ChIP-Seq: TB Example
description
Transcript of ChIP-Seq: TB Example
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ChIP-Seq: TB Example
James Galagan
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Acknowledgements
Broad, BUSeq and Analysis
Brian WeinerMatt PetersenDesmond Lun
SBRICHIP
Kyle MinchTige Rustad
David Sherman
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ChIP-Seq
Immuno-precipitation
(IP)
Control (no IP)
Sequence FragmentsAlign to Genome
Look for Enrichment
Target Site
IP
Control
(ChIP-chip) (ChIP-Seq)
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Dormancy RegulonGenes induced >2 fold after a 2-h shift from ambient to 0.2% O2.
~100 genes modulated47 upregulated = dormancy regulon
Sherman et al., 2001
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DosR Confirmation
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Hypoxic Response Depends on DosR
ORF Gene Rv ΔdosR Gene product*Rv0079 22.2 ± 6.9 0.9 ± 0.1 HPRv0080 7.8 ± 1.7 0.9 ± 0.1 HPRv0081 4.9 ± 1.2 0.5 ± 0.1 Transc. regulatorRv0082 3.1 ± 0.4 0.6 ± 0.1 Prob. oxidored. sub.Rv0083 2.1 ± 0.4 1.1 ± 0.2 Prob. oxidored. sub.Rv0569 9.0 ± 4.3 0.9 ± 0.1 CHPRv0570 nrdZ 5.5 ± 3.0 1.1 ± 0.1 Ribonuc. red. cl. II*Rv0571c 1.7 ± 0.5 1 ± 0.2 CHPRv0572c 3.0 ± 0.8 1 ± 0.1 HP*Rv0574c 2.0 ± 0.5 1.1 ± 0.1 CHPMT0639 2.0 ± 0.4 1 ± 0.2 HP***Rv1733c 4.1 ± 1.3 ND Poss. mem. prot.*Rv1734c 1.5 ± 0.1 1 ± 0.2 HPRv1736c narX 3.7 ± 0.7 1 ± 0.2 Fused nitrate red.***Rv1737c narK2 8.5 ± 2.0 1.1 ± 0.2 Nitrite extrus. prot.***Rv1738 22.8 ± 9.7 1.3 ± 0.1 CHPRv1812c 3.6 ± 0.6 1 ± 0.1 HP*Rv1813c 11.4 ± 3.0 0.8 ± 0.2 CHP*Rv1996 7.9 ± 4.6 0.7 CHPRv1997 ctpF 4.3 ± 2.2 0.8 ± 0.1 Cation trans.
ATPaseRv2003c 2.3 ± 0.6 0.9 ± 0.1 CHPRv2004c 4.2 ± 1.4 1.2 ± 0.2 HP*Rv2005c 7.3 ± 3.7 1.3 ± 0.5 CHP
The presence of one, two or three motif sequences (matrix score> 9.5) upstream of a gene is indicated by *, **or ***respectively.
ORF Gene Rv ΔdosR Gene product*Rv2006 otsB 2.2 ± 0.9 0.8 ± 0.1 Trehalose phos.*Rv2007c fdxA 25.9 ± 3.3 0.8 ± 0 FerredoxinRv2028c 6.0 ± 1.7 0.9 ± 0.2 CHPRv2029c pfkB 13.3 ± 5.7 0.9 ± 0.1 Phosphofruct. IIRv2030c 27.3 ± 6.3 ND CHP**Rv2031c acr 27.9 ± 7.6 ND α-Crystallin**Rv2032 15.1 ± 5.0 ND CHPRv2623 18.8 ± 4.1 ND CHPRv2624c 3.9 ± 1.3 0.6 CHPRv2625c 3.0 ± 1.1 1.3 ± 0.1 CHP**Rv2626c 24.5 ± 4.6 1.2 ± 0.1 CHP**Rv2627c 12.4 ± 4.9 0.8 CHP**Rv2628 13.6 ± 10.8 0.8 ± 0 HPRv2629 7.6 ± 7.4 1.4 ± 0.1 HPRv2630 6.5 ± 4.6 2 ± 0.5 HPRv2631 3.4 ± 2.1 1.4 ± 0.3 CHPRv2830c 2.6 ± 0.7 1.2 ± 0.1 HPRv3126c 1.7 ± 0.7 0.8 ± 0 HP**Rv3127 17.4 ± 2.4 0.8 ± 0.1 CHPRv3128c 1.5 ± 0.5 0.8 ± 0.1 CHPRv3129 2.7 ± 1.3 0.6 ± 0.1 CHP*Rv3130c 25.5 ± 9.4 ND CHP*Rv3131 34.1 ± 6.4 ND CHPRv3132c 5.7 ± 1.1 0.8 ± 0.1 Sensor hist. kinaseRv3133c dosR 9.1 ± 3.3 1.1 ± 0.2 Two-comp. resp.
reg.**Rv3134c 22.2 ± 17.9 1.2 ± 0.2 CHPRv3841 bfrB 5.2 ± 1.9 2.0 ± 1.3 Bacterioferritin
26 of 27 most induced genes depend on DosR
Park et al., 2001
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DosR Binding Motif
• Computational identification– YMF to search promotors of hypoxic
response genes
5’-TTSGGGACTWWAGTCCCSAA-3’
• Experimental validation– Binds both copies of motif in acr promotor– Mutation abolishes binding and induction
Park et al., 2001
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DosR Chip-Seq
• Native antibody to DosR– No tag
• DosR Control – no target for antibody– Control ChIP-Seq
• Runs at 2,4 and 8 Hours
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DosR ChIP-Seq Replicates
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Chip-Seq for TBDosR transcription factor binding at 4 hours post hypoxia
IP Enrichment
Known DosR Regulated Genes
hspxRv1733c
Desmond Lun, Kyle Minch
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DosR Binding (2 hours-IP channel)
= forward read= reverse readRv1733c
Rv1737cRv1738
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Window Enrichment Analysis
1. Divide genome into non-overlapping bins
2. Take reads from ChIP and control libraries
3. Calculate log-likelihood ratio for independence (based on chi-square)
1 2 3 4 5 6 7 8 9 10 11 12 …
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DosR ROC Curve
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Combined DosR Network
Matt Petersen, Brian Weiner
Previously known (from Park et al.)Green Dashed Park, CLR, and Chip-SeqGreen CLR and Park et al.Black Dashed Chip-Seq onlyBlack Not CLR or Chip-Seq
New Predictions (not in Park)Red Dashed CLR and Chip-SeqRed CLR
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IP Coverage vs Induction in Park et al.
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IP Coverage vs Motif MatchPeak Amplitude and Motif Match
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ChIP-Seq Peak Amplitude (Enrichment over Control)
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Location, Location, Location…
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Valouev et al (2008) Nature Methods
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DosR Binding (2 hours-IP channel)
= forward read= reverse readRv1733c
Rv1737cRv1738
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Chip-Seq Blind Deconvolution
Impulse
Function
Deconvolve
3 binding sites
Desmond Lun, Brian Weiner
single binding site
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Binding Site Resolution
Desmond Lun
Fit enrichment curve topeak from putative single site
Fit other peaks to single Site enrichment curve
Refit peaks for 2,3, etc sites
Re-estimate enrichment curvefrom all predicted site
Repeat
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Reconstruct Promoter Architecture
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CSDeconv on DosR data
Peak ID Position Amplitude
Position of motif match Difference
Absolute difference Location
1 88094.9 2.5 88124.5 -29.6 29.6 Upstream of Rv0079
2 665845.8 7.4 665858.5 -12.7 12.7 In Rv0573c
3 668495.8 2.7 668499.5 -3.7 3.7 Upstream of Rv0574c
4 1639601.5 7.4 1639626.5 -25.0 25.0 In Rv1453
5 1960517.8 13.1 1960519.5 -1.7 1.7 Upstream of Rv1733c
6 1960611.5 25.5 1960623.5 -12.0 12.0 Upstream of Rv1733c
7 1960692.3 10.4 1960692.5 -0.2 0.2 Upstream of Rv1733c
8 1965458.4 10.5 1965470.5 -12.1 12.1 Upstream of Rv1737c
9 1965543.4 14.4 1965532.5 10.9 10.9 Upstream of Rv1737c
10 2056358.2 3.1 2056374.5 -16.3 16.3 Upstream of Rv1813c, Rv1814
11 2238942.3 9.5 2238937.5 4.8 4.8 Upstream of Rv1996
12 2256458.2 12.9 2256495.5 -37.3 37.3 Upstream of Rv2007c
13 2278996.3 39.4 2279004.5 -8.2 8.2 Upstream of Rv2031c, Rv2032
14 2279046.9 22.8 2279061.5 -14.6 14.6 Upstream of Rv2031c, Rv2032
15 2949477.7 6.6 2949471.5 6.2 6.2 Upstream of Rv2623
16 2953045.1 8.3 2953073.5 -28.4 28.4 Upstream of Rv2626c
Continued…
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CSDeconv on DosR data
Peak ID Position Amplitude
Position of motif match Difference
Absolute difference Location
17 2954749.8 5.2 2954791.5 -41.7 41.7 Upstream of Rv2627c, Rv2628
18 2955065.3 9.5 2955030.5 34.8 34.8 Upstream of Rv2627c, Rv2628
19 2955479.4 9.4 2955475.5 3.9 3.9 Upstream of Rv2629
20 3492068.8 12.5 3492091.5 -22.7 22.7 In Rv3126c
21 3496439.2 50.6 3496450.5 -11.3 11.3 Upstream of Rv3130c, Rv3131
22 3500822.9 3.2 3500831.5 -8.6 8.6 Upstream of Rv3134c
• Identifies a total of 22 binding sites
• All have sequences that match a motif resembling that previously identified by Park et al.
• Motif recovered:
Park et al. (2003) Mol Microbiol.Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis.
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Rv2627c
Novel dosR Binding Site?
Rv2629Rv2628
dosR Binding Site
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Getting Induction –Inducible Promoter System
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Tagged Tet-Promotor Construct
• Named pEXNF-xxxx• EX = expression
vector• NF = N-terminal
FLAG tag• xxxx = Rv number
for gene of interest• pEXNF-3133c used
in subsequent slides
Gateway recombination sites
Tet Operator
FLAG tag
Gene of Interest
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Episomal vs KO Background
Transcription Factor Gene
Tet Promotor
Epitope Tag
Genome
X
Transcription Factor Protein
Target Promotor Region
Tet
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Induction Profiles – dosR background
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Episomal vs WT Background
Transcription Factor Gene
Tet Promotor
Epitope Tag
Genome
Transcription Factor Protein
Target Promotor Region
??
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Questions
• To drive or not to drive– Tet can drive– TF X – may not know the condition
• Can we know• Drugs• Lipids
• Induce– Qpcr – rna TO transcriptomics SBRI– Crosslink/save lysate for western debug if necessary– ChIP– Quantify that we have DNA and QC – SBRI/BU– Library prep (at BU – Chris Mahwinney)– Multiplex (10x) Solexa
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Induction Profiles – WT background
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Assaying for Tag/Untagged Ratio
Transcription Factor Gene
Tet Promotor
Epitope Tag
Genome
Transcription Factor Protein
Target Promotor Region
taggedmRNA
untaggedmRNA
qPCR mRNA levels
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Acknowledgements
Broad, BUSeq and Analysis
Brian WeinerMatt PetersenDesmond Lun
SBRICHIP
Kyle MinchTige Rustad
David Sherman
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