Comparison of CHiP-chip analysis methods
John Cumbers - Tatar lab
Diet restriction increases lifespan
No Insulin
Insulin
P P FOXO FOXO
http://www.vscht.cz/eds/knihy/uid_es-002/figures/receptor.01.jpg
FOXO is a Winged-Helix Transcription factor
•
Jin, C et al J.Mol.Biol. 1999
Research goals
• To understand how FOXO:
– Controls multiple phenotypes – Extends lifespan
Strategy
• To model FOXO’s insulin signaling network • To find the genes regulated by FOXO
Investigating FOXO
DNA binding sites
Computational Predictions Biological Validation
Transcription factors bind to DNA regulating transcription of genes.
FOXO
Image: Wikipedia
ChIP chip Technique Chromatin Immuno-Precipitation
FOXO
FOXO
Formaldehyde crosslinking
FOXO
FOXO
Sonication
FOXO
500 base pair fragments
FOXO
Antibody
FOXO
Glass beads
Precipitation
FOXO
Elution
Antibody
Glass bead
FOXO
Uncrosslink
FOXO
Purify DNA
What is the sequence?
1. PCR for individual genes 2. Sequence all fragments 3. Use tiled micro arrays
Genomic location
Example results
Fold enrichment
Richard Young, MIT, Yeast ChIP chip results
Multiscale analysis
MAT
Mixture Model
Plan
• Pick dataset
– Isogai et al, RNA Pol III – Cawley et al, cMyc, p53, Sp1
• Get a list of unbiased genes from computational predictions. • Use TiMAT to put into correct format • Put the dataset through method