scholarly journals IGF2BP1 Reverses Hemoglobin Switching in Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 639-639
Author(s):  
Laxminath Tumburu ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Jaira F. de Vasconcellos ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adult Stage ◽  
Globin Gene ◽  
Expression Patterns ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Empty Vector ◽  
Cd34 Cells ◽  
Gene And Protein Expression

Abstract During human ontogeny, high-level transcription within the beta-globin gene cluster switches sequentially from embryonic-to-fetal-to-adult genes. Beta-thalassemias and sickle-cell disease are manifested by reduced or mutated expression of the adult-stage, beta-globin gene. Research is aimed toward the eventual therapeutic goal of safely preventing or reversing the fetal-to-adult hemoglobin switch among these patient populations. To identify genes that may be involved in regulation of the fetal-to-adult erythroid switch, purified CD34(+) cells from six umbilical cord (fetal) and six adult peripheral blood samples were cultured in serum-free medium, and gene expression libraries were prepared and sequenced from CD71(+), CD235a(+) erythroblast mRNA. In total, 546 million paired-end reads with a length of 101bp were generated for a comparison of cord and adult erythroblast transcriptomes. Reads were aligned to the human reference genome (hg19), and differential gene expression was identified [false discovery rate ≤ 0.05, fold change ≥ 1.5, and reads per kilobase per million mapped reads (RPKM) ≥ 0.5]. A total of 145 genes were differentially expressed according to these criteria, with four of the top five encoding targets of the let-7 family of microRNAs. The topmost gene was insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which is normally involved in transcriptome regulation and developmental timing. IGF2BP1 expression was 770-fold increased in the fetal erythroblasts (RPKM > 3.0) compared with low background levels in adult erythroblasts (RPKM < 0.01). IGF2BP1 protein is present in fetal tissues including fetal liver; however, it is not detected in adult human bone marrow. A potential role for adult-stage IGF2BP1 over-expression (IGF2BP1-OE) in the regulation of globin genes and proteins was explored using lentiviral vectors designed for let-7 resistant, erythroid-specific expression of IGF2BP1 protein. IGF2BP1-OE transduced CD34(+) cells expressed the transgenic protein and maintained their ability to differentiate, accumulate hemoglobin, and enucleate ex vivo in the presence of erythropoietin. Globin mRNA and protein levels were investigated. While alpha-globin mRNA remained unchanged, gamma-globin mRNA became predominant [90% of (gamma + beta) mRNA] in IGF2BP1-OE samples [Control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p < 0.05], and beta-globin mRNA decreased to minor levels [Control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p < 0.05]. IGF2BP1-OE caused a pan-cellular HbF distribution by flow cytometry. Cellular fetal hemoglobin percentages [HbF/(HbF + HbA)] were measured as 5.3 ± 0.4% in donor matched control cells versus 80.3 ± 3.7% in IGF2BP1-OE cells (p < 0.05). HPLC tracings revealed that the minor HbA2 peak, composed of alpha and delta globin chains, was reduced or absent in IGF2BP1-OE. Also, IGF2BP1-OE suppressed the expression of related genes including the transcription factor BCL11A. These data demonstrate that erythroblast IGF2BP1 is silenced in humans during fetal-to-adult ontogeny, and that IGF2BP1 in adult erythroblasts reverses the developmentally related switch in beta-like globin gene and protein expression patterns. Disclosures No relevant conflicts of interest to declare.

Ineffective Erythropoiesis and Production of Normoblasts with a Beta Thalassemia Major Phenotype Using CD34+ Cells From Healthy Donors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1085-1085
Author(s):  
Y.Terry Lee ◽  
Colleen Byrnes ◽  
Emily Riehm Meier ◽  
Antoinette Rabel ◽  
Jeffery L. Miller
Keyword(s):  
Ex Vivo ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Ineffective Erythropoiesis ◽  
Globin Mrna ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Gene And Protein Expression

Abstract Abstract 1085 Reversal of anemia is the major target of thalassemia research, but studies of the molecular and cellular basis of the ineffective erythropoiesis of thalassemia are limited by access to donor progenitor cells. Here we demonstrate that thalassemic erythropoiesis may be recapitulated ex vivo by reducing the expression of hemoglobin in cultured CD34+ cells. Using lentiviral transduction of progenitor cells obtained from three healthy adult human donors, shRNA molecules were screened for their ability to reduce beta-globin gene and protein expression over 21 days in culture. Cells transduced with a scrambled vector served as donor-matched controls. Among the screened shRNA, one named HBB caused a consistent and significant reduction in beta-globin mRNA and protein. Beta-globin mRNA was reduced to levels <10% (p<0.001) compared to that of the controls (day14/21), while maintaining expression of gamma- and alpha-globin mRNA. HPLC was performed on an equivalent number of cells sampled on culture day 21 for hemoglobin type (HbA vs. HbF) and quantitation (area under each HPLC peak). The HbA peak was reduced by 96%, and there was a minor increase in the HbF peak (1.6 fold) after HBB transduction. Based upon these quantitative changes in hemoglobin, HbF represented 49.3±9.3% in the HBB transduced population compared with 2.9±0.7% (p<0.01) in controls. On culture day 14, there was no significant difference in glycophorin A (CD235), transferrin receptor (CD71), or cellular morphology despite the reduction in beta-globin mRNA. However, impaired terminal differentiation was detected by retainment of surface CD71 and a lack of enucleation during the third week of culture. Cell counts were lower in HBB transduced cells during the final stages of erythroid differentiation with a 61% (p=0.03) reduction in total cell counts by day 21 when compared to controls. Annexin V assay on day 21 also demonstrated increased phosphatidylserine expression in the HBB transduced cells [HBB=55.7±14.4% vs. Control=25.0±3.0%] in association with the decreased terminal differentiation. GDF15 quantitation demonstrated a significant (p=0.006) increase in the culture supernatants of HBB transduced cells. Sorted cytospin preparations revealed a distinct population of mature normoblasts containing a highly condensed nucleus surrounded by a thin ring of hypochromic cytoplasm. Reduction of erythroblast beta-globin gene and protein expression to levels associated with beta thalassemia major in humans causes ineffective erythropoiesis ex vivo by reducing cell production, increasing surface expression of phosphatidylserine, and impairing enucleation during terminal maturation. Efforts are now underway to use the culture system to explore mechanisms whereby reduced hemoglobin synthesis causes normoblast defects, and for screening of chemical and genetic rescue therapies for the thalassemic erythroid phenotype. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

1991 ◽  
Vol 11 (9) ◽  
pp. 4690-4697 ◽  
Author(s):  
J G Glauber ◽  
N J Wandersee ◽  
J A Little ◽  
G D Ginder
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Adult Chicken ◽  
Beta Globin Gene ◽  
Flanking Sequences ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

Role of the Krüppel-Type Zinc Finger Transcription Factor ZBP-89 In Human Globin Gene Regulation and Erythroid Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2067-2067
Author(s):  
Andrew J. Woo ◽  
Jonghwan Kim ◽  
Jian Xu ◽  
Hui Huang ◽  
Alan Cantor
Keyword(s):  
Globin Gene ◽  
Regulatory Elements ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Histone 3 ◽  
Cd34 Cells ◽  
Alpha Globin ◽  
Globin Gene Regulation ◽  
Erythroid Development

Abstract Abstract 2067 The molecular mechanisms underlying developmental globin gene regulation remain incompletely understood. Prior studies have identified key cis-regulatory elements within the beta globin locus that contain core regions of closely spaced functional binding sites for GATA, NF-E2p45/maf and GT/GC box binding transcription factors. We recently identified the GT/GC-box binding transcription factor ZBP-89 as a novel GATA-1 interacting partner, and showed that it is involved in erythroid development in mice (Woo et al. 2008. Mol. Cell Bio. 28:2675-2689). Brand et al. independently isolated ZBP-89 in NF-E2p45/mafk complexes from induced mouse erythroid leukemia (MEL) cells (Brand et al. 2004. Nat. Struct. Mol Biol. 11:73-80). In the current study, we show that ZBP-89 protein levels increase during in vitro erythroid differentiation of human bone marrow derived CD34+ cells. This correlates with the onset of alpha and beta globin gene transcription. ChIP-chip studies using ENCODE v2.0 arrays demonstrate that ZBP-89 occupies key cis-regulatory elements within both the beta globin (locus control regions HS3, HS2; delta and beta proximal promoters; and an intergenic region between gamma1 and delta globin) and alpha globin (HS-48, HS-40, HS-10 and alpha globin proximal promoters) loci in primary human erythroid precursors. Comparative analysis across the entire ENCODE array reveals a strong positive correlation between ZBP-89 occupancy, RNA polymerase II occupancy, and the activating histone marks acetylated histone 3 (AcH3) and trimethylated histone 3 lysine 4 (H3K4me3); and a negative correlation with the repressive mark trimethylated histone 3 lysine 27 (H3K27me3). Motif analysis under the ZBP-89 occupancy peaks indicates a preference for GGGG(G/A)NGGGG in vivo binding sites. Lentiviral shRNA mediated knock down of ZBP-89 in the in vitro differentiated CD34+ cells results in 30–50% reduction of alpha-, gamma-, and beta-globin gene expression, as well as modestly decreased expression of a number of additional erythroid-specific genes. Co-immunoprecipitation experiments demonstrate physical association between ZBP-89 and the GCN5/Trapp histone acetyltransferase complex. Based on these findings, we propose that ZBP-89 participates with GATA-1 and NF-E2 in the final epigenetic changes required for high-level expression of globin and other erythroid genes in terminally differentiating human erythroid cells. Disclosures: No relevant conflicts of interest to declare.

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1725-1735
Author(s):  
M A Bender ◽  
A D Miller ◽  
R E Gelinas
Keyword(s):  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
New Gene ◽  
Normal Human ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

Mitosis Is Not Required for Coordinated Changes in Iron Metabolism and High-Level Globin Gene Transcription in Erythropoietin-Responsive CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1776-1776
Author(s):  
Ajoy V. Bhupatiraju ◽  
Nicole M. Josleyn ◽  
Y. Terry Lee ◽  
Jeffery L. Miller
Keyword(s):  
Cell Division ◽  
Globin Gene ◽  
Beta Globin ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Cell Divisions ◽  
Adult Human ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Very High

Abstract Within the erythroid lineage, erythropoietin (EPO) responsiveness is manifested by cell division and growth as well as specific changes in heme and globin production that ultimately result in the production of erythrocytes. However, the nascent relationship between EPO-associated mitosis and globin gene regulation has not been fully defined. In this study, cultured adult human CD34+ cells from peripheral blood were used to investigate early cellular responses to erythropoietin in the context of mitosis. Matched cultures were performed in replicate using human cells from at least two healthy adults. To detect mitosis, one million cells were labeled with 2uM carboxyfluorescein diacetate succinamidyl ester (CFSE). The CFSE-labeled cells were then cultured in the presence [EPO(+)] or absence [EPO(−)] of 4U/mL EPO, and analyzed using flow cytometry. No cell divisions were detected in either condition during the first 24 hours in culture, and multiple cell divisions were noted on subsequent culture days only in the EPO(+) cultures. Remarkably, dual-staining with CFSE and CD71 revealed a small (&lt;1%) population of the undivided cells at 24 hours with very high surface levels of transferrin receptor [CD71(++++)] exclusively in the EPO(+) cultures. Further analysis of those rare EPO-responsive, pre-mitotic cells revealed DNA synthesis and entry into the cell cycle in 62.5±4.7% compared with 1.5±1.8% among the cells with lower CD71 expression. None expressed glycophorin A. Based upon their distinct phenotype, single cells were sorted into 96-well plates, with sorting confirmation by quantitative RT-PCR of GAPDH mRNA (20 copies/cell detection limit). Next, gamma- and beta-globin transcripts were amplified for comparison (4 separate experiments). Among the pre-mitotic, CD71(++++) population, 20 of 182 total sorted cells (11%) lacked detectable levels of gamma- and beta-globin mRNA. Only 1 of 182 cells (0.5%) expressed gamma-globin mRNA and no detectable beta globin mRNA. 138 of 182 cells (76%) expressed only beta-globin mRNA, and 23 of 182 cells (13%) expressed both globin mRNAs. The median and mean levels of gamma-globin mRNA among the 24 gamma(+) cells were 92 and 245±1015 copies/cell respectively. In contrast, the median and mean levels of beta-globin mRNA among the 161 beta(+) cells were 1624 and 3999±5892 copies/cell respectively. By comparison, the CD34+ cells with low levels of surface CD71 in either EPO(+) or EPO(−) cultures demonstrated detectable levels of gamma globin mRNA in only 7 of 145 sorted cells (5%), and beta-globin mRNA in 85 of 145 cells (59%), with total (gamma+beta) globin mRNA under 200 copies/cell in &gt;90% of those cells. These novel results suggest that the frequency and levels of gamma-globin transcripts are quite low at the earliest stages of an EPO response among adult human CD34+ cells. However, it is clearly demonstrated that the cells can increase their capacity to import iron and transcribe beta-globin mRNA at very high levels prior to their first EPO-dependent cell division.

scholarly journals Effect of in vitro DNA methylation on beta-globin gene expression.

1988 ◽  
Vol 85 (13) ◽  
pp. 4638-4642 ◽  
Author(s):  
J. Yisraeli ◽  
D. Frank ◽  
A. Razin ◽  
H. Cedar
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Globin Gene ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Globin Gene Expression

scholarly journals Fetal hemoglobin silencing in humans

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2081-2086 ◽  
Author(s):  
Patricia A. Oneal ◽  
Nicole M. Gantt ◽  
Joseph D. Schwartz ◽  
Natarajan V. Bhanu ◽  
Y. Terry Lee ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Postnatal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Age Related

Abstract Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

scholarly journals Two novel beta-thalassemia mutations in the 5' and 3' noncoding regions of the beta-globin gene [see comments]

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1342-1346 ◽  
Author(s):  
SP Cai ◽  
B Eng ◽  
WH Francombe ◽  
NF Olivieri ◽  
AG Kendall ◽  
...  
Keyword(s):  
Premature Termination ◽  
Globin Gene ◽  
Polyadenylation Signal ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Noncoding Regions ◽  
Beta Globin Gene ◽  
Italian Family ◽  
Irish Family

Abstract Two novel beta-thalassemia mutations are described. The first mutation, found in an Italian family, is a G----A substitution in nucleotide (nt) +22 relative to the beta-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3′ downstream. The second mutation, found in an Irish family, is a T----C substitution in nt +1570, or 12 bp 5′ upstream of the AATAAA polyadenylation signal in the 3′ noncoding region. It is postulated that this mutation leads to destabilization of the encoded beta-globin mRNA.

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