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from B-cell Biology to Data Integration

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from B-cell Biology to Data Integration

  1. 1. From  B-­‐cell  Biology  to  Data   Integra3on   Gopuraja  Dharmalingam   15-­‐02-­‐2013,  Barcelona    
  2. 2. Transcrip3on  Factor  -­‐  Ikaros  •  Founding  member  of  a  family  of  transcrip3on  factor  •  Expressed   in   haematopoie3c   stem   cells,   lymphoid   cells  and  some  myeloid  cells  •  Required  for  Lymphocyte  development  •  Muta3ons  in  IKZF1  observed  in  Human  Leukemia   Merkenschlager  review,  2010  
  3. 3. B  lymphocyte  development   mul:-­‐lineage   progenitors   lineage-­‐restricted  lymphoid  progenitors   common  haematopoie:c   lymphoid   cycling     Res3ng     Immature     stem  cells   progenitors   pre-­‐pro-­‐B  cell   pro-­‐B  cell   pre-­‐B  cell   pre-­‐B  cell   B  cell   MLP   Fr.  A   Fr.  B/C   Fr.  C’   Fr.  D   Fr.  E  HSC   MPP   CLP   LMPP   T  cell  progenitors   B3  Cell  line  
  4. 4. Challenges!!  •  Single  gene  to  genome  wide  -­‐  no  high  throughput   technologies  were  available  •  How  to  make  sense  of  the  data?  •  Linking  with  published  resources  •  Analysis   challenges:   Different   soVware   &   method   provides   different   results   (Ex:   different   peak   callers   provide  different  set  of  peaks  for  Ikaros)  •  Comparing   and   Integra3ng   data   from   different   methods  (Ex:  expression  and  binding)  •  Biological  relevance  
  5. 5. B  lymphocyte  development   mul:-­‐lineage   progenitors   lineage-­‐restricted  lymphoid  progenitors   common  haematopoie:c   lymphoid   cycling     Res3ng     Immature     stem  cells   progenitors   pre-­‐pro-­‐B  cell   pro-­‐B  cell   pre-­‐B  cell   pre-­‐B  cell   B  cell   MLP   Fr.  A   Fr.  B/C   Fr.  C’   Fr.  D   Fr.  E  HSC   MPP   CLP   LMPP   T  cell  progenitors   B3  Cell  line   mRNA expression (array counts) Ikaros  target  genes?   differentiation Vidal  et  al,  2013  
  6. 6. Roles  of  Ikaros  in  B-­‐cell  development?  •  Global   iden3fica3on   of   Ikaros   targets   and   regulated   genes  •  Iden3fying   pre-­‐B-­‐cell   differen3a3on   pathways   enriched  for  Ikaros  targets  •  Co-­‐occurrence   of   Ikaros   binding   with   other   hematopoie3c  transcrip3on  factors  •  Integra3ng   Ikaros   binding   and   gene   expression   with   gene   expression   changes   during   hematopoie3c   development  (using  Immgen  data)   Vidal  et  al,  2013  
  7. 7. Inducible  Ikaros  construct   HA  Ikaros IKAROS   ERt2   IRES   GFP   Cytoplasm   Nucleus   HA-­‐IK-­‐ER  fusion  protein   Heat-­‐Shock  protein  in  cytoplasm   4-­‐hydroxytamoxifen  (4-­‐OHT)  
  8. 8. Ikaros  induced  gene  expression  changes   Affymetrix  microarrays:   •  Mouse  Genome  430  2.0   Array  –  48h   •  Mouse  Gene  1.0  ST   Array  –  2h,  6h   Challenges:   •  Combining  two  microarray  plaborms   •  Limited  gene  annota3on  for  Mouse  Genome  430  2.0  !!   Isabel  Ferreiros  Vidal,  Tom  Carroll  
  9. 9. Genome  wide  iden3fica3on  of  Ikaros   binding  sites  endogenous  Ikaros    ChIP-­‐Seq    HA-­‐tagged  Ikaros  ChIP-­‐Seq    Common:  11349  high  confidence  peaks  11349  peaks                    6746  genes                5732  in  affy  arrays     Isabel  Ferreiros  Vidal,  Anna  Terry,  Tom  Carroll  
  10. 10. Integra3ng  Ikaros  binding  and  gene   expression     All   Upreg.   Downreg.  Ikaros-­‐responsive  genes  at  2h   79   28   51  Ikaros  binding  observed   65  (82.3%)   22  (78.6%)   43  (84.3%)  Ikaros  binding  expected   28   10   18  p  value   9.90x10-­‐19   4.55x10-­‐7   5.08x10-­‐14  odds  ra3o   8.55   6.75   9.9  Ikaros-­‐responsive  genes  at  6h   401   159   242  Ikaros  binding  observed   286  (71.3%)   112  (70.4%)   174  (71.9%)  Ikaros  binding  expected   141   56   85  p  value   5.88x10-­‐51   2.35x10-­‐20   2.39x10-­‐32  odds  ra3o   4.58   4.39   4.71  Ikaros-­‐responsive  genes  at  48h     4471   1982   2486  Ikaros  binding  observed   2497  (55.8%)   1195  (60.3%)   1302  (41.5%)  Ikaros  binding  expected   1573   697   875  p  value   2.16x10-­‐246   1.43x10-­‐131   7.52x10-­‐82  odds  ra3o   2.33   2.8   1.398   Vidal  et  al,  2013  
  11. 11. Ikaros  de-­‐novo  mo3f   •  Top  1000  Ikaros  peaks     •  NestedMICA   Vidal  et  al,  2013  
  12. 12. Mo3fs  of  other  transcrip3on  factors  close  to  Ikaros  binding  sites   Vidal  et  al,  2013  
  13. 13. Ikaros  co-­‐localiza3on  with  other   hematopoie3c  transcrip3on  factors  Overlap  between  binding  sites  of   Overlap  between  genes  bound  by  karos  and  Ikaros  and  other  TFs(%)   other  TFs(%)   Vidal  et  al,  2013  
  14. 14. Ikaros  targets  in  B-­‐cell     differen3a3on  pathways    Pre-­‐BCR  signaling   Cell  cycle   V(D)J  recombina3on   Vidal  et  al,  2013  
  15. 15. Ikaros  target  genes  in  hematopoiesis   and    B-­‐cell  lineage  specifica3on   Goals  of  Immunological  Genome  Project  (Immgen)   •  To  study  whole-­‐genome  expression  of  all  known   Immune  cell  types  in  Mouse     •  To  predict  the  gene  regulatory  network  in  immune   cells  using  computa3onal  tools   •  Data  deposited  to  GEO  /  ArrayExpress    www.Immgen.org  
  16. 16. Immgen   Igll1  gene  expression  in  B-­‐cell  popula:on  www.Immgen.org  
  17. 17. Ikaros  target  genes  in  hematopoiesis   and    B-­‐cell  development    gene  expression                                                                                                                      Ikaros  binding  Cycling  to  resNng  pre-­‐B-­‐cell  transiNon          gene  expression                                                                                                                      Ikaros  binding   Vidal  et  al,  2013  
  18. 18. Increased  Ikaros  expression  in  B3  cells   resembles  res3ng  pre-­‐B  cell  (Fr.D)   MLP   CLP   Fr.B/C   Fr.C’   Fr.D  B3  Ikaros   -1.5 0 +1.5 log2 ratio
  19. 19. Conclusion  •  Integra3ve  analysis  allowed  us  to  address  the   role  of  Ikaros  in  B-­‐cell  biology  •  Ikaros  targets  were  highly  enriched  in  key  pre-­‐ B-­‐cell  differen3a3on  pathways  •  Inducible   Ikaros   expression   in   cycling   pre-­‐B   cells  was  sufficient  to  drive  the  transcrip3onal   changes  that  resembled  the  differen3a3on  of   cycling  to  res3ng  pre-­‐B  cells.  
  20. 20. Acknowledgements  Lymphocyte  Development  Group:   University  of  Cambridge:      Manhias  Merkenschlager   Mikhail  Spivakov  Amanda  Fisher    Isabel  Ferreiros  Vidal   University  of  California,  Los  Tom  Carroll   Angeles  Ben  Taylor    Anna  Terry   Stephen  T.  Smale  Ziwei  Liang    Sanjay  Khadayate  Prashant  Srivastava  Enrico  Petreno  Lee  Cooper    
  21. 21. Thank  you      Ques3ons?        

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