Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres Ashwini Oke School of Informatics, Indiana University

Drosophila compound eye 2

Rhabdomeres carry out the light reaction Montell, C

Formation ? ? InitiationElongationMaintenance 4

hzy is required for rhabdomere formation and phototransduction Development 130, (2003) 5

hzy is a transcription factor 6

The phototransduction pathway The EMBO Journal (2001) 20, 4414–4422, doi: /emboj/

Known targets of hzy 8

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 9

Identifying regulatory targets of hzy 10

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 11

DGRC-2 arrays ooooooooooooooooooo Probes ~14,400 probes representing 93.5% of the genome Negative Controls Blank Spotting_Solution Exogenous targets Positive Controls Housekeeping genes 12

tiff image from the scanner 13

GenePix Pro records spot intensities 14

GenePix Pro creates.gpr file 15

Analysis workflow R2.5.1 Bioconductor Packages: LIMMA, OLIN, MARRAY 16

Preprocessing Scanned Image Heat PlotDensity plot 17

Normalization A = log 2 average intensity for the two channels M = log ratio of intensities for the two channels Genes Blank Spotting Solution Arabidopsis 18

Normalization within array LOESS Local polynomial regression fit 19

Normalization between arrays Rawwithin array normalization between arrays normalization LIMMA: Linear models for Microarray Data 20

Density Plots after normalizations 21

Differential analysis 22

Volcano plot 23

at 72 hr APFat 96 hr APF (12 total)(28 total) GeneMP Gbeta76C ninaC Arr Trp CG CG TotB-2.50 nimB CG PIP Osi CG GeneMP Rh E-05 Rh E-05 Arr E-05 Trp Gbeta76C CG E-05 Ank Trpl E-05 CG CG rdga Sr-CI

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 25

Classifying genes by GO At 72 hr APF At 96 hr APF 26

otd : Another transcription factor regulating rhabdomere formation cnbw hzy;otd otdhzy cnbw 27

Results from microarray analysis of hzy;otd double mutant 28

hzy is required during elongation 29 Time point48 hr APF72 hr APF96 hr APF # down regulated genes EM data No difference between cnbw and hzy hzy mutant has irregular rhabdomeres hzy mutant has malformed rhabdomeres

Identifying DNA binding site for hzy 30

hzy has been shown to bind to a motif upstream of eyeG  Development 130, (2003) 31 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 32

Motif search : 72 APF targets meme 72.fas -dna -nmotifs 5 -mod zoops -revcomp -minw 3 -maxw 15 Down regulated genes 33

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 34

Motif is conserved across orthologs 35

Understanding the regulation of hzy 36

Locating the promoter region 1.4 kb 0.8 kb Apitz, H Dros.Inf.Serv.85:

Promoter testing with Gal4-UAS system X Gal4UASReporterPromoter 38 ElementResult 0.8 kb upstream of hzyEye specific GFP expression 1.4 kb upstream of hzyGFP in eye and some other organs

Conserved regions 39

Future Directions 40  RT PCR to confirm targets  Search hzy promoter region for known TFBS

Thank you 41  Dr. Andrew Zelhof  Dr. Haixu Tang  Dr. Qunfeng Dong  Monalisa Mishra PhD  Andreas Rechsteiner PhD  Jacqueline Lopez