Genetic Studies of Human Hematopoiesis

Transcription

1 Genetic Studies of Human Hematopoiesis Vijay G. Sankaran, M.D., August 23, 2018 SWISSTRANSFUSION 2018

2 A Perspective on Hematopoiesis Laurenti & Gottgens, Nature, 2018

3 Limitations for Understanding Observations in Patients

4 Genetic Studies of Human Hematopoiesis Population Variation Rare Blood Disorders

5 Biological Themes from Genetic Studies Translation Signal Transduction Transcriptional Regulation

6 A Wonderful Sabbatical Visit by Beat Frey

7 Genetic Studies of Human Hematopoiesis Population Variation Rare Blood Disorders

8 Amelioration of Severity in the β-hemoglobin Disorders with Increased Fetal Hemoglobin Production β-thalassemia Sickle Cell Disease Sankaran & Nathan, NEJM, 2010

9 Effects of HbF on Natural History in SCD 1.8 pain acute chest death Hb F > 9% Hb F <= 9% %F %F age Platt, NEJM, 1991 Castro, Blood, 1994 Platt, NEJM, 1994

10 The Search for Molecular Regulators of the Fetal-to- Adult Globin Switch β-globin locus Fetal Globin Activators Adult Globin Repressors

11 Genome-wide Association Studies of HbF Levels in Humans Reveal Novel Loci

12 BCL11A Regulates the Fetal-to-Adult Hemoglobin Switch and Silences HbF in Adults Sankaran et al., Science, 2008; Sankaran et al., Nature, 2009

13 BCL11A As a Rheostat that Regulates Fetal Hemoglobin Sankaran et al., Science, 2008 Sankaran et al., Nature, 2009 Xu et al., Genes Dev, 2010 Sankaran et al., NEJM, 2011 Basak et al., JCI, 2015

14 What Underlies the Developmental Regulation of BCL11A Expression? Anindita Basak Sankaran et al., Science, 2008; Sankaran et al., Nature, 2009

15 Consistent Variation in BCL11A Protein, but Not mrna During Human Ontogeny

16 BCL11A mrna Assembly is Similar Between Developmental Stages

17 What Explains Different Expression of BCL11A Between Adult and Newborn Stages? Is degradation different? Is protein synthesis different? Faster in newborn than in adult? Could protein translation vary between the adult and newborn stages? Cycloheximide(CHX)-chase

18 Similar BCL11A Protein Stability Between Adult and Newborn Erythroid Cells

19 What Explains Different Expression of BCL11A Between Adult and Newborn Stages? Is degradation different? Is protein synthesis different? Faster in newborn than in adult? Could protein translation vary between the adult and newborn stages? Cycloheximide(CHX)-chase

20 Variation in BCL11A Protein Synthesis Between Adult and Newborn Stages of Erythropoiesis

21 Polysome mrna Occupancy As An Indication of Association with Actively Translating Ribosomes

22 Despite Impaired Protein Synthesis, BCL11A mrna Continues to Occupy Polysomes

23 Could a Ribosome-Associated RNA Binding Protein Mediate the Observed Developmental Regulation of BCL11A mrna Translation?

24 Survey of Ribosome-Interacting Proteins Using RNA-Antisense Purification/ Mass Spectrometry

25 RNA Binding Protein Associates with the Ribosome

26 Expression is Regulated During Hematopoietic Development

27 A Strategy For Obtaining Levels of That More Closely Mirror Physiology a b c PE BCL11A NA control GFP low GFP GFP high N.S. GFP low GFP high d e f HBG2(%) Relative mrna *** Fetal Newborn Adult low GFP high let-7 micrornas Control LIN Di mrna

28 low GFP f high 70 Lower Expression in Adult Erythroid Cells Reduces GFP high GFP high 65 BCL11A Protein, but not mrna Levels60 c let-7 micrornas Control mrna targets β-actin Direct mrna binding +/- Translation BCL11A a PE d Relative BCL11A mrna g control GFP low GFP Control hsa-let-7a-1 hsa-let-7a-2 hsa-let-7a-3 hsa-let-7b hsa-let-7c hsa-let-7d hsa-let-7e hsa-let-7f-1 hsa-let-7f-2 N.S. GFP low e HBG1&HBG2(%) Control Seed U G A G G U A G U A G U G A G G U A G U A G U G A G G U A G U A G U G A G G U A G U A G U G A G G U A G U A G A G A G G U A G U A G U G A G G U A G G A G U G A G G U A G U A G U G A G G U A G U A G b Relative mrna *** h Relative HMGA2 mrna Fetal Newborn Adult low GFP f Newborn high let-7 microrn mrna tar *

29 Pleotropic Functions of

30 Let-7 MicroRNAs Share the Same Seed Sequence

31 microrna Mimics

32 8B *** 7b 7c 7d 7e g 7i 98 rn N.S. et7g dult Relative HMGA2 mrna HB Newborn 3 Relative HMGA2 mrna 0 BCL11A Let-7 Mimic Expression in Newborn 0.0 CD34 + Cells h i 1.5 g Newborn Newborn Seed f 2* 1 0 Control Seed GFP U G A G G U A G U A G U G A G G U A G U A G U G A G G U A G U A G U G A G G U A G U A G U G A G G U A G U A G let-7 A G A G G U A G U A G micrornas U G A G G U A G G A G U G A G G U A G U A G U G A G G U A G U A G U G A G G U mrna A G U Atargets G U G A G G U A G U A G U G A G G U A G U A A h Fetal Newborn Adult low k high l Control 50 i GAPDH Relative HMGA2 mrna 1.0 Direct mrna 0.5 binding +/- 0.0 Translation Adult Newborn mimic Adult β-actin mrna targets BCL11A GAPDH * l HBG1 & HBG2 (%) Relative mr GAPDH j & HBG2 (%) HBG1 Control /- Translation Control Adult Newborn HBG1& Relative HMGA2 mrna Control hsa-let-7a-1 U G A G G U A G U A G hsa-let-7a-2 U G A G G U A G U A G hsa-let-7a-3 U G A G G U A G U A G hsa-let-7b BCL11A U G A G G U A G U A G hsa-let-7c U G A G G U A G U A G let7 hsa-let-7d A G A G G U A G U A G GAPDH hsa-let-7e U G A G G U A G G A G hsa-let-7f-1 U G A G G U A G U A G hsa-let-7f-2 HMGA2 U G A G G U A G U A G hsa-let-7g U G A G G U A G U A G hsa-let-7i U G A G G U A G U A G hsa-mir-98 mimic U G A G G U A G U A A N.S. k Control Adult h scrambrelative HMGA2 mrna hsa-

33 & HBG2 (%) HBG1 let-7f-1 hsa-let-7f-1 U G A G GU GU A G UG AU GA G U A G let-7f-2 hsa-let-7f-2 U G A G GU GU A G UG AU GA G U A G a-let-7g hsa-let-7g a-let-7i hsa-let-7i U G A G GU GU A G UG AU GA G U A G a-mir-98 hsa-mir-98 U G A G GU GU A G UG AU A G U A A U G A G GU GU A G UG AU GA G U A G Regulates HbF Through a let7-independent Pathway Newborn Newborn 100 N.S. N.S. 80 bled k Relative HMGA2 mrna k Relative HMGA2 mrna Adult Relati Adult Relat Control Control l HBG1 & HBG2 (%) GAPDH l HBG1 & HBG2 (%) mimic GAPDH Adult mimic Adult Control Control let7 HMGA2

34 Pleotropic Functions of Could BCL11A mrna be a direct target?

35 Direct Binding of BCL11A mrna by

36 Primary Target of -Mediated Induction of HbF is BCL11A

37 Regulation of BCL11A is Independent of let-7

38 Conclusions is expressed in a graded manner at the earlier stages of hematopoietic development and controls hemoglobin switching. regulates BCL11A mrna translation through a direct RNA-protein interaction. The ability of to induce HbF is independent of its role in let-7 microrna biogenesis and activities in regulating other mrnas. Our findings provide a simplified model of human hemoglobin switching and suggest new therapeutic avenues for sickle cell disease and beta-thalassemia.

39 Acknowledgements Sankaran Lab Collaborators Nour Abdulhay Erik Bao Anindita Basak Aaron Cheng Claudia Fiorini Rajiv Khajuria Ah Ram Kim Caleb Lareau Leif Ludwig Sean McFarland Kara Montbleau Satish Nandakumar Jacob Ulirsch Jeffrey Verboon Mathias Munschauer Beat Frey Eric Lander Stu Orkin Helen Christou Monica Schenone Steven Carr Jane-Jane Chen Lee Gehrke John Lian Xiuli An Mohandas Narla Charles Rice