Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres Ashwini Oke Ashwini Ok School of Informatic Indiana Universi
Jan 11, 2016
Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres
Ashwini Oke
Ashwini OkeSchool of Informatics,
Indiana University
Drosophila compound eye2
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
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
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
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
Volcano plot23
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
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
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
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
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
Conserved regions 39
Future Directions40
RT PCR to confirm targets
Search hzy promoter region for known TFBS
Thank you41
Dr. Andrew Zelhof Dr. Haixu Tang Dr. Qunfeng Dong
Monalisa Mishra PhD
Andreas Rechsteiner PhD
Jacqueline Lopez