2 SNPs

1
2 SNPs

• Nov 2007

2
Fastas
gtrs6257Deb-0161121T/Ccommon CCCTGTAGCAGGGCCTC
TCCCTCTGTCTGTCTCTGACATGTCCCTACTCAGCTTTGTTTGT TTTCT
CTTTCTGATAGAGTGCCCAYGACCCTCCGGCTGTCCACCTCAGCAATGGC
CCAGG gtrs6257Deb-0161121T/Crare CCCTGTAGCAGGG
CCTCTCCCTCTGTCTGTCTCTGACATGTCCCTACTCAGCTTTGTTTGC T
TTCTCTTTCTGATAGAGTGCCCAYGACCCTCCGGCTGTCCACCTCAGCAA
TGGCCCAGG gtrs727428Deb-0261121G/Acommon CCT
GCTGCTTCTTCCCTGGGCAGCAATGGCCAGTCCAAGGACCAGATGACTGA
GATGTCAG AATCCCATCTCTTCCCTTCCTCCTGATCTCTCTGGACCAGC
CACAGAGGCAGGAAACTGG gtrs727428Deb-0261121G/Ara
re CCTGCTGCTTCTTCCCTGGGCAGCAATGGCCAGTCCAAGGACCAGAT
GACTGAGATGTCAA AATCCCATCTCTTCCCTTCCTCCTGATCTCTCTGG
ACCAGCCACAGAGGCAGGAAACTGG
3
Splicing rainbow no change Deb-01
4
Splicing rainbow Deb-02rare allele abrogates
binding
SRp40 ASF/SF2 Sites diminished In rare allele
5
ESE finder (splice factors)
Plenty of splice factors close to SNP but nothing
over Deb-01 No difference with rare allele
6
ESE finder (splice factors) Deb-02
One splice factor SC25 over SNP Binding
abolished with rare allele
7
Exonic Splice Enhancers for Deb-02 only2 in
common replaced by 1 in rare
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Transcription factors (genomatix) Deb-01
SNP position 61
p53 within 40bp and 5bp
TFs bind over SNP
4 TFs (two forkhead/FOX) replaced bu 3 TFs (one
different forkhead) One forkhead and a src-type
Tyrosine kinase binding abrogated by rare allele
9
Transcription factors (genomatix) Deb-02
SNP position 61
Oct1 Next to SNP
TFs bind over SNP
No difference in TF binding between alleles,
contrast splice and ESE binding changes
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Chr 35 positions (Premod)

• SNP Position Chr Allele Consequence - rs6257
  7474442 17 T/C INTRONIC rs727425 41551274 17 C/T
  INTRONIC
• Closest binding Modules
• chr177495122-7496108 , 20kb away, contains c-myc
  and forkhead 01, 04 boxes
• chr1741327428-41328284, 220kb away
• Contains Ah R (aryl hydrocarbon/dioxin receptor),
  Myc, Myb, POU/Oct binding sites
• Possibly too far away to be meaningful

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Conclude

• Deb-01 rs6257
• No change hn binding
• Or splice factors
• Or ESE (enhancers)
• P53 site upstream of SNP
• Changed TF binding with 2 forkheads (ER) on SNP
  replaced by one

• Deb-02 rs727428
• Changed binding of splice factors
• SC35
• SRp20
• AF2/
• Abrogated by rare SNP
• No change in TF binding
• Oct1 binding next to SNP (ER)

12
More techniques

• Nov 2007

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PreMod build35 cis-regulatory transcription
factor modules of 400-2000 bp
Gene desertBCAC3-11
rs2121629 HOX? RB/E2F-1/DP-1 complex
Wilms tumor suppressor, Forkhead/SMAD interactors
TATA N-Myc Myc associated
rs1447295
rs13281615 and linked SNPs
NSE1
cMYC
ER T T p53
56kbp 1738bp 199bp
Breast cancer membrane protein 101
breast 500kb
prostate 81kb
Chr8128616000
Chr8128535000
Chr8128350000
5 cis-regulatory modules in prostate block
11 cis-regulatory modules in BrC extended
block (r2p0.1)
Module collection of TFs - more likely to be a
functional unit than a single TF
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PreMod outputReview cis-regulatory modules,
click through for details
15
Genomatix.de Matinspector

• Determine changes in predicted TF binding sites
  caused by SNP alleles - input a file with a fasta
  for each allele (slide 2)

16
UCSC for conservation
1600bp from next cis-regulatory module
17
Good conservation of module and SNP
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Island of conservation within 30bp of
Transcription factor on SNP

• Combining evidence
• Island of conservation so could be TF binding
  site
• PreMod (limited) shows no suitable collection of
  TFs - ignore
• Matinspector predicts CUTL1 (CASP8) binding over
  SNP
• no altered binding with allele
• lots of potential TF binding sites neighbouring

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Vista genome browserConservation across species
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Detailed conservation across 85bpEnsembl
Alignslicewith conservation of SNP nucleotide
(3bp) sequence
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Alignslice results (count conserved bases)Heat
map of Conservation across SNP
Top conserved Red 90-100 conserved Pink
70-80 conserved Orange 50-80 conserved Yellow
lt50 conserved Bottom SNP
conserved Orange rare (high risk) homolog
conserved Green common homolog conserved (good
allele) Blue NOT conserved
1common, 2rare, ()Not conserved, 0wrong
base,-1gap,-no alignment
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BLAST if you cant find a predicted gene in other
speciesGood homology in mouse for LOC643714
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TNRC9-LOC643714

• 4256bp gap between 5UTR of TNRC9 and end of LOC
  in human Chr16
• Structure preserved in chimp (Ensembl)
• A mouse homolog to the LOC transcript sequence is
  found in the same position relative to mouse
  TNRC9 (Chr8) with 5202bp between the genes

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Mouse homolog conserved upstream of TNRC9 (and
Chimp)
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Ensembl gt protein function via conserved motifs
High Mobility group HMG found in transcription
factors e.g. FACT (facilitating chromatin
transcription complex ssrp1-B), histones, SOX
transcription factors, myeloid/lymphoid (mixed
lineage) leukaemia protein 2 (ALL1-related
protein)
CAGF9
Nuclear localisation signal
Q (Gln) rich region for binding DNA
Coiled coil domains for dimerisation formation
of active complexes
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BLAST
Evolutionary relations
HMG box protein 1 (Granulosa cell) etc. - HMG-box
homology only (20) - eukaryotic chromosomal
proteins - transcription factors
Epidermal Langerhans cell protein LCP1 - in human
cell signalling pathway - potent
antigen-presenting cells
Transcription factors MGC84449 protein
zebrafish and purple sea urchin -HMG containing
Thymus high mobility group box protein TOX
(thymocyte selection) TOX superfamily of HMG
containing proteins - expression induces changes
in coreceptor expression - beta-selection and CD8
(T cell receptor) gene demethylation.
TNRC9 zebrafish purple sea urchin
TNRC9 mouse cow rat chicken
Primate TNRC9 (incl Human)
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Ensembl for all ESTs in region
HMG box (ex5,6) CAGF9 (Epidermal Langerhans Cell
LCP1 family)
TNRC9
orf
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Chr16 51029418 - 51178307
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Bioinformatics for SNP function

• Conservation (good for splice site, island
  within 30bp for transcription factor TF)
• Vista for diagram
• Ensembl/UCSC alignments for homology,
  conservation of SNP site
• Splice factor and hn binding factors for introns
• Rainbow, ESE finder, Rescue-ese
• TFs for introns or intergenic regions
• Genomatix Matinspector for SNPs, PreMod for
  regions
• Use tissue filters
• Classic tools for protein function and evolution
• BLAST, phylogeny diagrams for DNA homologues
• ProSite from Ensembl for protein