1. ChIP-Seq: TB Example James Galagan

2. Acknowledgements Broad, BU Seq and Analysis Brian Weiner Matt Petersen Desmond Lun SBRI CHIP Kyle Minch Tige Rustad David Sherman

3. ChIP-Seq Immuno- precipitation (IP) Control (no IP) Sequence Fragments Align to Genome Look for Enrichment Target Site IP Control (ChIP-chip)(ChIP-Seq)

4. Dormancy Regulon Genes induced >2 fold after a 2-h shift from ambient to 0.2% O 2. ~100 genes modulated 47 upregulated = dormancy regulon Sherman et al., 2001

5. DosR Confirmation

6. Hypoxic Response Depends on DosR ORFGeneRvΔdosRGene product *Rv0079 22.2 ± 6.90.9 ± 0.1HP Rv0080 7.8 ± 1.70.9 ± 0.1HP Rv0081 4.9 ± 1.20.5 ± 0.1Transc. regulator Rv0082 3.1 ± 0.40.6 ± 0.1Prob. oxidored. sub. Rv0083 2.1 ± 0.41.1 ± 0.2Prob. oxidored. sub. Rv0569 9.0 ± 4.30.9 ± 0.1CHP Rv0570nrdZ5.5 ± 3.01.1 ± 0.1Ribonuc. red. cl. II *Rv0571c 1.7 ± 0.5 1 ± 0.2CHP Rv0572c 3.0 ± 0.8 1 ± 0.1HP *Rv0574c 2.0 ± 0.51.1 ± 0.1CHP MT0639 2.0 ± 0.4 1 ± 0.2HP ***Rv1733c 4.1 ± 1.3NDPoss. mem. prot. *Rv1734c 1.5 ± 0.1 1 ± 0.2HP Rv1736cnarX3.7 ± 0.7 1 ± 0.2Fused nitrate red. ***Rv1737cnarK28.5 ± 2.01.1 ± 0.2Nitrite extrus. prot. ***Rv1738 22.8 ± 9.71.3 ± 0.1CHP Rv1812c 3.6 ± 0.6 1 ± 0.1HP *Rv1813c 11.4 ± 3.00.8 ± 0.2CHP *Rv1996 7.9 ± 4.60.7CHP Rv1997ctpF4.3 ± 2.20.8 ± 0.1Cation trans. ATPase Rv2003c 2.3 ± 0.60.9 ± 0.1CHP Rv2004c 4.2 ± 1.41.2 ± 0.2HP *Rv2005c 7.3 ± 3.71.3 ± 0.5CHP The presence of one, two or three motif sequences (matrix score> 9.5) upstream of a gene is indicated by *, **or ***respectively. ORFGeneRvΔdosRGene product *Rv2006otsB2.2 ± 0.90.8 ± 0.1Trehalose phos. *Rv2007cfdxA25.9 ± 3.30.8 ± 0Ferredoxin Rv2028c 6.0 ± 1.70.9 ± 0.2CHP Rv2029cpfkB13.3 ± 5.70.9 ± 0.1Phosphofruct. II Rv2030c 27.3 ± 6.3NDCHP **Rv2031cacr27.9 ± 7.6NDα-Crystallin **Rv2032 15.1 ± 5.0NDCHP Rv2623 18.8 ± 4.1NDCHP Rv2624c 3.9 ± 1.30.6CHP Rv2625c 3.0 ± 1.11.3 ± 0.1CHP **Rv2626c 24.5 ± 4.61.2 ± 0.1CHP **Rv2627c 12.4 ± 4.90.8CHP **Rv2628 13.6 ± 10.80.8 ± 0HP Rv2629 7.6 ± 7.41.4 ± 0.1HP Rv2630 6.5 ± 4.6 2 ± 0.5HP Rv2631 3.4 ± 2.11.4 ± 0.3CHP Rv2830c 2.6 ± 0.71.2 ± 0.1HP Rv3126c 1.7 ± 0.70.8 ± 0HP **Rv3127 17.4 ± 2.40.8 ± 0.1CHP Rv3128c 1.5 ± 0.50.8 ± 0.1CHP Rv3129 2.7 ± 1.30.6 ± 0.1CHP *Rv3130c 25.5 ± 9.4NDCHP *Rv3131 34.1 ± 6.4NDCHP Rv3132c 5.7 ± 1.10.8 ± 0.1Sensor hist. kinase Rv3133cdosR9.1 ± 3.31.1 ± 0.2Two-comp. resp. reg. **Rv3134c 22.2 ± 17.91.2 ± 0.2CHP Rv3841bfrB5.2 ± 1.92.0 ± 1.3Bacterioferritin 26 of 27 most induced genes depend on DosR Park et al., 2001

7. 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

8. 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

9. DosR ChIP-Seq Replicates

10. Chip-Seq for TB DosR transcription factor binding at 4 hours post hypoxia IP Enrichment Known DosR Regulated Genes hspx Rv1733c Desmond Lun, Kyle Minch

11. DosR Binding (2 hours-IP channel) = forward read = reverse read Rv1733c Rv1737c Rv1738

12. 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) 123456789101112…

13. DosR ROC Curve

14. Combined DosR Network Matt Petersen, Brian Weiner Previously known (from Park et al.) Green Dashed Park, CLR, and Chip-Seq Green CLR and Park et al. Black Dashed Chip-Seq only Black Not CLR or Chip-Seq New Predictions (not in Park) Red Dashed CLR and Chip-Seq Red CLR

15. IP Coverage vs Induction in Park et al.

16. IP Coverage vs Motif Match Increasing Match to Logo (Negative LL Meme)

17. Location, Location, Location…

18. Valouev et al (2008) Nature Methods

19. DosR Binding (2 hours-IP channel) = forward read = reverse read Rv1733c Rv1737c Rv1738

20. Chip-Seq Blind Deconvolution Impulse Function Deconvolve 3 binding sites Desmond Lun, Brian Weiner single binding site

21. Binding Site Resolution Desmond Lun Fit enrichment curve to peak from putative single site Fit other peaks to single Site enrichment curve Refit peaks for 2,3, etc sites Re-estimate enrichment curve from all predicted site Repeat

24. Reconstruct Promoter Architecture

25. CSDeconv on DosR data Peak IDPositionAmplitude Position of motif matchDifference Absolute differenceLocation 188094.92.588124.5-29.629.6Upstream of Rv0079Rv0079 2665845.87.4665858.5-12.712.7In Rv0573cRv0573c 3668495.82.7668499.5-3.73.7Upstream of Rv0574cRv0574c 41639601.57.41639626.5-25.025.0In Rv1453Rv1453 51960517.813.11960519.5-1.71.7Upstream of Rv1733cRv1733c 61960611.525.51960623.5-12.012.0Upstream of Rv1733cRv1733c 71960692.310.41960692.5-0.20.2Upstream of Rv1733cRv1733c 81965458.410.51965470.5-12.112.1Upstream of Rv1737cRv1737c 91965543.414.41965532.510.9 Upstream of Rv1737cRv1737c 102056358.23.12056374.5-16.316.3Upstream of Rv1813c, Rv1814Rv1813cRv1814 112238942.39.52238937.54.8 Upstream of Rv1996Rv1996 122256458.212.92256495.5-37.337.3Upstream of Rv2007cRv2007c 132278996.339.42279004.5-8.28.2Upstream of Rv2031c, Rv2032Rv2031cRv2032 142279046.922.82279061.5-14.614.6Upstream of Rv2031c, Rv2032Rv2031cRv2032 152949477.76.62949471.56.2 Upstream of Rv2623Rv2623 162953045.18.32953073.5-28.428.4Upstream of Rv2626cRv2626c Continued…

26. CSDeconv on DosR data Peak IDPositionAmplitude Position of motif matchDifference Absolute differenceLocation 172954749.85.22954791.5-41.741.7Upstream of Rv2627c, Rv2628Rv2627cRv2628 182955065.39.52955030.534.8 Upstream of Rv2627c, Rv2628Rv2627cRv2628 192955479.49.42955475.53.9 Upstream of Rv2629Rv2629 203492068.812.53492091.5-22.722.7In Rv3126cRv3126c 213496439.250.63496450.5-11.311.3Upstream of Rv3130c, Rv3131Rv3130cRv3131 223500822.93.23500831.5-8.68.6Upstream of Rv3134cRv3134c 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.

27. Rv2627c Novel dosR Binding Site? Rv2629Rv2628 dosR Binding Site

28. Getting Induction –Inducible Promoter System

29. 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

30. Episomal vs KO Background Transcription Factor Gene Tet Promotor Epitope Tag Genome X Transcription Factor Protein Target Promotor Region Tet

31. Induction Profiles –  dosR background

32. Episomal vs WT Background Transcription Factor Gene Tet Promotor Epitope Tag Genome Transcription Factor Protein Target Promotor Region ??

33. 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

34. Induction Profiles – WT background

35. Assaying for Tag/Untagged Ratio Transcription Factor Gene Tet Promotor Epitope Tag Genome Transcription Factor Protein Target Promotor Region tagged mRNA untagged mRNA qPCR mRNA levels

36. Acknowledgements Broad, BU Seq and Analysis Brian Weiner Matt Petersen Desmond Lun SBRI CHIP Kyle Minch Tige Rustad David Sherman