GENOME-WIDE IDENTIFICATION OF
REGULATORS OF TRANSCRIPTIONAL
NETWORKS DURING HEMATOPOIETIC
DEVELOPMENT
or
Genetical Genomics in Stem Cell Research
Hematopoietic differentiation
(Bryder et al., 2006)
Four hematopoietic cell populations
B cell
T cell
Macrophage
Lin-Sca-1+c-Kit+
Granulocyte
Gr-1+
Platelets
9 25 BXD strains
9 4 cell populations
Lin-Sca-1-c-Kit+
9 Sentrix Mouse-6 BeadChips
(Illumina)
Erythrocyte
Ter-119+
3 dimensional analyses
(which genes are differentially expressed?)
25 samples x 47, 000 probes x 4 cell populations analyzed
Cell type A
Cell type B
(What are the cell-stage specific genes?)
>1000
transcripts
1. Are equally expressed transcripts
identically regulated during development?
2. Are the differentially expressed transcripts
under common control (ie master
regulators)?
3.If there are master regulators, where are
they and which genes do they control?
Genetically regulated stem cell traits
• C57BL/6:
• DBA/2:
Low frequency
Low proliferation
No loss during aging
Low mobilization
High in vivo self renewal
High in vitro expansion
High frequency
High proliferation
Strong loss during aging
High mobilization
Low in vivo self renewal
Low in vitro expansion
BXD recombinant inbred strains of mice
D2
B6
F1
F2
BXD
phenotype
phenotype
Mapping Quantitative
Trait Loci
Lod score
BXD strain
QTL
Genomic position (bp)
Genetical Genomics in BXD recombinant inbred mice
Bone
marrow cells
from 25 BXD
strains
Sort Lin-Sca1+ c-kit+,
Lin-Sca1- c-kit+,
Gr1+,
Ter119+ cells and isolate RNA
Illumina 47K chips
Measure variation in
gene expression
Candidate
Quantitative Trait Locus
Compare QTLs with
gene position
QTL and gene position
coincide
Cis regulation
QTL and gene position
do not coincide
Trans regulation
845 Consistent eQTLs
DBA allele
Mean values
Expression level
B6 allele
618 stem cell specific eQTLs
DBA allele
Mean values
B6 allele
Expression levels log2
9 transcripts with strong effects in HSC
...... and many more.....to be functionally characterized.....
mRNA level
Examples of cell type-dependent, dynamic, eQTLs
Stem
Progenitor
Erythroid
Myeloid
B6
D2
Transbands
204Mb, Chr2 :
182 genes
1103Mb, Chr7 :
89 genes
2075Mb, Chr15:
181 genes
2148Mb, Chr16:
117 genes
(Numbers given for threshold logp >= 6)
Networks, clusters, transbands
Combining differential expression and variation in expression
Variation between cell types
Variation across strains
stem cells
progenitors
myeloid cells
Genome-wide DNA methylation analysis using MeDIP
Methylated DNA Immuno Precipitation
myeloid cells
Bi-potent
progenitors
Gr-1+
LineageSca-1c-kit+
TER119+
erythroid cells
Nimblegen CpG island-Plus-Promoter array
- all UCSC-annotated CpG islands
- all promoter regions for all RefSeq genes
- promoter region covered is 1.8 kb
- small CpG islands are extended to a total coverage of 700 bp
(Weber et al., 2005)
Genetics of epigenetics
Tile 1
Tile 2
Tile 3
Number of methylated promoters
D2
pFP = 0.05
B6
D2
pFP = 0.01
B6
LSK
ter
gr1
Categorizing differential methylation profiles among B6 cell populations
(1)
gr1
3.08
Prog
(2)
0.38
(3)
0.75
(4)
0.37
83.04
(5)
(6)
(7)
(8)
ter
2.35
6.35
3.65
Categorizing differential methylation profiles among D2 cell populations
(1)
(2)
0.12
(3)
0.25
(4)
0.20
Prog
ter
74.79
(5)
(6)
gr1
3.58
3.22
(7)
8.29
(8)
9.50
Genetics of epigenetics
gr1
Prog
1200
B6
D2
1000
Number of promoters
ter
overlap
800
600
400
200
N.D.
0
1
methylated
2
3
demethylated
4
5
unmethylated
6
7
8
differentially methylated
Challenges
• how to confirm networks?
we can perturb expression of single candidates but not (so easy) of
multiple genes
• what is the molecular nature of cis- and trans
eQTLs?
do epigenetic modifications play a role?
do miRNAs play a role?
• how to integrate all layers of genetical
genomics data
• how to integrate/relate to other experimental
platforms
how 'private' are our data?
how to relate to human genetics?
Are eQTLs irrelevant noise to buffer the system,
or is the system stable because eQTLs are
there?
Stem Cell Biology, Groningen
the Netherlands
Groningen Bioinformatics
Center
UT at Memphis, TN
Sandra Rizo-Crcareva
Alice Gerrits
Marta Walasek
Evgenia Verovskaya
Leonid Bystrykh
Ronald van Os
Vincent van den Boom
Brad Dykstra
Ellen Weersing
Bertien Ausema
Olya Kalmykova
Bert Dontje
Gerald de Haan
Ritsert Jansen
Rainer Breitling
Bruno Tesson
Yang Li
Frank Johannes
Robert Williams
Xusheng Wang
PhD positions open!
www.genenetwork.org