Talk at Developmental Workshop NIH
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In adult erythroid cells, the LCR normally loops to the active adult β-globin gene, while the fetal γ-globin genes are epigenetically silenced and excluded from looping. Experiments showed that re-targeting the LCR to the γ-globin genes by using an LDB1-based artificial protein or inhibiting the H3K9 methyltransferase G9a led to relief of γ-globin silencing, redistribution of LCR looping, and increased γ-globin expression to potentially therapeutic levels for β-hemoglobinopathies. These experiments suggest that modulating chromosome looping may be a therapeutic target for gene activation in diseases like sickle cell anemia and β-thalas
Talk at Developmental Workshop NIH
- 1. Reversal of fetal globin developmental silencing in adult erythroid cells Ivan (Vanya) Krivega, LCDB, NIDDK Developmental Biology Workshop, NIH
- 2. Developmental switch in globin genes expression 9 50 10 20 30 40 α γ β HbF HbA percentageoftotalglobinsynthesis Birth-9 -6 -3 3 6 Month
- 3. Developmental switch in globin genes expression 9 50 10 20 30 40 α γ β HbF HbA percentageoftotalglobinsynthesis Birth-9 -6 -3 3 6 Month After the γ to β switch, β-thalassemia and sickle cell disease become manifest (β gene expression) s s
- 4. Reactivation of γ-globin gene expression as a therapeutic approach 4321 LCR εγγδβ FetalAdult s Adult Cells (HbS) 4321 LCR εγγδβ FetalAdult Reactivation Adult Cells (HbF) s 10kb
- 5. LDB1 regulates β-globin genes expression LMO2 GATA1 TAL1 LDB1 DD 4321 LCR εγγδβ FetalAdult Krivega et al., Genes Dev, 2014 Song et al., Mol Cell, 2007 Homodimerization Long-range interaction
- 6. LDB1 regulates β-globin genes expression Kiefer et al., 2011 Blood Palstra et al., 2003 Nat Genet 4321 LCR εγγδβ FetalAdult LDB1 LDB1 Adult Cells (HbA) Homodimerization
- 7. LDB1 regulates β-globin gene expression 4321 LCR εγγδβ FetalAdult LDB1 LDB1 Homodimerization 4321 LCR εγγδβ FetalAdult LDB1 LDB1 Homodimerization Adult Cells (HbA) Fetal Cells (HbF) Kiefer et al., 2011 Blood Palstra et al., 2003 Nat Genet
- 8. 4321 LCR εγγδβ FetalAdult LDB1 LDB1 Strategy of reactivating γ-globin gene by targeting LDB1 DD domain Adult Cells (HbA) Deng et al., 2014 Cell γZnF DD
- 9. Strategy of reactivating γ-globin gene by targeting LDB1 DD domain Adult Cells (HbF/HbA) 4321 LCR εγγδβ FetalAdult LDB1 LDB1 Deng et al., 2014 Cell γZnF DD Homodimerization
- 10. adult CD34+ erythroid progenitor cells expansion 5-6 days differentiation 10-12 days lentiviral infection GFP sort γZnF DD Experimental outline
- 11. 0 0.2 0.4 0.6 0.8 1 1.2 5220000 5230000 5240000 5250000 5260000 5270000 5280000 5290000 5300000 5310000 5320000 EcoRI Control γZnF-DD LCR Targeting LDB1 DD domain to γ-globin gene forced de-novo looping to LCR relativecrosslinkingfrequency β δ εγ γ
- 12. 0 0.2 0.4 0.6 0.8 1 1.2 5220000 5230000 5240000 5250000 5260000 5270000 5280000 5290000 5300000 5310000 5320000 EcoRI Control γZnF-DD Targeting LDB1 DD domain to γ-globin gene forced de-novo looping to LCR relativecrosslinkingfrequency β δ εγ γ LCR
- 13. Targeting LDB1 DD domain to γ-globin gene stimulates γ-globin gene expression 0 0.2 0.4 0.6 0.8 1 γ/(γ+β) relativeexpression 0 10 20 30 40 50 60 70 80 Control γZnF-DD %HbF HbF/(HbF+HbA)
- 14. Model of controlling looping by artificial tethering LDB1 DD domain Adult ++ LCR +++ Adult LCR LDB1 LDB1 ++ DD γZnF Deng et al., 2014 Cell
- 15. 4321 LCR εγγδβ FetalAdult Human adult Strategy of reactivating γ-globin genes by inhibiting G9a G9a H3K9me2 Sripichai et al., 2011 Blood
- 16. 4321 LCR εγγδβ FetalAdult Human adult Strategy of reactivating γ-globin genes by inhibiting G9a G9a H3K9me2 MT G9a UNC0638 Fetal Adult?
- 17. G9a inhibition activates fetal and represses adult β-globin genes Krivega et al., 2015 Blood 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 5 10 15 20 25 30 35 40 Control UNC0638 γ/(γ+β) relativeexpression %HbF HbF/(HbF+HbA)
- 18. 0 0.05 0.1 0.15 0.2 0.25 HS4 HS3 HS2 HS1 γ pro δ pro β pro H3K9me2 Control H3K9me2 Phase II relativeenrichment G9a inactivation reduces H3K9me2 at the β-globin locus * - p<0.05 ** - p<0.01 * * * ** ** ** ** UNC0638
- 19. 0 0.05 0.1 0.15 0.2 0.25 IgG Control IgG Phase II Ldb1 Control Ldb1 Phase II * * * LDB1 LDB1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 IgG Control IgG Phase II GATA-1 Control GATA-1 Phase II γ δ β Gapdh * * * UNC0638 UNC0638 LDB1 GATA-1 enrichmentenrichment LDB1 complex occupies reactivated γ-globin genes * - p<0.05 UNC0638 UNC0638
- 20. 0 0.5 1 1.5 2 2.5 3 3.5 5225000 5235000 5245000 5255000 5265000 5275000 5285000 5295000 5305000 EcoRI Control Phase II β δ εγ γ LCR relativecrosslinkingfrequency Reactivated γ-globin genes interact with the LCR UNC0638 Krivega et al., 2015 Blood
- 21. 0 0.5 1 1.5 2 2.5 3 3.5 5225000 5235000 5245000 5255000 5265000 5275000 5285000 5295000 5305000 EcoRI Control Phase II β δ εγ γ LCR relativecrosslinkingfrequency Reactivated γ-globin genes interact with the LCR UNC0638 Krivega et al., 2015 Blood
- 22. MT Adult ++ LCR G9a +++ Adult H3K9me2 LCR LDB1 LDB1 ++ Epigenetic mechanism of G9a regulation of β-globin gene expression Krivega et al., 2015 Blood
- 23. Summary In adult erythroid cells, the LCR loops to the active adult -globin gene and the fetal -globin genes are epigenetically silenced and excluded from looping. • Re-targeting the LCR to the β-globin genes in adult cells by using an LDB1-based artificial protein • Relief of γ-globin silencing and re-distribution of LDB1 and LCR looping by inhibiting the G9a Increase γ-globin expression to levels potentially therapeutic in β-thalassemia and sickle cell disease cab be achieved by: These experiments suggest that chromosome looping can be considered a therapeutic target for gene activation in β-hemoglobinopathies.
- 24. Acknowledgements LCDB, NIDDK • Ann Dean • Jennifer Plank • Soledad Ivaldi • Jun Zhang • Francine Katz • Matthew Miller Molecular Medicine Branch, NIDDK • Jeffery Miller • Colleen Byrnes • Jaira F. de Vasconcellos • Y. Terry Lee • Megha Kaushal Perelman School of Medicine, UPEN • Gerd Blobel • Wulan Deng • Jeremy Rupon Perelman School of Medicine, UPEN • Stefano Rivella • Laura Breda • Irene Motta