1. Cis-regulatory evolution of duplicate genes in yeasts
Gaurav Moghe
January-February 2009

2. Background S. cerevisiae S. bayanus S. castellii C. glabrata
S. kluyveri
K. lactis
E. gossypii

3. Goal
Scer
Scas
Klac
Pre-WGD species
Ago

4. Sequences used for the study
Genome sequences downloaded from GenBank and SGD
ORF sequences for Post-WGD species downloaded from SGD. Upstream sequences extracted using the location information of the ORF
Upstream sequences for Pre-WGD species downloaded from RSAT
PWMs obtained for 124 TFs from a study by MacIsaac et al, 2006

5. Motif Searches Search genome using MAST 106 million sites
Are MacIsaac sites being predicted?
Each site has a confidence (p-value) associated with it
Generate p-value threshold based on these sites
Filter the other predictions using these thresholds
1.4 million sites
Map the filtered predictions to intergenic regions

6. Motif Searches PWM used by MAST to scan genomes
Are MacIsaac sites being predicted?

7. Nature of PWMs
Split PWMs into 6 groups, based on their length and alphabet
Length
Class
Example
>6
Best large
gCATGTGAA
<6
Best small
GATAA
Better large
tGCTGg..
OK large
.tCGG.YsWATGGRr
OK small
wGACkC
Poor large
wwwwsyGGGG

8. Does size/nature have to do anything with False Positives?
PHO2
AYTAAr
OK small
RCS1
tgCACCy
Better large
SWI6
rACGCG
Best small
MSN2
mAGGGG.
Best large
SUT1
.gCsGgg
OK large
SWI5
tGCTGg..
SKN7
kCyrgsCc
Poor large
YAP5
ARrCAT
CST6
tgCATTT.
SOK2
.cAGGmAm No
10 TFs account for ~1.1 million sites out of 1.4 million sites
No relation between size/nature and False Positives
No good for many other TFs

9. Then…
We decided to use only the MacIsaac sites for searching across species
Map the MacIsaac sites onto the intergenic region
Look at their loss patterns in other species in orthologous promoters

10. Orthologous genes
Orthologous genes obtained through Yeast Genome Browser (YGOB)
The gene names of YGOB do not correspond to gene names provided by SGD for the sensu stricto species
BLASTp to find out which YGOB annotation corresponds to which SGD annotation
Some genes are being lost in this process

11. Then…
We decided to use only the MacIsaac sites for searching across species
Map the MacIsaac sites onto the intergenic region
Look at their loss patterns in other species in orthologous promoters

12. Using MAST Ideal case Also observed for some TFs Scer Macisaac sites
Sbay MAST predictions
Scer: Saccharomyces cerevisiae
Sbay: Sacharomyces bayanus

13. Using MAST Scer Macisaac sites Sbay MAST predictions
Scer: Saccharomyces cerevisiae
Sbay: Sacharomyces bayanus

14. Using Phylogeny based methods
Many programs available
PhyloCon
Morph
Phylogibbs
FootPrinter
Gibbs Sampler
All for motif discovery, not motif search using phylogenetic principles

15. Using Phylogeny based methods
Conserved Regulatory Elements anchored Alignment (CONREAL)
Monkey (Mike Eisen)
PhyloScan
Conditional Shadowing via Multi-resolution Evolutionary Trees (CSMET)

18. Plans for the next month
Test MONKEY/PhyloScan on the intergenic elements
Estimate the False positive/False negative rate under the specified parameters, based on known TFBS

19. Novel RNA genes project
January-February 2009

20. Download EST sequences corresponding to PUTs
737301
Map them to the genome using GMAP (L>50bp,Cov>70,Idt>90%)
605624
1353
Yes?
Map to AT RNA genes
7357
Map to protein-coding regions
No?
Map to other AT features
BLASTn against all known CDS sequences +
GeneWise to confirm alignment on translated CDS sequences
2500
3260
No match?
2431
BLASTn against Repetitive Sequence Database
No match?
Coding Index to double-verify absence of protein-like seq
1893
No match?
BLASTx against all known proteins to verify absence of any protein in the sequences
1867
No match?

21. BLASTn against all RNA family sequences in RFAM
1867
BLASTn against all RNA family sequences in RFAM
1837 30
Manual filtering on NCBI by Andy giving ~13% False Positive Rate
~1600 novel ESTs
Conservation in lyrata using GMAP
RNA structure prediction
Expression conservation
Wet lab confirmation
Substitution rate
Tiling array
817 at 60% coverage and 75% Idt, nhits<=3
Shan helping out with this