ChIP-Seq: TB Example

James Galagan

Acknowledgements

Broad, BUSeq and Analysis

Brian WeinerMatt PetersenDesmond Lun

SBRICHIP

Kyle MinchTige Rustad

David Sherman

ChIP-Seq

Immuno-precipitation

(IP)

Control (no IP)

Sequence FragmentsAlign to Genome

Look for Enrichment

Target Site

IP

Control

(ChIP-chip) (ChIP-Seq)

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

DosR Confirmation

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

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

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

DosR ChIP-Seq Replicates

Chip-Seq for TBDosR transcription factor binding at 4 hours post hypoxia

IP Enrichment

Known DosR Regulated Genes

hspxRv1733c

Desmond Lun, Kyle Minch

DosR Binding (2 hours-IP channel)

= forward read= reverse readRv1733c

Rv1737cRv1738

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)

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DosR ROC Curve

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Fasle Positive Rate

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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

IP Coverage vs Induction in Park et al.

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Park et al. Differential Expression

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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…

Valouev et al (2008) Nature Methods

DosR Binding (2 hours-IP channel)

= forward read= reverse readRv1733c

Rv1737cRv1738

Chip-Seq Blind Deconvolution

Impulse

Function

Deconvolve

3 binding sites

Desmond Lun, Brian Weiner

single binding site

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

Reconstruct Promoter Architecture

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…

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.

Rv2627c

Novel dosR Binding Site?

Rv2629Rv2628

dosR Binding Site

Getting Induction –Inducible Promoter System

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

Episomal vs KO Background

Transcription Factor Gene

Tet Promotor

Epitope Tag

Genome

X

Transcription Factor Protein

Target Promotor Region

Tet

Induction Profiles – dosR background

Episomal vs WT Background

Transcription Factor Gene

Tet Promotor

Epitope Tag

Genome

Transcription Factor Protein

Target Promotor Region

??

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

Induction Profiles – WT background

Assaying for Tag/Untagged Ratio

Transcription Factor Gene

Tet Promotor

Epitope Tag

Genome

Transcription Factor Protein

Target Promotor Region

taggedmRNA

untaggedmRNA

qPCR mRNA levels

Acknowledgements

Broad, BUSeq and Analysis

Brian WeinerMatt PetersenDesmond Lun

SBRICHIP

Kyle MinchTige Rustad

David Sherman