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![Page 1: Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres Ashwini Oke School of Informatics, Indiana University.](https://reader036.fdocuments.us/reader036/viewer/2022070407/56649e295503460f94b17120/html5/thumbnails/1.jpg)
Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres
Ashwini Oke
Ashwini OkeSchool of Informatics,
Indiana University
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Drosophila compound eye2
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Rhabdomeres carry out the light reaction
Montell, C. 1999
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Formation
? ?
Initiation Elongation Maintenance
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hzy is required for rhabdomere formation and phototransduction
Development 130, 4383-4392 (2003)
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hzy is a transcription factor6
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The phototransduction pathway
The EMBO Journal (2001) 20, 4414–4422, doi:10.1093/emboj/20.16.4414
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Known targets of hzy8
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Goals
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
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Microarray design
www.scq.ubc.ca/wp-content/cDNAarray.gif
DGRC-2 Oligonucleotide Arrays
Genotypes:WildType - cnbwMutant - hzy
- otd;hzyTime Points:
48 hr APF72 hr APF96 hr APF
Replicates:4 independent samples for each time point
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DGRC-2 arrays
oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo
Probes~14,400 probes representing 93.5% of the genome
Negative ControlsBlankSpotting_SolutionExogenous targets
Positive ControlsHousekeeping genes
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tiff image from the scanner 13
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GenePix Pro records spot intensities
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GenePix Pro creates .gpr file
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Analysis workflow
R2.5.1 Bioconductor
Packages: LIMMA, OLIN,
MARRAY
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Preprocessing
ScannedImage
Heat Plot Density plot
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Normalization
A = log 2 average intensity for the two channels
M = log ratio of intensities for the two channels
GenesBlankSpotting SolutionArabidopsis
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Normalization within array
LOESSLocal polynomial regression fit
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Normalization between arrays
Raw within arraynormalizati
on
between arraysnormalization
LIMMA: Linear models for Microarray Data
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Density Plots after normalizations
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Differential analysis22
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Volcano plot23
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at 72 hr APF at 96 hr APF (12 total) (28 total)
Gene M P
Gbeta76C -3.75 0
ninaC -3.65 0
Arr2 -3.41 0.00002
Trp -2.16 0.00011
CG8889 -2.68 0
CG31313 -2.35 0.00002
TotB -2.5 0
nimB2 -2.25 0.00002
CG5172 -2.84 0.00001
PIP82 -2.12 0.00002
Osi18 -2.45 0.00004
CG10953 -2.35 0.00038
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Gene M P
Rh6 -4.94 1.00E-05
Rh2 -5.13 3.00E-05
Arr1 -3.25 7.00E-05
Trp -2.75 0.00015
Gbeta76C -4.12 0.00021
CG2650 -3.56 5.00E-05
Ank2 -2.21 0.00018
Trpl -6.51 3.00E-05
CG8889 -2.64 0.00029
CG42330 -10.75 0.00329
rdga -2.55 0.00452
Sr-CI -2.65 0.00028
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Gene Ontology
GO Database provides a vocabulary to describe gene and gene products
Cellular Component Eg : Cytoplasmic, Nuclear, plasma membrane
Molecular FunctionEg : Enzyme activity, protein binding
Biological ProcessEg : Apoptosis, cell adhesion, phototransduction
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Classifying genes by GO
At 72 hr APF At 96 hr APF
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otd : Another transcription factor regulating rhabdomere formation
cnbw
hzy;otd
otdhzy
cnbw
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Results from microarray analysis of hzy;otd double mutant28
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hzy is required during elongation
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Time point
48 hr APF
72 hr APF
96 hr APF
# down regulated genes
0 12 28
EM dataNo difference between cnbw and hzy
hzy mutant has irregular rhabdomeres
hzy mutant has malformed rhabdomeres
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Identifying DNA binding site for hzy
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hzy has been shown to bind to a motif upstream of eyeG
Development 130, 4383-4392 (2003)
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TAATNNNATTA
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MEME
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
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Motif search : 72 APF targets
meme 72.fas -dna -nmotifs 5 -mod zoops -revcomp -minw 3 -maxw 15
Down regulated genes
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A true motif should be conserved
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
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Motif is conserved across orthologs
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Understanding the regulation of hzy
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Locating the promoter region
1.4 kb0.8 kb
Apitz, H. 2002. Dros.Inf.Serv.85:118-120.
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Promoter testing with Gal4-UAS system
XGal4 UAS ReporterPromoter
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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
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Conserved regions 39
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Future Directions40
RT PCR to confirm targets
Search hzy promoter region for known TFBS
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Thank you41
Dr. Andrew Zelhof Dr. Haixu Tang Dr. Qunfeng Dong
Monalisa Mishra PhD
Andreas Rechsteiner PhD
Jacqueline Lopez