1 / 37

ChIP-Seq: TB Example

ChIP-Seq: TB Example. James Galagan. Acknowledgements. SBRI CHIP Kyle Minch Tige Rustad David Sherman. Broad, BU Seq and Analysis Brian Weiner Matt Petersen Desmond Lun. ChIP-Seq. Sequence Fragments Align to Genome Look for Enrichment. Target Site. IP. Control. Immuno-

nuru
Download Presentation

ChIP-Seq: TB Example

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. ChIP-Seq: TB Example James Galagan

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

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

  4. Dormancy Regulon Genes induced >2 fold after a 2-h shift from ambient to 0.2% O2. ~100 genes modulated 47 upregulated = dormancy regulon Sherman et al., 2001

  5. DosR Confirmation

  6. Hypoxic Response Depends on DosR 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 • DDosR 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 Rv1733c hspx Known DosR Regulated Genes Desmond Lun, Kyle Minch

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

  12. Window Enrichment Analysis • Divide genome into non-overlapping bins • Take reads from ChIP and control libraries • Calculate log-likelihood ratio for independence (based on chi-square) 1 2 3 4 5 6 7 8 9 10 11 12 …

  13. DosR ROC Curve

  14. Combined DosR Network 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 Matt Petersen, Brian Weiner

  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 Rv1737c Rv1738 Rv1733c

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

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

  22. Reconstruct Promoter Architecture

  23. CSDeconv on DosR data Continued…

  24. CSDeconv on DosR data • 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.

  25. Rv2628 Rv2629 dosR Binding Site Novel dosR Binding Site? Rv2627c

  26. Getting Induction –Inducible Promoter System

  27. Tagged Tet-Promotor Construct Tet Operator FLAG tag Gene of Interest Gateway recombination sites Named pEXNF-xxxx EX = expression vector NF = N-terminal FLAG tag xxxx = Rv number for gene of interest pEXNF-3133c used in subsequent slides

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

  29. Induction Profiles – DdosR background

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

  31. 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

  32. Induction Profiles – WT background

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

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

More Related