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Similar to from B-cell Biology to Data Integration
from B-cell Biology to Data Integration
- 1. From B-‐cell Biology to Data Integra3on Gopuraja Dharmalingam 15-‐02-‐2013, Barcelona
- 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. 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. 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. 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. 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. 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. 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. 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. 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. Ikaros de-‐novo mo3f • Top 1000 Ikaros peaks • NestedMICA Vidal et al, 2013
- 12. Mo3fs of other transcrip3on factors close to Ikaros binding sites Vidal et al, 2013
- 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. Ikaros targets in B-‐cell differen3a3on pathways Pre-‐BCR signaling Cell cycle V(D)J recombina3on Vidal et al, 2013
- 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. Immgen Igll1 gene expression in B-‐cell popula:on www.Immgen.org
- 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. 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. 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. 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. Thank you Ques3ons?