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Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres. Ashwini Oke School of Informatics, Indiana University. Ashwini Oke. Drosophila compound eye. Montell, C. 1999. Rhabdomeres carry out the light reaction. ?. ?. Formation. Initiation.
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Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres Ashwini Oke School of Informatics, Indiana University Ashwini Oke
Montell, C. 1999 Rhabdomeres carry out the light reaction
? ? Formation Initiation Elongation Maintenance
hzy is required for rhabdomere formation and phototransduction Development 130, 4383-4392 (2003)
The phototransduction pathway The EMBO Journal (2001) 20, 4414–4422, doi:10.1093/emboj/20.16.4414
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 hzyitself
Microarray design DGRC-2 Oligonucleotide Arrays Genotypes: WildType - cnbw Mutant - hzy - otd;hzy Time Points: 48 hr APF 72 hr APF 96 hr APF Replicates: 4 independent samples for each time point www.scq.ubc.ca/wp-content/cDNAarray.gif
DGRC-2 arrays Probes ~14,400 probes representing 93.5% of the genome Negative Controls Blank Spotting_Solution Exogenous targets Positive Controls Housekeeping genes ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo
Analysis workflow R2.5.1 Bioconductor Packages: LIMMA, OLIN, MARRAY
Preprocessing Scanned Image Heat Plot Density plot
Normalization 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 LOESS Local polynomial regression fit
Normalization between arrays LIMMA: Linear models for Microarray Data Raw within array normalization between arrays normalization
Gene Ontology 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 At 72 hr APF At 96 hr APF
otd : Another transcription factor regulating rhabdomere formation cnbw hzy otd cnbw hzy;otd
hzy has been shown to bind to a motif upstream of eyeG TAATNNNATTA Development 130, 4383-4392 (2003)
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
Motif search : 72 APF targets Down regulated genes meme 72.fas -dna -nmotifs 5 -mod zoops -revcomp -minw 3 -maxw 15
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
Locating the promoter region 1.4 kb 0.8 kb Apitz, H. 2002. Dros.Inf.Serv.85:118-120.
Promoter testing with Gal4-UAS system X Promoter Gal4 UAS Reporter
Future Directions • RT PCR to confirm targets • Search hzy promoter region for known TFBS
Thank you • Dr. Andrew Zelhof • Dr. Haixu Tang • Dr. Qunfeng Dong • MonalisaMishra PhD • Andreas Rechsteiner PhD • Jacqueline Lopez