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Dynamics of Epigenetic States in Erythroid Differentiation Study

Investigating the changes in epigenetic states during erythroid differentiation using in vitro hematopoietic modeling with the G1E-ER4+estradiol system. Examining GATA1 function, gene expression alterations, ChIP-seq, RNA-seq assays, and chromatin state dynamics are key aspects of this research.

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Dynamics of Epigenetic States in Erythroid Differentiation Study

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  1. Dynamics of epigenetic states during erythroid differentiation Ross Hardison July 20, 2010 Barcelona

  2. Investigators on one mouse ENCODE project • Penn State • Hardison • Stephan Schuster • Frank Pugh • Robert Paulson • Francesca Chiaromonte • Yu Zhang • Webb Miller • Anton Nekrutenko • Childrens’ Hospital of Philadelphia • Mitch Weiss • Gerd Blobel • Emory Univ. • James Taylor • Univ. Massachusetts • Job Dekker • Duke Univ. • Greg Crawford, consultant • Terry Furey, consultant • Cal Tech • - Barbara Wold

  3. Hematopoiesis

  4. Erythroid differentiation

  5. GATA1, partners, teammates

  6. add back GATA-1, hybrid protein with ER G1E-ER4 estradiol Differentiated erythroblasts G1E-ER4+estradiol Somatic cell model to study GATA1 function in vitro hematopoietic differentiation erythropoietin stem cell factor Gata1– ES cells immortalize G1E Erythroid progenitors BFU-e, CFU-e

  7. Changes in transcription factors and cell morphology during eythroid differentiation

  8. Changes in gene expression during erythroid differentiation

  9. Features interrogated by ChIP-seq and RNA-seq assays DNase hypersensitive sites CTCF

  10. + + + + + - - - - - ChIP-seq finds previously known distal CRMs Known CRMs + DHSs + GATA1 TAL1 CTCF H3K4me1 H3K4me3 PcG: H3K27me3 - = no GATA1 + = GATA1-ER activated

  11. + + + + + - - - - - GATA1 activates Zfpm1 by displacing GATA2 and retaining TAL1 Known CRMs + DHSs + GATA1 TAL1 GATA2 H3K4me1 H3K4me3 PcG: H3K27me3

  12. + + + + + - - - - - GATA1 represses Kit by displacing GATA2 and TAL1 Known CRMs + DHSs + GATA1 TAL1 GATA2 H3K4me1 H3K4me3 PcG: H3K27me3

  13. GATA1 action via TAL1 is common but not universal

  14. Chromatin states from integrative analysis: limited change during differentiation chromHMM segmentations: Jason Ernst and Manolis Kellis, MIT Broad

  15. GATA1-occupied segments of DNA: Vast majority are in active chromatin, few change states Dominant mark H3K4me3 H3K4me1 H3K27me3 None H3K4me3 H3K4me1 H3K27me3 None

  16. Changes in chromatin state at Btg2

  17. Quantum change in chromatin state distinguishes expressed from non-expressed loci Enrichment of chromatin state

  18. Chromatin state precedes GATA1-induced TF changes Chromatin state established (mostly): Active Repressed Dead zones Chromatin condenses Nucleus removed Induction and repression: Dynamics of transcription factor binding within the already-established chromatin context.

  19. Thanks Weisheng Wu, Yong Cheng, Demesew Abebe, Cheryl Keller Capone,Ying Zhang, Ross, Swathi Ashok Kumar, Christine Dorman, David King ….Tejaswini Mishra, Nergiz Dogan Collaborating labs: Mitch Weiss and Gerd Blobel (Childrens’ Hospital of Philadelphia), James Taylor (Emory) Webb Miller, Francesca Chiaromonte, Yu Zhang, Stephan Schuster, Frank Pugh, Bob Paulson (PSU), Greg Crawford (Duke), Jason Ernst, Manolis Kellis (MIT) Funding: NIH NIDDK, NHGRI (ARRA), Huck Institutes of Life Sciences and Institute for Cyberscience, PSU

  20. + + + + + - - - - - Big changes at Hbb-b1 Known CRMs + DHSs + GATA1 TAL1 Pol2 H3K4me1 H3K4me3 PcG: H3K27me3

  21. GATA1 causes TAL1 to move at some CRMs, but retains it at others TAL1 occupancy, ER4 TAL1 occupancy, G1E TAL1 occupancy, ER4 GATA1 occupancy, ER4

  22. All GATA1-occupied segments active as enhancers are also occupied by SCL and LDB1 Tripic et al. (2009) Blood

  23. Distinguishing genes induced vs repressed by GATA1

  24. Kinetics of GATA1-regulated Gene Expression GATA1-induced (>2-fold) 1048 genes • known targets • new gene discovery GATA1-repressed (>2-fold) 1568 genes • stem cell/progenitor markers • proto-oncogenes (Kit/Myc/Myb) • function unknown Affy 430 2.0

  25. Globin RNA dominates the transcription profile RNA-seq Change in expression Swathi A. Kumar, Tejaswini Mishra, et al.

  26. Induction and repression occurs within active chromatin

  27. Histone modification profiles across TSS+/ 4kb High Level of expression Low

  28. H3K4me3 at TSS correlates with expression level

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