Transcript Identification & characterization of regulatory targets of

Microarray data analysis towards the
understanding the role of hzy in the
formation of rhabdomeres
Ashwini Oke
Ashwini Oke
School of Informatics,
Indiana University
Drosophila compound eye
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Rhabdomeres carry out the light
reaction
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Montell, C. 1999
Formation
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Initiation
Elongation
?
?
Maintenance
hzy is required for rhabdomere formation and
phototransduction
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Development 130, 4383-4392 (2003)
hzy is a transcription factor
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The phototransduction pathway
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The EMBO Journal (2001) 20, 4414–4422, doi:10.1093/emboj/20.16.4414
Known targets of hzy
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Goals
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

Identify targets of hzy using oligonucleotide
expression arrays
Determine the stage of rhabdomere formation
that requires hzy

Identify DNA binding site for hzy

Understand the regulation of hzy itself
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Identifying regulatory targets of hzy
Microarray design
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DGRC-2 Oligonucleotide Arrays
Genotypes:
WildType - cnbw
Mutant - hzy
- otd;hzy
Time Points:
48 hr APF
72 hr APF
96 hr APF
www.scq.ubc.ca/wp-content/cDNAarray.gif
Replicates:
4 independent samples
for each time point
DGRC-2 arrays
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Probes
~14,400 probes representing 93.5%
of the genome
Negative Controls
Blank
Spotting_Solution
Exogenous targets
Positive Controls
Housekeeping genes
tiff image from the scanner
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GenePix Pro records spot intensities
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GenePix Pro creates .gpr file
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Analysis workflow
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R2.5.1 Bioconductor
Packages: LIMMA, OLIN, MARRAY
Preprocessing
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Scanned
Image
Heat Plot
Density plot
Normalization
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Genes
Blank
Spotting Solution
Arabidopsis
A = log 2 average intensity
for the two channels
M = log ratio of intensities
for the two channels
Normalization within array
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LOESS
Local polynomial regression fit
Normalization between arrays
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LIMMA: Linear models for Microarray Data
Raw
within array
normalization
between arrays
normalization
Density Plots after normalizations
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Differential analysis
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Volcano plot
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at 72 hr APF
(12 total)
at 96 hr APF
(28 total)
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Gene
M
Gene
P
M
P
Gbeta76C
-3.75
0
Rh6
-4.94
1.00E-05
ninaC
-3.65
0
Rh2
-5.13
3.00E-05
Arr2
-3.41
0.00002
Arr1
-3.25
7.00E-05
Trp
-2.16
0.00011
Trp
-2.75
0.00015
CG8889
-2.68
0
Gbeta76C
-4.12
0.00021
CG31313
-2.35
0.00002
CG2650
-3.56
5.00E-05
TotB
-2.5
0
Ank2
-2.21
0.00018
nimB2
-2.25
0.00002
Trpl
-6.51
3.00E-05
CG5172
-2.84
0.00001
CG8889
-2.64
0.00029
PIP82
-2.12
0.00002
CG42330
-10.75
0.00329
Osi18
-2.45
0.00004
rdga
-2.55
0.00452
CG10953
-2.35
0.00038
Sr-CI
-2.65
0.00028
Gene Ontology
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GO Database provides a vocabulary to describe
gene and gene products
 Cellular
Component
Eg : Cytoplasmic, Nuclear, plasma membrane
 Molecular
Function
Eg : Enzyme activity, protein binding
 Biological
Process
Eg : Apoptosis, cell adhesion, phototransduction
Classifying genes by GO
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4
4
phototransduction, rhabdomere
development
phagocytosis, defense
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9
structural components
enzymes with unknown biological
process
other
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1
2
At 72 hr APF
unknown
3
5
2
2
At 96 hr APF
otd : Another transcription factor regulating
rhabdomere formation
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cnbw
hzy
cnbw
hzy;otd
otd
Results from microarray analysis of
hzy;otd double mutant
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otd;hzy 72 hr APF
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structural components
15
4
5
3
4
otd;hzy 96 hr APF
phototransduction,
rhabdomere development
phagocytosis, defense
15
18
enzymes with unknown
biological process
other
5
unknown
3
8
1
hzy is required during elongation
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Time point 48 hr APF 72 hr APF 96 hr APF
# down
regulated genes
EM data
0
No difference
between cnbw and
hzy
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hzy mutant has
irregular
rhabdomeres
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hzy mutant has
malformed
rhabdomeres
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Identifying DNA binding site for hzy
hzy has been shown to bind to a motif
upstream of eyeG
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TAATNNNATTA
Development 130, 4383-4392 (2003)
MEME
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MEME is a tool for discovering motifs in a group of
related DNA or protein sequences
Primary targets of hzy should have a conserved
binding site in their promoter region
Motif search : 72 APF targets
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Down regulated genes
meme 72.fas -dna -nmotifs 5 -mod zoops -revcomp -minw 3 -maxw 15
A true motif should be conserved
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


For each of the hzy targets at
72 hr APF, find orthologs in
other groups of Drosophila
Retrieve 500 b upstream of
each gene
Run MEME on promoter
regions from each specie
Motif is conserved across orthologs
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Understanding the regulation of hzy
Locating the promoter region
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1.4 kb
0.8 kb
Apitz, H. 2002. Dros.Inf.Serv.85:118-120.
Promoter testing with Gal4-UAS system
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Promoter Gal4
X
UAS Reporter
Element
Result
0.8 kb upstream of hzy
Eye specific GFP expression
1.4 kb upstream of hzy
GFP in eye and some other organs
Conserved regions
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Future Directions
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
RT PCR to confirm targets

Search hzy promoter region for known TFBS
Thank you
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


Dr. Andrew Zelhof
Dr. Haixu Tang
Dr. Qunfeng Dong



Monalisa Mishra PhD
Andreas Rechsteiner PhD
Jacqueline Lopez