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1
Ikuta, Tohru, Thalia Papayannopoulou, George Stamatoyannopoulos, and Yuet Wai Kan. "Globin Gene Switching." Journal of Biological Chemistry 271, no. 24 (1996): 14082–91. http://dx.doi.org/10.1074/jbc.271.24.14082.
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Wood, WG. "Haemoglobin Switching." Physiology 3, no. 1 (1988): 33–35. http://dx.doi.org/10.1152/physiologyonline.1988.3.1.33.
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Haemoglobin switching involves changes in production of the globin chains at specific times during vertebrate development. The hereditary haemolytic anaemias known as thalassaemias, which result from decreased or no synthesis of one of the globins, are some of the most common genetic diseases of humans.
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Perrine, Susan P. "Switching globin, raising red cells." Blood 118, no. 4 (2011): 834–36. http://dx.doi.org/10.1182/blood-2011-06-354373.
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Johnson, Robert M., Deborah Gumucio, and Morris Goodman. "Globin gene switching in primates." Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 133, no. 3 (2002): 877–83. http://dx.doi.org/10.1016/s1095-6433(02)00205-2.
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Hsia, Nelson, Jennifer L. Axe, Noelle Paffett-Lugassy, Yi Zhou, and Leonard I. Zon. "Globin switching in the zebrafish." Blood Cells, Molecules, and Diseases 38, no. 2 (2007): 145. http://dx.doi.org/10.1016/j.bcmd.2006.10.062.
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Kingsley, Paul D., Jeffrey Malik, Rachel L. Emerson, et al. "“Maturational” globin switching in primary primitive erythroid cells." Blood 107, no. 4 (2006): 1665–72. http://dx.doi.org/10.1182/blood-2005-08-3097.
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Mammals have 2 distinct erythroid lineages. The primitive erythroid lineage originates in the yolk sac and generates a cohort of large erythroblasts that terminally differentiate in the bloodstream. The definitive erythroid lineage generates smaller enucleated erythrocytes that become the predominant cell in fetal and postnatal circulation. These lineages also have distinct globin expression patterns. Our studies in primary murine primitive erythroid cells indicate that βH1 is the predominant β-globin transcript in the early yolk sac. Thus, unlike the human, murine β-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a βH1- to ϵy-globin switch, up-regulate adult β1- and β2-globins, and down-regulate ζ-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions. Furthermore, the ϵy- and βH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. These data suggest that this “maturational” βH1- to ϵy-globin switch is dynamically regulated at the transcriptional level. Globin switching during ontogeny is due not only to the sequential appearance of primitive and definitive lineages but also to changes in globin expression as primitive erythroblasts mature in the bloodstream.
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Roberts, N. A., J. A. Sloane-Stanley, J. A. Sharpe, S. J. Stanworth, and W. G. Wood. "Globin Gene Switching in Transgenic Mice Carrying HS2-Globin Gene Constructs." Blood 89, no. 2 (1997): 713–23. http://dx.doi.org/10.1182/blood.v89.2.713.
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Abstract We have examined the pattern of human globin gene switching in transgenic mice containing three different γ and β gene constructs (HS2GγAγδβ, HS2Aγβneo, and HS2Aγenβ) and compared the results with previously described transgenics (HS2Aγβ, HS2GγAγ-117δβ, and LCRεGγAγδβ). Developmental regulation was observed in all cases with identical patterns in lines bearing the same construct. Three different patterns of switching were observed: LCRεGγAγδβ and HS2Aγβneo mice switched rapidly, HS2GγAγδβ and HS2GγAγ-117δβ at an intermediate rate, and HS2Aγβ and HS2Aγenβ mice showed delayed switching, with a plateau in late fetal-early neonatal life and readily detectable levels of γ mRNA in adults. No difference was observed in the time of switching of the HS2GγAγδβ mice compared with those with the Aγ-117 hereditary persistence of fetal hemoglobin mutation, but adult levels of γ mRNA were significantly higher (≈5%) in lines carrying the mutation than in those without (≈1%). Reversion to the rapid switch of the LCRεGγAγδβ mice was observed in three lines with the HS2Aγβ neo construct in which expression of the tk-neo gene was approximately equal to that of the globin genes. The inclusion of the Aγ enhancer in HS2Aγβ mice did not alter the pattern of switching, or reduce the relatively high levels of γ mRNA in these lines. However, unlike other HS2 mice, the combination of HS2 and the Aγ enhancer resulted in copy number-dependent expression in HS2Aγenβ lines, with intrauterine death at ≈12.5 days gestation at high copy numbers. These results demonstrate that numerous elements throughout the β globin gene cluster interact to produce the correct pattern of developmental regulation of these genes. Furthermore, extinction of γ gene expression in adult life is not completely autonomous and is incomplete when HS2 is the only LCR element present.
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Palis, James, Jeff Malik, Rachael L. Emerson, et al. "“Maturational” Globin Switching in Primary Primitive Erythroid Cells." Blood 106, no. 11 (2005): 3634. http://dx.doi.org/10.1182/blood.v106.11.3634.3634.
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Abstract Mammals have two distinct erythroid lineages. The “definitive” erythroid lineage generates small, enucleated erythrocytes that constitute the predominant cell type in the fetal and postnatal circulation. It is preceded by the “primitive” erythroid lineage, which originates in the yolk sac and generates a semi-synchronous wave of large erythroblasts that terminally differentiate in the bloodstream. This feature provides a unique opportunity to investigate changes in gene expression during erythroid maturation. Here, we have examined expression of the various α- and β-globin genes in purified populations of primary primitive erythroid cells isolated from progressive developmental time-points of mouse embryogenesis. Our studies, using both in situ hybridization and quantitative PCR, indicate that βH1 is the predominant β-globin transcript in the early yolk sac. Thus, unlike the human, the murine β-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a βH1- to εy-globin switch, up-regulate low levels of the adult β1- and β2-globins, and down-regulate ζ-globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions as assayed by ChIP assays. Furthermore, we found that the εy- and βH1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. Taken together, these results are consistent with the notion that this βH1- to εy-globin “maturational” switch is dynamically regulated at the transcriptional level. We conclude that the embryonic to adult globin switch that occurs during murine ontogeny is due not only to the sequential appearance of primitive and definitive erythroid lineages but also to striking changes in globin gene expression that occur as primitive erythroblasts mature in the bloodstream.
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Xu, Jian, Vijay G. Sankaran, Yuko Fujiwara, and Stuart H. Orkin. "Control of Hemoglobin Switching by BCL11A." Blood 114, no. 22 (2009): 5. http://dx.doi.org/10.1182/blood.v114.22.5.5.
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Abstract Abstract 5 All vertebrates switch expression of globin chains during development. In humans b-like globins switch from embryonic to fetal to adult, whereas in the mouse a single switch from embryonic to adult occurs. The switch from human fetal (g) to adult (b) expression is especially critical in the b-hemoglobin disorders, such as sickle cell anemia and the b-thalassemias. Delay of the switch or reactivation of the fetal gene in the adult stage greatly ameliorates clinical severity. Despite intensive molecular studies of the human b-globin cluster over more than two decades, the proteins regulating the switch, and the mechanisms controlling the process, have been largely elusive. Recently, genome-wide association studies identified genetic variation at a chromosome 2 locus that correlates with the level of HbF in different populations. The most highly associated single nucleotide polymorphisms (SNPs) reside in an intron of the BCL11A gene, which encodes a zinc-finger repressor protein. Previously we showed that shRNA-mediated ex vivo knockdown of BCL11A in cultured human CD34-derived erythroid precursors leads to robust HbF expression, consistent with a role for BCL11A in maintaining g-genes in a silenced state in adult cells. To address in vivo roles of BCL11A either in development or in globin gene silencing in an intact individual, we have employed stringent genetic tests of function in mice that carry a complete human b-globin gene cluster as a yeast artificial chromosome transgene (b-locus mice). Knockout of BCL11A in mice leads to failure to silence the endogenous b-like embryonic genes in adult erythroid cells of the fetal liver (>2500-fold derepression). The ratio of human g to b globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that g constitutes >90% of the b-like human expression at embryonic day (E)14.5 and >75% at E18.5. These quantitatively striking findings indicate that BCL11A controls developmental silencing of g-globin gene expression. To address by formal genetics the contribution of BCL11A to g silencing in adult animals we have employed conditional inactivation of BCL11A through hematopoietic- and erythroid-specific Cre-alleles. These experiments reveal that BCL11A is also required in vivo for g-gene silencing in adults. We observed that human g-globin expression is persistently derepressed >2000-fold (as compared to littermate controls) in bone marrow erythroblasts of 15-20 week old b-locus mice upon erythroid-specific deletion of BCL11A. Taken together, these findings establish BCL11A as the first genetically validated transcriptional regulator of both developmental control of globin switching and silencing of g-globin expression in adults. The recognition of these roles for BCL11A now permits focused mechanistic studies of the switch. In human erythroid cells, BCL11A physically interacts with at least two corepressor complexes, Mi-2/NuRD and LSD1/CoREST, as well as the erythroid transcription factor GATA-1 and the HMG-box protein SOX6. Rather than binding to the promoters of the g- or b-globin genes as do these latter factors, BCL11A protein occupies the upstream locus control and g-d-intergenic regions of the b-globin cluster (as determined by high resolution ChIP-Chip analysis), suggesting that BCL11A mediates long-range interactions and/or reconfigures the locus during different stages. An in-depth mechanistic understanding of globin switching offers the prospect for design of target-based activation of HbF in adult erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.
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Grosveld, F., M. Antoniou, M. Berry, et al. "Regulation of Human Globin Gene Switching." Cold Spring Harbor Symposia on Quantitative Biology 58 (January 1, 1993): 7–13. http://dx.doi.org/10.1101/sqb.1993.058.01.004.
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Hebbes, Tim R., Alan W. Thorne, Alison L. Clayton, and Colyn Crane-Robinson. "Histone acetylation and globin gene switching." Nucleic Acids Research 20, no. 5 (1992): 1017–22. http://dx.doi.org/10.1093/nar/20.5.1017.
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Chan, Fung-Yee, Judith Robinson, Alison Brownlie та ін. "Characterization of Adult α- and β-Globin Genes in the Zebrafish". Blood 89, № 2 (1997): 688–700. http://dx.doi.org/10.1182/blood.v89.2.688.
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Abstract Developmental switching of hemoglobins (Hbs) occurs in most vertebrates, yet the cellular and molecular basis for this process remains elusive. The zebrafish is a new genetic and developmental system that can be used to study embryogenesis, and mutants with a variety of defects in hematopoiesis have recently been derived. To initiate our studies on Hb switching in this organism, we have characterized the globins expressed in the adult. Reversed-phase high performance liquid chromatography and mass spectrometric analyses of adult peripheral blood hemolysates showed that there are three major α globins and two β globins in circulating erythroid cells. In addition, we have isolated and characterized zebrafish adult α- and β-globin cDNA clones that encode some of these globins. High levels of α- and β-globin gene expression were detected in adult erythroid cells, whereas embryonic erythroid cells expressed little, if any, of these RNAs. We have also shown that the α- and β-globin genes are tightly linked on the same chromosome and are arrayed in a 3′-5′ to 5′-3′ configuration, respectively. The characterization of these genes and regulatory elements in this globin locus will provide insight into the process of globin gene transcription. With these reagents, future studies of Hb switching in zebrafish mutants with defective hematopoiesis will be possible.
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Tuan, Dorothy. "ERV-9 LTR Retrotransposon Modulates Globin Gene Switching." Blood 132, Supplement 1 (2018): 2341. http://dx.doi.org/10.1182/blood-2018-99-117280.
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Abstract In the human beta-globin gene locus, an LTR retrotransposon derived from ERV-9 human endogenous retrovirus is located near the locus control region (LCR) far upstream of the globin genes. In transgenic mice carrying the 100 kb human globin gene locus, deleting the ERV-9 LTR by cre-loxP mediated in situ recombination inactivates transcription of beta-globin gene by ~50% and activates that of gamma-globin gene by up to 5 fold, to at least 20% the level of beta-globin mRNA in both fetal and adult erythroid cells. Chromosome-conformation-capture (3C) shows that the ERV-9 LTR preferentially loops with beta-globin gene, even in fetal erythroid cells where gamma-globin gene is predominantly transcribed. Unique in the locus, the ERV-9 LTR contains high density of CCAAT motifs that strongly bind transcription factor (TF) NF-Y critical for assembling the LTR enhancer complex but no CCNCNCCC motifs that bind master erythroid TF KLF1, while the beta-globin promoter contains two tandem CCNCNCCC motifs that bind KLF1 but a CCAAT motif that only weakly bind NF-Y. However, both the LTR and beta-globin promoter associate in vivo with high levels of NF-Y and KLF1 in ChIP assays. Protein immunoprecipitation shows that neither NF-Y nor KLF1 associates with transcription co-regulator Ldb1, which has been reported to mediate chromosome looping of the LCR with the globin genes. Thus, the Ldb1/LMO2/GATA -1, -2 complex potentially assembled by the GATA motifs distributed throughout the locus does not appear to mediate the preferential looping of ERV-9 LTR with beta-globin promoter. Through interaction of NF-Y and KLF1 mediated by the to be identified co-regulator, the ERV-9 LTR interacts with and activates beta-globin gene, and suppresses gamma-globin gene by a competitive mechanism. Deletion of the ERV-9 LTR removes the competitive advantage of beta-globin gene, thus re-activating gamma-globin gene. Our findings suggest that deleting the ERV-9 LTR from the globin locus by genome-editing could provide a potential new therapy for beta-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.
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Beauchemin, Hugues, and Marie Trudel. "Evidence for a Bigenic Chromatin Subdomain in Regulation of the Fetal-to-Adult Hemoglobin Switch." Molecular and Cellular Biology 29, no. 6 (2008): 1635–48. http://dx.doi.org/10.1128/mcb.01735-08.
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ABSTRACT During development, human β-globin locus regulation undergoes two critical switches, the embryonic-to-fetal and fetal-to-adult hemoglobin switches. To define the role of the fetal Aγ-globin promoter in switching, human β-globin-YAC transgenic mice were produced with the Aγ-globin promoter replaced by the erythroid porphobilinogen deaminase (PBGD) promoter (PBGDAγ-YAC). Activation of the stage-independent PBGDAγ-globin strikingly stimulated native Gγ-globin expression at the fetal and adult stages, identifying a fetal gene pair or bigenic cooperative mechanism. This impaired fetal silencing severely suppressed both δ- and β-globin expression in PBGDAγ-YAC mice from fetal to neonatal stages and altered kinetics and delayed switching of adult β-globin. This regulation evokes the two human globin switching patterns in the mouse. Both patterns of DNA demethylation and chromatin immunoprecipitation analysis correlated with gene activation and open chromatin. Locus control region (LCR) interactions detected by chromosome conformation capture revealed distinct spatial fetal and adult LCR bigenic subdomains. Since both intact fetal promoters are critical regulators of fetal silencing at the adult stage, we concluded that fetal genes are controlled as a bigenic subdomain rather than a gene-autonomous mechanism. Our study also provides evidence for LCR complex interaction with spatial fetal or adult bigenic functional subdomains as a niche for transcriptional activation and hemoglobin switching.
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Bank, Arthur. "Regulation of human fetal hemoglobin: new players, new complexities." Blood 107, no. 2 (2006): 435–43. http://dx.doi.org/10.1182/blood-2005-05-2113.
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AbstractThe human globin genes are among the most extensively characterized in the human genome, yet the details of the molecular events regulating normal human hemoglobin switching and the potential reactivation of fetal hemoglobin in adult hematopoietic cells remain elusive. Recent discoveries demonstrate physical interactions between the β locus control region and the downstream structural γ- and β-globin genes, and with transcription factors and chromatin remodeling complexes. These interactions all play roles in globin gene expression and globin switching at the human β-globin locus. If the molecular events in hemoglobin switching were better understood and fetal hemoglobin could be more fully reactivated in adult cells, the insights obtained might lead to new approaches to the therapy of sickle cell disease and β thalassemia by identifying specific new targets for molecular therapies.
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Lowrey, Christopher. "Another piece of the globin-switching puzzle." Blood 107, no. 5 (2006): 1744. http://dx.doi.org/10.1182/blood-2005-12-4896.
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ENVER, T., and D. GREAVES. "Globin gene switching: A paradigm or what?" Current Opinion in Biotechnology 2, no. 6 (1991): 787–95. http://dx.doi.org/10.1016/s0958-1669(05)80108-9.
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Enver, Tariq, and David R. Greaves. "Globin gene switching — a paradigm or what?" Current Biology 2, no. 3 (1992): 145. http://dx.doi.org/10.1016/0960-9822(92)90262-9.
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Choi, Ok-Ryun Baik, та James Douglas Engel. "Developmental regulation of β-globin gene switching". Cell 55, № 1 (1988): 17–26. http://dx.doi.org/10.1016/0092-8674(88)90005-0.
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Orkin, Stuart H. "Globin gene regulation and switching: Circa 1990." Cell 63, no. 4 (1990): 665–72. http://dx.doi.org/10.1016/0092-8674(90)90133-y.
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McConnell, Sean C., Yongliang Huo, Shan-Run Liu, Ting-Ting Zhang, Clayton L. Ulrey, and Thomas M. Ryan. "Human Gamma Globin Gene Regulation in Knock-In Mouse Models of Anemia." Blood 110, no. 11 (2007): 1779. http://dx.doi.org/10.1182/blood.v110.11.1779.1779.
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Abstract The generation of transgenic and gene targeted mouse models of human hemoglobinopathies provides valuable opportunities to test mechanisms of human globin gene regulation and experimental therapies. Yet mice do not naturally have a fetal hemoglobin, challenging our ability to adequately model the developmental onset of disease. Transgenic model systems that contain the entire human β-globin locus present obstacles to the study of human globin gene switching, including a fetal to adult globin gene switch that occurs too early in development. The generation of genetically engineered mice with a delayed human γ to β hemoglobin switch has been a major topic of interest for our laboratory. Delayed γ globin gene expression improves the clinical progression in patients as well as animal models with hemoglobinopathies. However, molecular mechanisms involved in globin gene switching are not well understood. In this study the transcriptional and epigenetic regulation of human γ to β hemoglobin switching are analyzed in novel human knock-in (KI) mouse models that complete the switch from fetal to adult hemoglobin after birth. These KI mice were generated by replacement of the adult mouse β-globin genes by homologous recombination in embryonic stem cells with a delayed switching human γ to β globin gene construct. Quantitative real-time PCR and HPLC were used to measure mouse and human embryonic, fetal, and adult globin genes through development and show that we have given the mouse a true fetal hemoglobin. Heterozygous mice express human β-like globin genes at a high level comparable to the adult mouse β globin genes. Mutations responsible for hereditary persistence of fetal hemoglobin (HPFH) in the γ globin promoter recapitulate the human phenotype in KI mice, with over 50 fold γ globin gene upregulation in adults. These HPFH KI mice also display higher γ globin levels at birth and markedly delayed γ globin gene downregulation in the weeks following birth. These studies in KI mice demonstrate that human β-like globin genes interacting with the mouse LCR are regulated in a manner similar to what is seen in humans and may be used to study the mechanisms of globin gene switching. Greater understanding of γ-globin gene regulation will be required for achieving the therapeutic goal of reactivating silenced γ-globin genes to ameliorate severe human hemoglobinopathies.
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Sabatino, Denise E., Amanda P. Cline, Patrick G. Gallagher та ін. "Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice". Molecular and Cellular Biology 18, № 11 (1998): 6634–40. http://dx.doi.org/10.1128/mcb.18.11.6634.
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ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.
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Esteghamat, Fatemehsadat, Nynke Gillemans, Ivan Bilic, et al. "Erythropoiesis and Globin Switching in Compound Klf1::Bcl11a mutant mice." Blood 120, no. 21 (2012): 1019. http://dx.doi.org/10.1182/blood.v120.21.1019.1019.
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Abstract Abstract 1019 Reactivation of fetal γ-globin is of outstanding demand in patients with β-hemoglobinopathies. B-Cell/Lymphoma 11A (BCL11A) is a well-known repressor of γ-globin, and its expression is directly activated by Kruppel-Like Factor 1 (KLF1). KLF1 is a major regulator of human fetal to adult hemoglobin switching and reduced expression of KLF1 due to mutations is associated with hereditary persistence of fetal hemoglobin (HPFH). Analysis of the HPFH phenotype has led to the proposal that KLF1 has a dual role in γ-globin suppression, through its preferential activation of the β-globin gene and as a key activator of expression of the BCL11A repressor protein. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11a in an in vivo model, we used mice carrying a human β-globin locus transgene with combinations of Klf1 haploinsufficiency, and Bcl11a floxed and EpoRCre knockin alleles. We measured hematological parameters of the mutant mice. With the exception of a small reduction in MCV (mean corpuscular volume), parameters of Klf1wt/ko animals were similar to those observed in the control animals. Bcl11acko/cko animals displayed a small but significant reduction of HCT (Hematocrit), RBC (red blood cell count) and HGB (hemoglobin) values. The reductions in these values were more pronounced in the Klf1wt/ko::Bcl11acko/cko animals. In addition, Klf1wt/ko::Bcl11acko/cko mice displayed small but significantly increased values for MCV, MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration). We observed a higher concentration of erythropoietin in Bcl11acko/cko and compound Klf1wt/ko::Bcl11acko/cko animals suggesting a mild compensated anemia. To extend these observations, we analyzed embryonic blood and fetal livers at day E18.5, just prior to birth. Flow cytometry analysis of E18.5 blood revealed no difference in the CD71+Ter119+ population in peripheral blood of Bcl11acko/cko embryo's. This percentage was increased in Klf1wt/ko blood samples and was highest in blood from the compound Klf1wt/ko::Bcl11acko/cko embryos. Similar results were obtained following flow cytometry of E18.5 fetal liver cells. Consequently, the percentage of mature CD71−/Ter119+ cells in fetal liver and peripheral blood of E18.5 Klf1wt/ko::Bcl11acko/cko embryos was significantly lower than that observed in Klf1wt/ko, Bcl11acko/cko and control embryos. Analysis of Klf1wt/ko, Bcl11acko/cko and Klf1wt/ko::Bcl11acko/cko mutant embryos demonstrated increased expression of mouse embryonic α - and β-like globins during fetal development. Expression of human γ-globin remained high in Bcl11acko/cko embryos during fetal development, and this was further augmented in Klf1wt/ko::Bcl11acko/cko embryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11acko/cko and Klf1wt/ko::Bcl11acko/cko mice, but the levels remained much higher than those observed in control animals. We find that haploinsufficiency for KLF1 delays γ- to β-globin switching leading to a ∼2-fold increase in the γ/(γ+β) ratio at E14.5 and E18.5. Part of this increase can be explained by diminished BCL11A expression in embryos with KLF1 insufficiency. Collectively, these data support the proposed role of the KLF1-BCL11A axis in γ-globin regulation. In conclusion, our results suggest that haploinsufficiency for KLF1 prolongs reticulocyte maturation, and that this phenotype is further exacerbated in combination with BCL11A deficiency. Despite this, the impact on erythropoiesis is modest and none of the compound mutant mice suffer from overt anemia even at prenatal stages when the demand for erythroid expansion is high. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression and importantly in the absence of BCL11A, KLF1 still preferentially activates the β-globin gene. Disclosures: No relevant conflicts of interest to declare.
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Ganis, Jared J., Elizabeth B. Riley, James Palis, and Leonard I. Zon. "A Screen for Regulators of Globin Switching in the Zebrafish Embryo." Blood 120, no. 21 (2012): 826. http://dx.doi.org/10.1182/blood.v120.21.826.826.
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Abstract Abstract 826 The switching of the globin genes involves critical transcriptional regulators such as BCL11A, EKLF and SOX6, and the induction of fetal globin has been shown to ameliorate the symptoms of diseases such as sickle cell anemia. Recently, there has been interest in driving iPS cells to produce mature red cells that express adult globin genes in an attempt to make these cells therapeutically useful. Here, to understand hemoglobin switching and the molecular pathways that allow the establishment of an adult fate in embryonic tissues, we utilized a screening approach in the zebrafish model. The concept of the screen is to find transcription factors that are expressed in a stage-specific manner, and manipulate the expression of these genes to alter the cell fate of embryonic erythroid cells. In order to generate a candidate list of genes, microarray analysis was performed on murine yolk sac, fetal liver and adult derived red blood cells and red blood cell precursors, which express unique sets of globin genes. Pair-wise comparison of these populations yielded 879 unique differentially regulated genes. GO term analysis was used to narrow the list to 49 transcription factors. We focused on the transcription factors that might increase adult globin expression in the embryo based on their differential expression in the microarrays. Morpholinos were used to knock down these 24 genes by individually injecting each into one-cell stage embryos, allowing the embryos to reach 24 hpf and performing in situ hybridization for the adult globin gene αa1. The number of adult globin positive cells present in each embryo was counted for a clutch control group, which on average has 2–4 positive cells per embryo, and three doses of morpholino. We identified 4 genes, Tcf7l2, Ncoa1, Hif1al and E2F5, the knock down of which results in a significant increase in the number of adult globin positive cells in at least one dose of morpholino (control [n=53, mean=6.34], 6ng [n=56, mean=15.07], p=<0.0001; control [n=35, mean=1.543], 4ng [n=56, mean=2.75], p=<0.01; control [n=19, mean=1.368], 12ng [n=16, mean=6.188], p=<0.0001; control [n=44, mean=1.091], 4ng [n=30, mean=2.7], p=<0.05, respectively). Pair-wise knock down of these genes were also tested, and the combinations of Ncoa1 and E2F5, Tcf7l2 and E2F5 and Tcf7l2 and Ncoa1 were found to synergistically increase the number of adult globin expressing cells (control [n=49, mean=0.5306], knock down [n=38, mean=9.895], p=<0.0001; control [n=49, mean=7.633], knock down [n=54, mean=17.41], p=<0.0001; control [n=20, mean=2.95], knock down [n=28, mean=too numerous to count], p=<0.0001, respectively). The combined knock down of Tcf7l2 and Ncoa1 was both the strongest inducer of adult globin expression and had the lowest toxicity of the pair-wise combinations. Further characterization of this phenotype shows that, while many globin genes are up regulated, both of the adult globin genes, αa1 and βa1, are upregulated to a higher degree than other globin genes. In order to determine if the Wnt pathway is responsible for phenotype observed with the Tcf7l2 morpholino, we tested the Wnt pathway inhibitors IWR1 and XAV939. Both drugs phenocopied the Tcf7l2 knockdown response. In addition, XAV939 synergies with the Ncoa1 morpholino to enhance the increase in adult globin observed in a similar manner to Tcf7l2 knockdown. These results indicate that modulation of Wnt signaling, rather than a Wnt-independent function of Tcf7l2, is responsible for the phenotype and regulation of globin gene expression. Chip-Seq analysis of Ncoa1 occupancy in the erythroid cell line K562 was performed to examine potential mechanisms of action. Significant binding was observed at the enhancers of the α- and β-globin loci, indicating that the nuclear hormone receptor pathway may be acting directly on the globin loci to modulate globin expression patterns. These results indicate that Wnt signaling in combination with alterations of other pathways regulated by Ncoa1 are responsible for stage-specific globin expression. Our studies have impact on the understanding of globin switching in vertebrates, and could establish new methods to activate specific globins clinically, and to make iPS cells form adult-type tissues. Disclosures: Zon: Fate Therapeutics: Founder Other.
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Zitnik, G., Q. Li, G. Stamatoyannopoulos, and T. Papayannopoulou. "Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids." Molecular and Cellular Biology 13, no. 8 (1993): 4844–51. http://dx.doi.org/10.1128/mcb.13.8.4844-4851.1993.
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The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.
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Zitnik, G., Q. Li, G. Stamatoyannopoulos, and T. Papayannopoulou. "Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids." Molecular and Cellular Biology 13, no. 8 (1993): 4844–51. http://dx.doi.org/10.1128/mcb.13.8.4844.
Full textAbstract:
The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.
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Marongiu, Maria F., Susanna Porcu, Daniela Poddie та ін. "Different Hemoglobin Switching Pattern of β-Thalassemia Mutations at the Proximal and Distal Human β-Globin CACCC Box." Blood 110, № 11 (2007): 1780. http://dx.doi.org/10.1182/blood.v110.11.1780.1780.
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Abstract The CACCC box is duplicated in the β globin gene promoter of humans and other mammals. While the function of the proximal element as a binding site for EKLF has already been well established, the role of the distal element remains unclear The distal CACCC box has been previously reported not to bind EKLF in vitro. A minor role of the distal CACCC element in β globin gene promoter function is suggested by the observation that naturally occurring β thalassemia mutations affecting the proximal CACCC box are far more severe than those affecting the distal element. Nevertheless recent evidences demonstrate: that EKLF does indeed bind to the distal CACCC motif, although with low affinity. that the CCTCACCC is required for maximal stimulation of the β-globin gene by EKLF and that silent β-thalassemia due to mutations of the distal CACCC box affects the binding and responsiveness to EKLF of the β-globin gene promoter. Our interest in the function of the distal CACCC element springs from the observation that β thalassemia mutation affecting the distal box show an age related pattern of expression being more severe in the childhood than in the adulthood. In order to get light inside the role of this element in the function of the β globin gene and in the γ to β hemoglobin switching we have analyzed the effect of mutations at the proximal and distal element “in vivo”. We have engineered, by site specific mutagenesis, the β-101 (distal CACCC element) and β-87 (proximal CACCC element) mutations inside the “minilocus “ γ-β construct. The minilocus construct has been widely used to study hemoglobin switching in vivo. This construct contains the full β-globin Locus Control Region (LCR), the Aγ globin gene, the β-globin gene and the 3′ hypersensitive site (HS) of the β-globin cluster. Three mice transgenic lines have been produced. The pattern of g versus β-globin switching has been analyzed during the development by S1 analysis and real time PCR. We have dissected the yolk sac at 10 days post conception (pc) to asses the embryonic stage of erythopoiesis; the fetal liver at 12, 14 and 16 days pc to asses the fetal stage or erythropoiesis when the g to b competitive switching take place; and the adult blood. Our results indicated that neither the β-101 nor the β-87 thalassemia mutations affect the competitive silencing of the b-globin gene in the yolk sac. During the fetal liver stage of erythropoiesis, were both human g and b human transgenes are expressed, the pattern of γ-β hemoglobin switching is striking different for the two different constructs. The b-87 minilocus γ-β construct shows a delayed switching patter mainly due to the low activation of the mutated β globin gene. The impairment of the expression of the β-87 globin gene became more severe during the fetal development compared to the control line. On the other hand the β-101 minilocus γ-β construct shows a γ-β hemoglobin switching pattern which is anticipated respect to the control line. In addition the effect of the β-101 mutation became less severe during the fetal development. These results highlight a possible role of the distal CACCC element in hemoglobin switching and in particular in the early stage of β-globin activation.
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Harju, Susanna, Kellie J. McQueen та Kenneth R. Peterson. "Chromatin Structure and Control of β-Like Globin Gene Switching". Experimental Biology and Medicine 227, № 9 (2002): 683–700. http://dx.doi.org/10.1177/153537020222700902.
Full textAbstract:
The human β-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional β-like globin genes, ε, Gγ, Aγ, δ, and β, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNasel-hypersensitive sites located 6-22 Kb upstream of the ε-globin gene. During ontogeny two switches occur in β-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic ε- to fetal γ-globin occurs at six weeks of gestation. The second switch from γ- to adult δ- and β-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching.
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Felice, Alexander E. "KLF1 Dependent Pathways In Developmental Globin Gene Switching." Blood 116, no. 21 (2010): 5162. http://dx.doi.org/10.1182/blood.v116.21.5162.5162.
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Abstract Abstract 5162 We provide additional data on members of the family from Malta with Hereditary Persistence of Fetal Hemoglobin (HPFH) due to KLF1 haplo-insufficiency. The data indicated a possible role of additional loci in the pathway of globin gene control. We showed that KLF1 functions as a master regulator of erythropoiesis and developmental globin gene switching (Borg et al., Nature Genetics doi: 10. 1038/ng.630, 2010), at least partly through BCL11A. Given the phenoytpes of the HPFH heterozygotes, the truncating KLF1 p.K288X was best described as a dominant mutation with variable penetrance; most likely due to interplay with other regulatory factors that we have been seeking. Genome-wide association analysis, in the context of genome-wide expression profiles from cultured Human Erythroid Progenitors (HEPs) of critically informative family members, revealed additional loci of potential interest. The effect of the Hb F inducer Hydroxyurea on the gamma globin profiles of the KLF1- (p.K288X) HPFH HEPs was enhanced compared to the wild type, and 74 loci were differentially expressed. It is anticipated that extensive re-sequencing of these new targets may reveal the extent of the molecular pathways under control of KLF1 in erythropoiesis and globin gene switching, and in particular those that may be targeted for therapeutics in patients. Disclosures: No relevant conflicts of interest to declare.
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Mookerjee, B., MO Arcasoy, and GF Atweh. "Spontaneous delta- to beta-globin switching in K562 human leukemia cells." Blood 79, no. 3 (1992): 820–25. http://dx.doi.org/10.1182/blood.v79.3.820.820.
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Abstract Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta- globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.
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Mookerjee, B., MO Arcasoy, and GF Atweh. "Spontaneous delta- to beta-globin switching in K562 human leukemia cells." Blood 79, no. 3 (1992): 820–25. http://dx.doi.org/10.1182/blood.v79.3.820.bloodjournal793820.
Full textAbstract:
Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta- globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.
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Grosveld, F. "Control of globin gene switching and the search for foetal globin activating compounds." Journal of Pharmacy and Pharmacology 50, S9 (1998): 25. http://dx.doi.org/10.1111/j.2042-7158.1998.tb02225.x.
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Guo, Xiang, Jennifer Plank-Bazinet, Ivan Krivega, Ryan K. Dale, and Ann Dean. "Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin organization." Nucleic Acids Research 48, no. 18 (2020): 10226–40. http://dx.doi.org/10.1093/nar/gkaa736.
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Abstract The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human β-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the β-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.
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Tumburu, Laxminath, Colleen Byrnes, Y. Terry Lee, Jaira F. de Vasconcellos, Antoinette Rabel, and Jeffery L. Miller. "IGF2BP1 Reverses Hemoglobin Switching in Adult Erythroblasts." Blood 126, no. 23 (2015): 639. http://dx.doi.org/10.1182/blood.v126.23.639.639.
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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.
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PERRINE, SUSAN P., DOUGLAS V. FALLER, PAUL SWERDLOW, et al. "Stopping the Biologic Clock for Globin Gene Switching." Annals of the New York Academy of Sciences 612, no. 1 Sixth Cooley' (1990): 134–40. http://dx.doi.org/10.1111/j.1749-6632.1990.tb24299.x.
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Esteghamat, Fatemehsadat, Nynke Gillemans, Ivan Bilic, et al. "Erythropoiesis and globin switching in compound Klf1::Bcl11a mutant mice." Blood 121, no. 13 (2013): 2553–62. http://dx.doi.org/10.1182/blood-2012-06-434530.
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Key Points Our data support an important role for the KLF1-BCL11A axis in erythroid maturation and hemoglobin switching. In adults, gamma-globin levels decline in Bcl11a and Klf1::Bcl11a mutants, suggesting an additional layer of gamma-globin silencing.
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Perkins, Andrew C., Michael Tallack, Ye Zhan, et al. "Ikaros Drives Human Haemoglobin Switching by Facilitating Active Chromatin Hub Formation." Blood 110, no. 11 (2007): 1772. http://dx.doi.org/10.1182/blood.v110.11.1772.1772.
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Abstract The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5′-ε-Gγ-Aγ- δ- β-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of γ-globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the δ-globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the δ-globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease.
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Caria, Cristian Antonio, Valeria Faà, and Maria Serafina Ristaldi. "Krüppel-Like Factor 1: A Pivotal Gene Regulator in Erythropoiesis." Cells 11, no. 19 (2022): 3069. http://dx.doi.org/10.3390/cells11193069.
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Krüppel-like factor 1 (KLF1) plays a crucial role in erythropoiesis. In-depth studies conducted on mice and humans have highlighted its importance in erythroid lineage commitment, terminal erythropoiesis progression and the switching of globin genes from γ to β. The role of KLF1 in haemoglobin switching is exerted by the direct activation of β-globin gene and by the silencing of γ-globin through activation of BCL11A, an important γ-globin gene repressor. The link between KLF1 and γ-globin silencing identifies this transcription factor as a possible therapeutic target for β-hemoglobinopathies. Moreover, several mutations have been identified in the human genes that are responsible for various benign phenotypes and erythroid disorders. The study of the phenotype associated with each mutation has greatly contributed to the current understanding of the complex role of KLF1 in erythropoiesis. This review will focus on some of the principal functions of KLF1 on erythroid cell commitment and differentiation, spanning from primitive to definitive erythropoiesis. The fundamental role of KLF1 in haemoglobin switching will be also highlighted. Finally, an overview of the principal human mutations and relative phenotypes and disorders will be described.
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Lloyd, J. A., J. M. Krakowsky, S. C. Crable, and J. B. Lingrel. "Human gamma- to beta-globin gene switching using a mini construct in transgenic mice." Molecular and Cellular Biology 12, no. 4 (1992): 1561–67. http://dx.doi.org/10.1128/mcb.12.4.1561-1567.1992.
Full textAbstract:
The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.
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Lloyd, J. A., J. M. Krakowsky, S. C. Crable, and J. B. Lingrel. "Human gamma- to beta-globin gene switching using a mini construct in transgenic mice." Molecular and Cellular Biology 12, no. 4 (1992): 1561–67. http://dx.doi.org/10.1128/mcb.12.4.1561.
Full textAbstract:
The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.
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Moi, Paolo, Loredana Porcu, Maria G. Marini та ін. "Differential Modulation of the β-Like Globin Genes by KLFs Isolated with a γ-Globin CACCC Bait." Blood 106, № 11 (2005): 3637. http://dx.doi.org/10.1182/blood.v106.11.3637.3637.
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Abstract The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globin genes. While the β-globin CACCC box binds EKLF/KLF1, a likely adult switching factor, analogous factors, interacting with the γ-globin gene and predicted to regulate the fetal stage of hemoglobin switching, have so far been elusive. By using yeast one hybrid assay, we have isolated four KLFs, KLF1, 2, 4, and 6, that bound the γ-CACCC bait. To establish their role in globin regulation and in the switching of hemoglobins, these factors were compared to four other KLFs already established or putative globin regulators, KLF3, 11, 13 and 16, mainly evaluating their ability to bind and transactivate the ε-, γ- and β-globin gene. γ-CACCC binding at variable intensities was confirmed in band shift assay for all four isolated KLFs, for KLF3 and, faintly, for KLF13. The ε- and β-CACCC were bound by the same factors with similar affinities with the exception of KLF3 and KLF13 that bound stronger to the β- and ε- than to the γ-CACCC box. On the other hand, KLF11 and 16 did not produce any specific complex in band shift assays with anyone of the globin CACCC boxes. More relevant differences were observed among the factors in the transactivation of single and dual luciferase reporters in both K562 and MEL cells. In these assays, most factors presented peculiar modulatory properties and specific promoter tropism. Several factors presented bidirectional activity displaying in the same time the capacity to stimulate and repress different globin promoters. KLF1 and 4 were the strongest stimulators of the β-globin promoter in both cell lines, whereas KLF2 activated the β-promoter only in K562 cells. KLF1 and especially KLF4 consistently repressed ε-globin expression especially in MEL cells. KLF3 behaved always as a general globin repressor in MEL cells, but acted as a weak stimulator of the γ- and ε-promoter in K562 cells. KLF4 was the strongest inhibitor of the ε-globin gene. KLF13 significantly stimulated the γ-promoter in both cell lines, whereas KLF3, 4 and 6 showed statistically significant stimulation only in MEL cells. By RT-PCR analysis we found that KLFs were highly variable in their tissue expression and that KLF1, 3 and 13 had the highest expression in erythroid tissues. Thus the level of tissue expression should ultimately determine which factors are really active in physiological conditions. Taken together our binding and expression studies suggest that several KLFs have the potential to modulate the activity of the globin genes and that the resulting globin expression will depend on the vectorial sum of the relative activities of the factors expressed at any given time of development. Furthermore, as some KLFs, like KLF1 and 4, exert opposite effects on fetal and adult globin genes, their role in hemoglobin switching may be direct and not only dependent on their ability to mediate promoter competition for the LCR.
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Russell, J. Eric, та Stephen A. Liebhaber. "Reversal of Lethal - and β-Thalassemias in Mice by Expression of Human Embryonic Globins". Blood 92, № 9 (1998): 3057–63. http://dx.doi.org/10.1182/blood.v92.9.3057.
Full textAbstract:
Abstract Genetic mutations that block - or β-globin gene expression in humans can result in severe and frequently lethal thalassemic phenotypes. Homozygous inactivation of the endogenous - or β-globin genes in mice results in corresponding thalassemic syndromes that are uniformly fatal in utero. In the current study, we show that the viability of these mice can be rescued by expression of human embryonic ζ- and -globins, respectively. The capacity of embryonic globins to fully substitute for their adult globin homologues is further demonstrated by showing that ζ- and -globins reverse the hemolytic anemia and abnormal erythrocyte morphology of mice with nonlethal forms of - and β-thalassemia. These results illustrate the potential therapeutic utility of embryonic globins as substitutes for deficient adult globins in thalassemic individuals. Moreover, the capacity of embryonic globins to functionally replace their adult homologues brings into question the physiologic basis for globin gene switching. © 1998 by The American Society of Hematology.
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Russell, J. Eric, та Stephen A. Liebhaber. "Reversal of Lethal - and β-Thalassemias in Mice by Expression of Human Embryonic Globins". Blood 92, № 9 (1998): 3057–63. http://dx.doi.org/10.1182/blood.v92.9.3057.421k57_3057_3063.
Full textAbstract:
Genetic mutations that block - or β-globin gene expression in humans can result in severe and frequently lethal thalassemic phenotypes. Homozygous inactivation of the endogenous - or β-globin genes in mice results in corresponding thalassemic syndromes that are uniformly fatal in utero. In the current study, we show that the viability of these mice can be rescued by expression of human embryonic ζ- and -globins, respectively. The capacity of embryonic globins to fully substitute for their adult globin homologues is further demonstrated by showing that ζ- and -globins reverse the hemolytic anemia and abnormal erythrocyte morphology of mice with nonlethal forms of - and β-thalassemia. These results illustrate the potential therapeutic utility of embryonic globins as substitutes for deficient adult globins in thalassemic individuals. Moreover, the capacity of embryonic globins to functionally replace their adult homologues brings into question the physiologic basis for globin gene switching. © 1998 by The American Society of Hematology.
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Kulozik, AE, A. Bellan-Koch, E. Kohne, and E. Kleihauer. "A deletion/inversion rearrangement of the beta-globin gene cluster in a Turkish family with delta beta zero-thalassemia intermedia." Blood 79, no. 9 (1992): 2455–59. http://dx.doi.org/10.1182/blood.v79.9.2455.2455.
Full textAbstract:
Abstract The most common forms of hereditary persistence of fetal hemoglobin synthesis (HPFH) and delta beta zero-thalassemia result from simple deletions of the beta-globin gene cluster or from point mutations in the gamma-globin gene promoters. These naturally occurring mutants extend our understanding of globin gene regulation and hemoglobin switching. Furthermore, they provide the opportunity to test in vivo hypothetical switching models that are based on the experimental approach. We report here a family with delta beta zero-thalassemia from Turkey with a complex rearrangement of the beta-globin gene cluster that involves two deletions of 11.5 kb and 1.6 kb, and an inversion of 7.6 kb. The larger deletion removes both the delta-and the beta-globin genes with 3′ flanking sequences, whereas the smaller deletion affects DNA of unknown function. The inversion contains the entire L1 repeat 3′ of the beta-globin gene. There are structural motifs near the breakpoints (introduction of an “orphan” nucleotide, multiple direct and inverted repeats) suggesting a nonhomologous type of recombination event. The hematologic phenotype and the molecular structure of the rearranged beta-globin gene cluster are consistent with a competitive relationship between the fetal and the adult globin genes and/or with the translocation of enhancer sequences into the gamma-globin gene region.
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Kulozik, AE, A. Bellan-Koch, E. Kohne, and E. Kleihauer. "A deletion/inversion rearrangement of the beta-globin gene cluster in a Turkish family with delta beta zero-thalassemia intermedia." Blood 79, no. 9 (1992): 2455–59. http://dx.doi.org/10.1182/blood.v79.9.2455.bloodjournal7992455.
Full textAbstract:
The most common forms of hereditary persistence of fetal hemoglobin synthesis (HPFH) and delta beta zero-thalassemia result from simple deletions of the beta-globin gene cluster or from point mutations in the gamma-globin gene promoters. These naturally occurring mutants extend our understanding of globin gene regulation and hemoglobin switching. Furthermore, they provide the opportunity to test in vivo hypothetical switching models that are based on the experimental approach. We report here a family with delta beta zero-thalassemia from Turkey with a complex rearrangement of the beta-globin gene cluster that involves two deletions of 11.5 kb and 1.6 kb, and an inversion of 7.6 kb. The larger deletion removes both the delta-and the beta-globin genes with 3′ flanking sequences, whereas the smaller deletion affects DNA of unknown function. The inversion contains the entire L1 repeat 3′ of the beta-globin gene. There are structural motifs near the breakpoints (introduction of an “orphan” nucleotide, multiple direct and inverted repeats) suggesting a nonhomologous type of recombination event. The hematologic phenotype and the molecular structure of the rearranged beta-globin gene cluster are consistent with a competitive relationship between the fetal and the adult globin genes and/or with the translocation of enhancer sequences into the gamma-globin gene region.
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Marini, Giuseppina M., Isadora Asunis, Annalisa Cabriolu, et al. "Delayed Embryonic to Adult Globin Switching in HMGB2 Knock Out Mice." Blood 118, no. 21 (2011): 2152. http://dx.doi.org/10.1182/blood.v118.21.2152.2152.
Full textAbstract:
Abstract Abstract 2152 Hypersensitive site 2 (HS2) of the locus control region (LCR) is required for the optimal regulation of the beta globin gene cluster. Screening a λgt11 cord blood cDNA library with the tandem NFE2 repeat of HS2 as recognition site probe, we isolated 14 cDNA clones of HMGB2, a chromatin non histone protein. Binding to the HS2 region was confirmed in vivo by ChIP assay. Transactivation analysis in K562 cells showed mild repression of a luciferase reporter driven by HS2 and the γ-promoter. The DNA bending capacity and the increased expression of HMGB2 during erythroid differentiation are properties well suited to facilitate LCR looping toward the β-globin genes, the mechanism thought to mediate globin gene activation. Hence, HMGB2 binding to HS2 may be relevant for the regulation of the β-globin gene cluster. To assess the function of HMGB2 as a possible regulator of the globin genes we analyzed the hematological phenotype of the HMGB2 knock out mice during erythroid differentiation. In peripheral blood from E18 fetus or adult mice no hematological differences were found among normal, heterozygous or homozygous HMGB2 mice. However, at earlier stages of development, at E12, β-minor and major were slightly reduced in HMGB2−/− mice, whereas embryonic εy and βH1 globin genes were overexpressed 4–5 times compared to normal littermate mice. These results support a repressing activity of HMGB2 on embryonic globin genes and a possible role in the switching of hemoglobins during early stages of differentiation. The lack of any hematological phenotype in the latter stages of development may be explained by a temporal restriction in the expression of HMGB2 or by the vicarious activity of other HMGB proteins during definitive erythropoiesis Disclosures: No relevant conflicts of interest to declare.
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Enver, T., J. W. Zhang, T. Papayannopoulou, and G. Stamatoyannopoulos. "DNA methylation: a secondary event in globin gene switching?" Genes & Development 2, no. 6 (1988): 698–706. http://dx.doi.org/10.1101/gad.2.6.698.
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BICHET, SANDRINE, ROLAND H. WENGER, GIERI CAMENISCH та ін. "Oxygen tension modulates β‐globin switching in embryoid bodies". FASEB Journal 13, № 2 (1999): 285–95. http://dx.doi.org/10.1096/fasebj.13.2.285.
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Iarovaia, O. V., A. P. Kovina, N. V. Petrova та ін. "Genetic and Epigenetic Mechanisms of β-Globin Gene Switching". Biochemistry (Moscow) 83, № 4 (2018): 381–92. http://dx.doi.org/10.1134/s0006297918040090.
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Tanimoto, K. "In vivo Modulation of Human β-globin Gene Switching". Trends in Cardiovascular Medicine 10, № 1 (2000): 15–19. http://dx.doi.org/10.1016/s1050-1738(00)00035-9.
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