comparative lactation biology - international milk genomics … · 2013-09-29 · comparative...
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Comparative Lactation BiologyMamExp: an interactive gene expression browser
for collaborative comparative lactation genomics
Christophe Lefèvre , ITRI, BioDeakin, GTP Deakin University
Kevin Nicholas
Julie Sharp
Karensa Menzies
Pragati Sharma
Sanjana Kuruppath
Amit Kumar
Philip Church
James Stillman
Shannu Palamuru
VPAC
Adam Wong
Zoology Uni.Melbourne
Matthew Digby
John Arnould
Sonia Mailer
Cate Pooley
Kylie Cane
Elizabeth Pharo
Coralie Reich
Alison DeLeo
Elie Khalil
Danny Topcic
Joly kwek
Rachael O’Dowd
Amelia Brennan
Karensa Menzies
Maurice Ling
V.B.C, monash
David Powell
Yvan Strahm
Torsten Seeman
Reprogen Uni. Sydney
Keith L. Macmilland
Paul A Sheehy
Peggy Rismiller, University of Adelaide, SA
Katherine Belov, Faculty of Veterinary Science, University of Sydney, NSW platypus genome consortium
Exploring lactation diversity
Comparative genomics & transcriptomics of the lactation system
Molecular interface between mother and child
Evolution/adaptation of the lactation programfrom echidna and wallaby to man...
Comparative lactation biology:
Mammalian diversity…
GENETIC RESOURCES - biodiversity approach
GENETIC RESOURCES - biodiversity approach
Using comparative genomics
Mammal evolution:
fossil dating
Mining 200 Million years of lactation …
Mammal evolution: Mammalian super-tree
The delayed rise of present-day mammals. Olaf R. P. et al. Nature Vol 446 | 29 March 2007
Mammal evolution: Mammalian super-tree
The delayed rise of present-day mammals. Olaf R. P. et al. Nature Vol 446 | 29 March 2007
Mammal evolution: Mammalian super-tree
The delayed rise of present-day mammals. Olaf R. P. et al. Nature Vol 446 | 29 March 2007
Research Platforms
Genomics
Bioinformatics
Proteomics Bioactives
metabolics
Lactation
program
gene discoverygene regulation
gene evolution
sequencing
Genome analysis
sequencing
Expression microarray
Protein prediction
signal prediction
Function prediction
Web query interface generate dynamic annotation (php scripts)
Gene index sequence annotation Bio-activity prediction gene expression regulation
Gene discovery
Raw sequence data
QC sequence
Assemblies Sequence data
Annotation
TRIM
PHRED base calling
CROSS MATCH vector removal
BLAST
Unigene
Nr protein
Genbank
Genomes
PHRAP
Predicted protein data
Signal
prediction
Ipsort,
Spscan,
hmm
Translate
ESTscan
consed
Function
prediction
sequence tags counts Microarray data
annotated
expression tags
annotated
differential expression
Mammary gland
EST sequencingMPSS cDNA Microarray
Tag to EST matching
Normalization
Analysis
- R/Bioconductor
Microarray layout
hmm
Pfam
Peptide analysis
Wallaby
databasemySQL
Discovery engine Machine Learning: marsupial models (hmm)
Pattern matching: gene networks
Data mining: candidate genes
Bioinformatics pipeline
data
Est
sequencesBlast
hits
Pfam
hits
Protein
sequences
Sequence
similarity
search
Blast
HMM
PfamProtein Domain
search
ESTscan2Protein Prediction
Est
sequences
Protein
sequences
contig
sequences
Sequence
assemblyLoad inte
rfa
ce
query
inte
rface
Job management interface
EST-PAC
EST-PAC a web package for EST annotation and protein sequence predictionYvan Strahm , David Powell and Christophe Lefèvre. Source Code for Biology and Medicine 2006, 1:2
Percentage identity
humandog
Distribution of similarity between assembled seal
EST’s
Using estpac…
Lefevre, C. M., Sharp, J. A., & Nicholas, K. R. (2010)Annual review of human genomics
Evolution of lactation
Monotreme
lactation
Monotreme lactation- short gestation period of 21-23 days
- egg incubation for 10-12 days
- lactation 120 to 200 days
milk is delivered to a highly altricial young.
Method
Milk cell cDNA sequencing
EST assembly
EST & contig annotation
Genome mapping
Contig clustering
EST to contig mapping (expression)
Comparative genomics
BLG
CSN2
CSN2b
C6orf58
?
CSN3
50%
8%
10%
23%
91%
Platypus milk cDNA library
87 ESTs (after removal of 20% rRNA +10% low quality)
1667 ESTs 58 genes
-Milk cell sequencing identifies milk protein sequences
-Milk cell gene expression
-High level of major milk protein
-Overall similar profiles in echidna and platypus
-Consistent with other mammals
-C8orf58:
-new monotreme milk protein ?
- alpha, beta, kappa and beta like caseins in echidna
- alpha-casein in platypus?
- gap in the platypus genome sequence
- CSN1 cDNA sequence confirmed by PCR
Caseins• three classes of phosphorylated micelles forming caseins:
- Related to Ca binding protein involved in bone and dentition
- alpha-caseins (CSN1s1, and CSN1s2 in bovine milk) . precipitated by very low calcium concentrations. have the highest phosphate content. no discernable secondary structure . the least conserved caseins
- Beta-casein: CSN2. precipitated by moderate calcium concentrations . have some secondary structure . moderately conserved
- kappa-caseins: CSN3. insensitive to calcium. essential for micelle formation. most conserved caseins
• rapidly evolving cluster of lactation specific genes • single CSN1 in marsupial• short exons, all in-frame• splice variants common in eutherians• phosphorylation motif straddle exon junction
Ca sensitive
in the platypus genome sequence
casein genes are tightly clustered
on 100kb of ultracontig362
Ensembl, assembly gaps: CSN1, beta 5’UTR & promoter …
short in-frame exons, low similarity with therian caseins
CSN2
CSN1
CSN2b (CSN1s2)
CSN3
Montreme CSN1: alpha-casein
80% identity
1 splice variant long variant platypus pcr is minor in echidna 1/6
>14 exons (gap) in-frame
3 cannonical P sites (exon junction)
Montreme CSN2: beta-casein
64% id.
10 exonscoding in-frame
Long exon VIII
3 echidna variants
2 P sitesP site junctions
Montreme CSN2b: beta-like casein
70% id.
15 exonscoding in-frame
Spice variantsexonIIb echidna
exons IV, VI, VIII, IX
3 P sites2xP sites in CSN1s2
Montreme CSN3: kappa-casein
70% id.
5 exons
1 splice variant
CSN1CSN1S2-like
CSN2
phyllogenetic tree of calcium sensitive caseins
Partial duplication of CSN2 / CSN2b in monotreme lineage
1
5
43
2
6
Sequence similarity in the duplicated region
covering the first 5 exons of CSN2
Presence of P site and in-frame exons in the remaining
of CSN2b suggest an ancestral casein (CSN1s2-like?)
was already present at that position
Comparison of mammalian casein gene clusters
0 0.05 0.1 0.15 0.2 0.25 0.3 Mb
CSN2
CSN2
CSN1S1 CSN3
CSN2
CSN1S2STAT HIS3 HIS1
NP_999876.1
ODAM
FDSCP
CSN1S2b
CSN1 CSN2b CSN3
CSN1 CSN3Odam
CSN1S1
CSN2
CSN3CSN1S2 OdamHIS STAT
csnkcsna Csn1s2a Csn1s2b OdamSTAT HIS
csnb
AK05291
Platypus ultra362
Opossum chr.5
Cattle chr.6
Mouse chr.5
Human chr.4
CSN1 CSN1S2 CSN3CSN2
CSN1 CSN3CSN2
Model 1
Model 2
Ancestral locus
1) CSN2 duplication
2) Homologous recombination CSN2/CSN1S2
1) Transposon invasion
2) CSN1S2 deletion
CSN1S2 duplication
1) CSN1S1 duplication
CSN-s2 duplication
Alternative models for the evolution of caseins(work in progress…)
Monotreme genomics:
- Ancient origin of the casein cluster
- Conserved genomic organisation-Beta-casein promoter un-characterised …
- lineage specific rearrangements
- role in lactation function?
-Comparative genomics and lactation evolution…
-Highly sensitive comparative sequence analysis will be
required together with high resolution genome sequence
EVOLUTION OF WAP GENE STRUCTURE
– proposal of an ancient WAP progenitor
• The apparently dispensable role of WAP in eutherians, in particular humans and domesticated animals such as
the cow, goat and sheep suggests the evolution of WAP may be related to diet of the young which is being
adequately compensated in these species or maybe linked to improved care and hygiene in domesticated life.
ŅMolecular evolution of Monotreme and marsup ial whey acidic protein genesÓ. Julie Anne
Sharp, Christophe Lefevre, Kevin R Nicholas. Evol Dev. 2007 Jul-Aug;9(4):378-92.
IIBIIII
tammar lactation program phases
Phase 1
Pregnant
Proliferation
Differentiation
Little or no milk
production
Phase 2A
Attached
to nipple
Production of:
Carbohydrates
Protein
Lipid
Bioactives
Phase 2B
In pouch
on and off
nipple
Production of:
Carbohydrates
Protein
Lipid
Bioactives
Phase 3
In and out
of pouch
Large increases in:
Protein productionLipid production
Large decreases in:
Carbohydrate production
WAPELP LLP-A, LLP-B, b-gal
b-1,3 galactosyltransferase
a-casein, b-casein, a-lactalbumin, b-lactoglobulin
-26 0 50 100 150 200 250 300 350
Asynchronous Concurrent Lactation:a model to identify systemic and local regulators of lactation
High Volume
High Lipids
Low Carbohydrates
Higher Protein
Low Volume
Low Lipids
High Carbohydrates
Lower Protein
•Adjacent glands secreting milk of very different composition
•Local control of milk composition
•Local and endocrine control interactions
Does the composition of milk effect
development of the young ?
-26 0 50 100 150 200 250 300 350Phase 1 Phase 3Phase 2A Phase 2B
60 day old pouch young were fostered onto
teats producing milk for 110 day old young
Animals were examined at 172 days of age by which
time they were receiving day 222 milk
Foster Pouch Young
172 days old
Receiving milk from day 222 of lactation
Control Pouch Young
172 days old
Receiving day 172 milk
Milk composition makes a big difference!
Nicholas and al. 1997
Quantitative gene expression from mammary gland EST sequencing
Proportional transcript composition of cDNA libraries
Complexity (singleton)
Good quantification of highly expressed transcripts only
Limitations: sequencing depth and complexity
Gene expression profiling from lactation stage specific
mammary gland transcriptome sequencing
EST library sequencing MPSS
differential expression of the secretome during lactation
Comparative biology
Gene identification
Bioinformatics
e-research platforms
Biotechnology
MammoSapiens
Gene regulation…
NormalizedUn-normalized
Array Fabrication Spotting
•Use PCR to amplify DNA
•Robotic "pen" deposits DNA at defined coordinates on glass slides
•approximately 1-10 ng per spot
Expression series
database
Oligo
microarray
MPSS
cDNA
microarray
Load inte
rfa
ce
query
inte
rface
management interface
eResource for expression data analysis
Preprocessing
Normalisation
Statistical test
EST-PAC
baab
-cas-cas-lac-LG
ELP WAP
100 12515
b -1,3-GAT
b -1,4 GATLLP-B
LLP-A
20017515050 7525 225 2500
b -gal
-26
Phase 2A
Permanent attachment to teat
Phase 2B
On and off teat
Always in pouch
Phase 1
Pregnant
Phase 3
In and out of Pouch
Beta casein
alpha casein
elp
20
40
60
95 96 106 144 144 169 172 174 198 202 231202 242 244 275 276276140
WAP
b-LG623955623955
Lactation (days)
0
WAP mRNA
Phase 2A Phase 2B Phase 3
Condition tree
Distance
1000 (5/70)
Clustering of microarrays
Liver
Brain
Seal Preg/Lact
LL
P
EL
3
2b
c
More than 80 secreted proteins during
lactation
2A 2B 31Phase
Sharp et al (2009) Milk proteins: from expression to food p55
Cross-fostering study
Mother at day
120 lactation
Control group
PY day 120 PY day 120 Mother at day
170 lactation
Fostered group
60 days period
Fostered PY had access to
day 170 - 230 milk
Control PY had access to
day 120 - 180 milk
Fostered PY had accelerated growth
Initial 200 days post-partum ~ 230 days post-partum
• Forestomach is
gastric-like
– Has gastric cells
• Forestomach matures
into cardia-like
– Lost gastric cells
Forestomach with
gastric cellsForestomach with
NO�gastric cells
Genes highly expressed /
“unique” to forestomach
with gastric cells
Genes highly expressed /
“unique” to forestomach
with NO gastric cells
ATP4A
GKN2
GHRL
NDRG2
MUC4
KRT20
CSTB
ITLN2
LPLUNC1Kwek et al. (2008) J Exp Zool B Mol Evol Dev
PY 170 PY 230
Gastric phenotype gene expression
* P < 0.001 compared to control PY 180 # P < 0.001 compared to control PY 230
D NDRG2C GHRLB GKN2A ATP4A
Down-regulated in the fostered PY forestomach
Cardia phenotype gene expression
* P < 0.001 compared to control PY 180 # P < 0.001 compared to control PY 230
A MUC4 B KRT20 C CSTB D ITLN2 E LPLUNC1
Remained unchanged in the fostered PY
forestomach
conclusion
Milk switching accelerates growth
And influences forestomach differentiation
• Small body size
• 10-12 month lactation
• Long foraging trips
• Milk production at 20% while foraging
• Controlled at the transcriptional level
• Milk volume increases with each trip
2-3 days
23 days
Otariids (fur seal)
– foraging strategy
Comparative biology
Bioinformatics
e-research platforms
Biotechnology
MammoSapiens
GENE LOSS MAY ACCOUNT FOR REDUCED INVOLUTION
STRATEGY TO REDUCE INVOLUTION / EXTEND LACTATION
Comparative Genomics
cDNA sequencing
canine microarray
Est
sequencesBlast
hits
Sequence
similarity
search
Blast
gene
Sequences
Unigene
ensemblLoad inte
rfa
ce
query
inte
rface
Job management interface
compEXP-PAC
Expression series
database
Oligo
Microarray
cDNA
Microarray
MPSS
EST-PAC
EXP-PAC
MamExp: online retrieval of expression data across experiments and species
Gene mapping
Comparative biology
Bioinformatics
e-research platforms
Biotechnology
Essential pathwaysMilk composition, synthesis and function
MammoSapiens
Comparative Transcriptomics
Folate receptor α
Phase 2B – 3 lactation
1229 1980
711
*9220
seal wallaby
cow
x
*2617 probes
~26%
Preg – lactation
Folate receptor identified:comparative microarray analysis
also up-regulated in
•human & mouse lactating
mammary glands
•IFP cultured cow & mouse
mammary explants
* Limitation: gene transcripts only identified if the gene probe is
present on both the canine and bovine Affymetrix genechips as
well as the wallaby EST array.
Preg – lactation
lactation bioinformatics
-Conclusion
Comparative milk transcriptomics
Developing�on line resources for analysis of genomic data:
-facilitate data storage, visualization and analysis on line
-across: partners, experimental platforms, species..
Learning from the data…
MamExpressionist: portal to gene expression
Challenges:
Integrating expression data analysis, pathway, genomes, other public data & literature…
Comparative genomics … collaborative analysis?
Comparative Lactation BiologyMamExp: an interactive gene expression browser
for collaborative comparative lactation genomics
Christophe Lefèvre , ITRI, BioDeakin, GTP Deakin University
Kevin Nicholas
Julie Sharp
Karensa Menzies
Pragati Sharma
Sanjana Kuruppath
Amit Kumar
Philip Church
James Stillman
Shannu Palamuru
VPAC
Adam Wong
Zoology Uni.Melbourne
Matthew Digby
John Arnould
Sonia Mailer
Cate Pooley
Kylie Cane
Elizabeth Pharo
Coralie Reich
Alison DeLeo
Elie Khalil
Danny Topcic
Joly kwek
Rachael O’Dowd
Amelia Brennan
Karensa Menzies
Maurice Ling
V.B.C, monash
David Powell
Yvan Strahm
Torsten Seeman
Reprogen Uni. Sydney
Keith L. Macmilland
Paul A Sheehy
Peggy Rismiller, University of Adelaide, SA
Katherine Belov, Faculty of Veterinary Science, University of Sydney, NSW platypus genome consortium
?
!
Cluster analysis of in vivo cow mammary and in vitro mammary
explant cultures expression data.
Involution lactation pregnancy in vitro
Separation in vivo / in vitro & sample source.
colostrum ?. sFPI(Karensa) versus IFP(Amelia)