Ashwini Oke

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

3

Formation

? ?

Initiation Elongation Maintenance

4

hzy is required for rhabdomere formation and phototransduction

Development 130, 4383-4392 (2003)

5

hzy is a transcription factor6

The phototransduction pathway

The EMBO Journal (2001) 20, 4414–4422, doi:10.1093/emboj/20.16.4414

7

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

9

Identifying regulatory targets of hzy

10

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

11

DGRC-2 arrays

oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo

Probes~14,400 probes representing 93.5% of the genome

Negative ControlsBlankSpotting_SolutionExogenous targets

Positive ControlsHousekeeping 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

ScannedImage

Heat Plot Density plot

17

Normalization

A = log 2 average intensity for the two channels

M = log ratio of intensities for the two channels

GenesBlankSpotting SolutionArabidopsis

18

Normalization within array

LOESSLocal polynomial regression fit

19

Normalization between arrays

Raw within arraynormalizati

on

between arraysnormalization

LIMMA: Linear models for Microarray Data

20

Density Plots after normalizations

21

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

24

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

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 mutant28

hzy is required during elongation

29

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

30

hzy has been shown to bind to a motif upstream of eyeG

Development 130, 4383-4392 (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 kb0.8 kb

Apitz, H. 2002. Dros.Inf.Serv.85:118-120.

37

Promoter testing with Gal4-UAS system

XGal4 UAS ReporterPromoter

38

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