Induction of adult levels of β-globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL

A major barrier to the clinical use of erythrocytes generated in vitro from pluripotent stem cells or cord blood progenitors is failure of these erythrocytes to express adult hemoglobin. The key regulators of globin switching KLF1 and BCL11A are absent or at a lower level than in adult cells in K562 and erythroid cells differentiated in vitro from induced pluripotent stem cells and cord blood progenitors. Transfection or transduction of K562 and cord blood erythroid cells with either KLF1 or BCL11A-XL had little effect on β-globin expression. In contrast, transduction with both transcription factors stimulated β-globin expression. Similarly, increasing the level of BCL11A-XL in the induced pluripotent stem cell-derived erythroid cell line HiDEP-1, which has levels of endogenous KLF1 similar to adult cells but lacks BCL11A, resulted in levels of β-globin equivalent to that of adult erythroid cells. Interestingly, this increase in β-globin was coincident with a decrease in ε− and ζ−, but not γ-globin, implicating BCL11A in repression of embryonic globin expression. The data show that KLF1 and BCL11A-XL together are required, but sufficient to induce adult levels of β-globin in induced pluripotent stem cell and cord blood-derived erythroid cells that intrinsically express embryonic or fetal globin.

[1]  K. Heesom,et al.  Qualitative and Quantitative Comparison of the Proteome of Erythroid Cells Differentiated from Human iPSCs and Adult Erythroid Cells by Multiplex TMT Labelling and NanoLC-MS/MS , 2014, PLoS ONE.

[2]  T. Townes,et al.  KLF1 regulates BCL11A expression and γ- to β-globin gene switching , 2010, Nature Genetics.

[3]  S. Orkin,et al.  Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A , 2013, Proceedings of the National Academy of Sciences.

[4]  Simon Heath,et al.  A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15 , 2007, Nature Genetics.

[5]  Cong Peng,et al.  Correction of Sickle Cell Disease in Adult Mice by Interference with Fetal Hemoglobin Silencing , 2011, Science.

[6]  J. Bieker,et al.  Non-random subcellular distribution of variant EKLF in erythroid cells. , 2008, Experimental cell research.

[7]  A. Toye,et al.  Ex-vivo generation of human red cells for transfusion , 2012, Current opinion in hematology.

[8]  Stuart H. Orkin,et al.  Developmental and species-divergent globin switching are driven by BCL11A , 2009, Nature.

[9]  Jaulang Hwang,et al.  Subcellular Transport of EKLF and Switch-On of Murine Adult βmaj Globin Gene Transcription , 2007, Molecular and Cellular Biology.

[10]  J. Bieker.  Isolation, genomic structure, and expression of human erythroid Krüppel-like factor (EKLF). , 1996, DNA and cell biology.

[11]  David C Williams,et al.  Mi2β-mediated silencing of the fetal γ-globin gene in adult erythroid cells. , 2012, Blood.

[12]  S. Orkin,et al.  Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6. , 2010, Genes & development.

[13]  D G Oscier,et al.  The BCL11 gene family: involvement of BCL11A in lymphoid malignancies. , 2001, Blood.

[14]  A. McDowall,et al.  A global role for EKLF in definitive and primitive erythropoiesis. , 2005, Blood.

[15]  P. Cullen,et al.  SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways , 2012, The Journal of cell biology.

[16]  S. Viville,et al.  Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine , 2010, Haematologica.

[17]  E. Massey,et al.  Maturing reticulocytes internalize plasma membrane in glycophorin A-containing vesicles that fuse with autophagosomes before exocytosis. , 2012, Blood.

[18]  D. Kaufman.  Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells. , 2009, Blood.

[19]  J. Thomson,et al.  Generation of red blood cells from human induced pluripotent stem cells. , 2011, Stem cells and development.

[20]  P. Sebastiani,et al.  BCL11A is a major HbF quantitative trait locus in three different populations with beta-hemoglobinopathies. , 2008, Blood cells, molecules & diseases.

[21]  F. Grosveld,et al.  The role of EKLF in human beta-globin gene competition. , 1996, Genes & development.

[22]  S. Thein,et al.  Binding patterns of BCL11A in the globin and GATA1 loci and characterization of the BCL11A fetal hemoglobin locus. , 2010, Blood cells, molecules & diseases.

[23]  Raymond K. Auerbach,et al.  A User's Guide to the Encyclopedia of DNA Elements (ENCODE) , 2011, PLoS biology.

[24]  M. Siatecka,et al.  The multifunctional role of EKLF/KLF1 during erythropoiesis. , 2011, Blood.

[25]  L. Staudt,et al.  Functional studies of BCL11A: characterization of the conserved BCL11A-XL splice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells , 2006, Molecular Cancer.

[26]  N. Burton,et al.  Mutations in EKLF/KLF1 form the molecular basis of the rare blood group In(Lu) phenotype. , 2008, Blood.

[27]  J. Hirschhorn,et al.  Supporting Online Material Materials and Methods Figs. S1 to S10 Tables S1 to S7 References Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-specific Repressor Bcl11a , 2022 .

[28]  J. Bieker,et al.  A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins , 1993, Molecular and cellular biology.

[29]  F. Grosveld,et al.  The Erythroid Phenotype of EKLF-Null Mice: Defects in Hemoglobin Metabolism and Membrane Stability , 2005, Molecular and Cellular Biology.

[30]  H. Dressman,et al.  Failure of Terminal Erythroid Differentiation in EKLF-Deficient Mice Is Associated with Cell Cycle Perturbation and Reduced Expression of E2F2 , 2008, Molecular and Cellular Biology.

[31]  Gonçalo R. Abecasis,et al.  Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of β-thalassemia , 2008, Proceedings of the National Academy of Sciences.

[32]  Kirby D. Johnson,et al.  Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs within a broad chromosomal region. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[33]  F. Grosveld,et al.  Erythropoiesis and globin switching in compound Klf1::Bcl11a mutant mice. , 2012, Blood.

[34]  F. Grosveld,et al.  Haploinsufficiency for the erythroid transcription factor KLF1 causes Hereditary Persistence of Fetal Hemoglobin , 2010, Nature Genetics.

[35]  Yukio Nakamura,et al.  Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells , 2012, PloS one.

[36]  T. Peyrard,et al.  Banking of pluripotent adult stem cells as an unlimited source for red blood cell production: potential applications for alloimmunized patients and rare blood challenges. , 2011, Transfusion medicine reviews.

[37]  M. Steinberg,et al.  BCL11A represses HBG transcription in K562 cells. , 2009, Blood cells, molecules & diseases.

[38]  J. Hirschhorn,et al.  DNA polymorphisms at the BCL11A, HBS1L-MYB, and β-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease , 2008, Proceedings of the National Academy of Sciences.

[39]  Merlin Crossley,et al.  Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells , 2007, Molecular and Cellular Biology.

[40]  F. Grosveld,et al.  The active spatial organization of the beta-globin locus requires the transcription factor EKLF. , 2004, Genes & development.

[41]  Chris Fisher,et al.  A functional element necessary for fetal hemoglobin silencing. , 2011, The New England journal of medicine.

[42]  M. Tsai,et al.  Tight regulation of a timed nuclear import wave of EKLF by PKCθ and FOE during Pro-E to Baso-E transition. , 2014, Developmental cell.

[43]  Zhigang Gao,et al.  Human stem-progenitor cells from neonatal cord blood have greater hematopoietic expansion capacity than those from mobilized adult blood. , 2002, Experimental hematology.