A
naly
sis
Can we determine the regulatory interactions driving photoreceptor development?
Identification of conserved regulatory interactions
Motif D
iscovery
Down-regulation in mutant context
II:Experimental Validation
Gene Le
vel
En
hance
r Le
vel Enhancer-reporter assays
Cro
ss-s
peci
es
Moti
f D
isco
very
(ci
sTarg
etX
)
I: Computational Predictions
Alignment to species genome
Amendments on genome annotation
Comparison of Differential expression methods
Identification of eye-enriched genes across Drosophila
species
TAG-seq Analysis
Can we determine the regulatory interactions driving photoreceptor development?
Identification of conserved regulatory
interactions
Motif Discovery
Down-regulation in mutant context
II:Experimental Validation
Gene Level Enhancer Level
Enhancer-reporter assays
TAG-seq Analysis: Alignment to non-model organisms
478 bp
Genome Annotation Amendments
Can we determine the regulatory interactions driving photoreceptor development?
I: Computational Predictions
TAG-seq Analysis
Identification of conserved regulatory
interactions
Cross-species Motif
Discovery (cisTargetX)
Identification of eye-enriched genes across
Drosophila species
I: Experimental Validations
Can we detect functional divergent regulatory interactions across species?
Presence of motif
Absence of motif
TF binding site
Can we detect functional divergent regulatory interactions across species?
Presence of motif
Absence of motif‣ TF binding intensity ‣ Chromatin accessibility‣ Gene expression
Can we detect functional divergent regulatory interactions across species?
Presence of motif
Absence of motif‣ TF binding intensity ‣ Chromatin accessibility
Can we detect functional divergent regulatory interactions across species?
Presence of motif
Absence of motif
TF binding site
‣ TF binding intensity ‣ Chromatin accessibility‣ Gene expression
Scoring TF binding site turnover
Random drift
= white noise
= intensity of random fluctuations
- 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
Detecting cis-regulatory changes causing chromatin accessibility alterations
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
Transcriptional Regulation
DNA GTGTGGTGCGCAGGGATGAGAAGGCAGAGGCGCGACTGGGGTTCATGAGGAAGGGCAGGAGGAGGGTGTGGGATGGTGGAGGGGTTTGAGAAGGCAGAGGCGCGACTGGGGTTCATGAGGAAAGGGAGGGGGAGGATGTGGGATGGTGGAGGGGCTGCAGACTCTGGGCTAGGGAAAGCTGGGATGTCTCTAAAGGTTGGAATGAATGGCCTAGAATCCGACCCAATAAGCCAAAGCCACTTCCACCAACGTTAGAAGGCCTTGGCCCCCAGAGAGCCAATTTCACAATCCAGAGTCCCCGTGCCCTAAAGGGTCTGCCCTGATTACTCCTGGCTCCTTGTGTGCAGGGGGCTCAGGCATGGCAGGGCTGGGAGTACCAGCAGGCACTCAAGCGGCTTAAGTGTTCCATGACAGACTGGTATGAAGGTGGCCACAATTCAGAAAGAAAAAAGAAGAGCACCATCTCCTTCCAGTGAGGAAGCGGGACCACCACCCAGCGTGTGCTCCATCTTTTCTGGCTGGGGAGAGGCCTTCATCTGCTGTAAAGGGTCCTCCAGCACAAGCTGTCTTAATTGACCCTAGTTCCCAGGGCAGCCTCGTTCTGCCTTGGATTACTGCCAGCCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCTTGTGTGCAGGGGGCTCAGGCATGGCAGGGCTGGGAGAATAATAAATACTTGACCACCTGTAGTACATAAAAACCTATATATTGC
- Transcription Factors (TFs)- Chromatin regulation
Transcriptional Regulation
Genetic RegionCorepromoter
Proximal Promoter
Enhancer
- Transcription Factors (TFs)- Chromatin regulation
Transcriptional Regulation
Genetic RegionCorepromoter
Proximal Promoter
- Transcription Factors (TFs)- Chromatin regulation
Transcriptional Regulation
Genetic RegionCorepromoter
Proximal Promoter
Enhancer
- Transcription Factors (TFs)- Chromatin regulation
Transcriptional Regulation
Genetic RegionCorepromoter
Proximal Promoter
Enhancer
Computational
Experimental
- Transcription Factors (TFs)- Chromatin regulation
Transcriptional Regulation
Genetic RegionCorepromoter
Proximal Promoter
EnhancerEnhancer
Nucleosome
Cell differentitation
In-house Cross-species data
‣ Gene Expression (RNA-seq)‣ 8 species
Lineage-specific motif evolution
Significant Overlap
Can we find correlations between lineage-specific CRM changes and other lineage-specific
traits?
In-house Cross-species data
‣ Gene Expression (RNA-seq)‣ 8 species
Lineage-specific motif evolution
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
I: Computational Predictions
Alignment to species genome
Amendments on genome annotation
Comparison of Differential expression methods
TAG-seq Analysis
Identification of conserved regulatory
interactions
Cross-species Motif
Discovery (cisTargetX)
Identification of eye-enriched genes across
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
I: Computational Predictions
II: Experimental Validations
Results
TAG-seq Analysis: Alignment to species genome
eye counts: 122
wing counts: 4
Last 3‘NlaII site
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
- Transcription Factors (TFs)- Chromatin regulation
Genetic RegionCorepromoter
Proximal Promoter
Enhancer
Introduction
Transcriptional Regulation
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