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1
Hodge, Denise, Elise Coghill, Janelle Keys, Tina Maguire, Belinda Hartmann, Alasdair McDowall, Mitchell Weiss, Sean Grimmond, and Andrew Perkins. "A global role for EKLF in definitive and primitive erythropoiesis." Blood 107, no. 8 (April 15, 2006): 3359–70. http://dx.doi.org/10.1182/blood-2005-07-2888.
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Abstract Erythroid Kruppel-like factor (EKLF, KLF1) plays an important role in definitive erythropoiesis and β-globin gene regulation but failure to rectify lethal fetal anemia upon correction of globin chain imbalance suggested additional critical EKLF target genes. We employed expression profiling of EKLF-null fetal liver and EKLF-null erythroid cell lines containing an inducible EKLF-estrogen receptor (EKLF-ER) fusion construct to search for such targets. An overlapping list of EKLF-regulated genes from the 2 systems included α-hemoglobin stabilizing protein (AHSP), cytoskeletal proteins, hemesynthesis enzymes, transcription factors, and blood group antigens. One EKLF target gene, dematin, which encodes an erythrocyte cytoskeletal protein (band 4.9), contains several phylogenetically conserved consensus CACC motifs predicted to bind EKLF. Chromatin immunoprecipitation demonstrated in vivo EKLF occupancy at these sites and promoter reporter assays showed that EKLF activates gene transcription through these DNA elements. Furthermore, investigation of EKLF target genes in the yolk sac led to the discovery of unexpected additional defects in the embryonic red cell membrane and cytoskeleton. In short, EKLF regulates global erythroid gene expression that is critical for the development of primitive and definitive red cells.
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Pilon, Andre M., Douglas G. Nilson, Dewang Zhou, Jose Sangerman, Tim M. Townes, David M. Bodine, and Patrick G. Gallagher. "Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice." Molecular and Cellular Biology 26, no. 11 (June 1, 2006): 4368–77. http://dx.doi.org/10.1128/mcb.02216-05.
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ABSTRACT Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the β-globin promoter, where it establishes local chromatin structure permitting β-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the β-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and β-globin genes and indicate that EKLF may play similar roles for other erythroid genes.
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Basu, Priyadarshi, Tina K. Lung, Wafaa Lemsaddek, Thanh Giang Sargent, David C. Williams, Mohua Basu, Latasha C. Redmond, Jerry B. Lingrel, Jack L. Haar, and Joyce A. Lloyd. "EKLF and KLF2 have compensatory roles in embryonic β-globin gene expression and primitive erythropoiesis." Blood 110, no. 9 (November 1, 2007): 3417–25. http://dx.doi.org/10.1182/blood-2006-11-057307.
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Abstract The Krüppel-like C2/H2 zinc finger transcription factors (KLFs) control development and differentiation. Erythroid Krüppel-like factor (EKLF or KLF1) regulates adult β-globin gene expression and is necessary for normal definitive erythropoiesis. KLF2 is required for normal embryonic Ey- and βh1-, but not adult βglobin, gene expression in mice. Both EKLF and KLF2 play roles in primitive erythroid cell development. To investigate potential interactions between these genes, EKLF/KLF2 double-mutant embryos were analyzed. EKLF−/−KLF2−/− mice appear anemic at embryonic day 10.5 (E10.5) and die before E11.5, whereas single-knockout EKLF−/− or KLF2−/− embryos are grossly normal at E10.5 and die later than EKLF−/−KLF2−/− embryos. At E10.5, Ey- and βh1-globin mRNA is greatly reduced in EKLF−/−KLF2−/−, compared with EKLF−/− or KLF2−/− embryos, consistent with the observed anemia. Light and electron microscopic analyses of E9.5 EKLF−/−KLF2−/− yolk sacs, and cytospins, indicate that erythroid and endothelial cells are morphologically more abnormal than in either single knockout. EKLF−/−KLF2−/− erythroid cells are markedly irregularly shaped, suggesting membrane abnormalities. EKLF and KLF2 may have coordinate roles in a common progenitor to erythroid and endothelial cells. The data indicate that EKLF and KLF2 have redundant functions in embryonic β-like globin gene expression, primitive erythropoiesis, and endothelial development.
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Pilon, Andre M., Murat O. Arcasoy, Holly K. Dressman, Serena E. Vayda, Yelena D. Maksimova, Jose I. Sangerman, Patrick G. Gallagher, and David M. Bodine. "Failure of Terminal Erythroid Differentiation in EKLF-Deficient Mice Is Associated with Cell Cycle Perturbation and Reduced Expression of E2F2." Molecular and Cellular Biology 28, no. 24 (October 13, 2008): 7394–401. http://dx.doi.org/10.1128/mcb.01087-08.
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ABSTRACT Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf −/− ) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf −/− embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild-type and Eklf −/− early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf −/− early erythroid progenitor cells, which showed a delay in the G1-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation.
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Pilon, Andre M., Subramanian S. Ajay, Swathi Ashok Kumar, Laurie A. Steiner, Praveen F. Cherukuri, Stephen Wincovitch, Stacie M. Anderson, et al. "Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation." Blood 118, no. 17 (October 27, 2011): e139-e148. http://dx.doi.org/10.1182/blood-2011-05-355107.
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Abstract Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map trans-factor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1.
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Perkins, Andrew C., Elise Coghill, Tina Maguire, Belinda Hartmann, Alasdair McDowall, Mitchell Weiss, Sean Grimmond, Janelle Keys, and Denise Hodge. "A Global Role for EKLF in Definitive and Primitive Erythropoiesis." Blood 106, no. 11 (November 16, 2005): 1745. http://dx.doi.org/10.1182/blood.v106.11.1745.1745.
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Abstract Erythroid Kruppel-like factor (EKLF or Klf1) is an erythroid specific C2H2 zinc-finger transcription factor which is essential for definitive erythropoiesis and β-globin gene expression. The absence of EKLF results in fatal anaemia but correction of globin chain imbalance does result in rescue, suggesting the existence of additional EKLF target genes. The aim of this study was to search for such genes by expression profiling. We performed profiling on fetal livers from wild-type versus EKLF null litter mates, and also EKLF null erythroid cell lines containing an inducible EKLF-ERTM fusion construct. Hybridisations were performed on microarray slides printed with a 23K oligo library from Compugen. Target gene validation was performed by real-time RT-PCR, chromatin immuno-precipitation (ChIP) and promoter-reporter assays. A large number of genes were down regulated in the absence of EKLF but few were up regulated, suggesting EKLF acts primarily as a transcriptional activator in vivo. One hundred genes were EKLF dependent in both systems. These include heme synthesis enzymes, red cell surface proteins including Rh and the transferrin receptor, and erythroid transcription factors. Two interesting highly EKLF-dependent genes are α-haemoglobin stabilising protein (AHSP), a key chaperone for free a-globin chains, and dematin (band 4.9) which links the cytoskeleton to the red cell membrane. A search for EKLF binding sites within the dematin and AHSP genes demonstrated a number of phylogenetically conserved CACC sites, and ChIP demonstrated in vivo EKLF occupancy at some but not all of these. Promoter-reporter assays showed EKLF directly activates dematin gene transcription through two promoters containing these sites. Lastly, investigation of EKLF target genes in the yolk sac lead to the discovery of unexpected defects in the embryonic red cell membrane and cytoskeleton. In conclusion, EKLF regulates global erythroid gene expression which is critical for development of primitive as well as definitive red cells.
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Coghill, Elise, Sarah Eccleston, Vanessa Fox, Loretta Cerruti, Clark Brown, John Cunningham, Stephen Jane, and Andrew Perkins. "Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice." Blood 97, no. 6 (March 15, 2001): 1861–68. http://dx.doi.org/10.1182/blood.v97.6.1861.
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Erythroid Kruppel-like factor (EKLF) is a transcription factor of the C2H2 zinc-finger class that is essential for definitive erythropoiesis. We generated immortal erythroid cell lines from EKLF−/− fetal liver progenitor cells that harbor a single copy of the entire human β-globin locus and then reintroduced EKLF as a tamoxifen-inducible, EKLF–mutant estrogen receptor (EKLF-ER™) fusion protein. Addition of tamoxifen resulted in enhanced differentiation and hemoglobinization, coupled with reduced proliferation. Human β-globin gene expression increased significantly, whereas γ-globin transcripts remained elevated at levels close to endogenous mouse α-globin transcript levels. We conclude that EKLF plays a role in regulation of the cell cycle and hemoglobinization in addition to its role in β-globin gene expression. The cell lines we used will facilitate structural and functional analyses of EKLF in these processes and provide useful tools for the elucidation of nonglobin EKLF target genes.
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Pilon, Andre M., Elliott H. Margulies, Hatice Ozel Abaan, Amy Werner Allen, Tim M. Townes, Abbie M. Frederick, Dewang Zhou, Patrick G. Gallagher, and David M. Bodine. "Genome-Wide Analysis of EKLF Occupancy in Erythroid Chromatin Reveals 5′, 3′ and Intragenic Binding Sites in EKLF Target Genes." Blood 112, no. 11 (November 16, 2008): 283. http://dx.doi.org/10.1182/blood.v112.11.283.283.
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Abstract Erythroid Kruppel-Like Factor (EKLF; KLF1) is the founding member of the Kruppel family of transcription factors, with 3 C2H2 zinc-fingers that bind a 9-base consensus sequence (NCNCNCCCN). The functions of EKLF, first identified as an activator of the beta-globin locus, include gene activation and chromatin remodeling. Our knowledge of genes regulated by EKLF is limited, as EKLF-deficient mice die by embryonic day 15 (E15), due to a severe anemia. Analysis of E13.5 wild type and EKLF-deficient fetal liver (FL) erythroid cells revealed that EKLF-deficient cells fail to complete terminal erythroid maturation (Pilon et al. submitted). Coupling chromatin immunoprecipitation and ultra high-throughput massively parallel sequencing (ChIP-seq) is increasingly being used for mapping protein-DNA interactions in vivo on a genome-wide scale. ChIP-seq allows a simultaneous analysis of transcription factor binding in every region of the genome, defining an “interactome”. To elucidate direct EKLF-dependent effects on erythropoiesis, we have combined ChIP-seq with expression array (“transcriptome”) analyses. We feel that integration of ChIP-seq and microarray data can provide us detailed knowledge of the role of EKLF in erythropoiesis. Chromatin was isolated from E13.5 FL cells of mice whose endogenous EKLF gene was replaced with a fully functional HA-tagged EKLF gene. ChIP was performed using a highly specific high affinity anti-HA antibody. A library of EKLF-bound FL chromatin enriched by anti-HA IP was created and subjected to fluorescent in situ sequencing on a Solexa 1G platform, providing 36-base signatures that were mapped to unique sites in the mouse genome, defining the EKLF “interactome.” The frequency with which a given signature appears provides a measurable peak of enrichment. We performed three biological/technical replicates and analyzed each data set individually as well as the combined data. To validate ChIP-seq results, we examined the locus of a known EKLF target gene, a-hemoglobin stabilizing protein (AHSP). Peaks corresponded to previously identified DNase hypersensitive sites, regions of histone hyperacetylation, and sites of promoter-occupancy determined by ChIP-PCR. A genome wide analysis, focusing on the regions with the highest EKLF occupancy revealed a set of 531 locations where high levels EKLF binding occurs. Of these sites, 119 (22%) are located 10 kb or more from the nearest gene and are classified as intergenic EKLF binding sites. Another 78 sites (14.6%) are within 10 kb of an annotated RefSeq gene. A plurality of the binding sites, 222 (42%), are within RefSeq coordinates and are classified as intragenic EKLF binding sites. Microarray profiling of mRNA from sorted, matched populations of dE13.5 WT and EKLF-deficient FL erythroid progenitor cells showed dysregulation of >3000 genes (p<0.05). Ingenuity Pathways Analysis (IPA) of the >3000 dysregulated mRNAs indicated significant alteration of a cell cycle-control network, centered about the transcription factor, E2f2. We confirmed significantly decreased E2f2 mRNA and protein levels by real-time PCR and Western blot, respectively; demonstrated that EKLF-deficient FL cells accumulate in G0/G1 by cell cycle analysis; and verified EKLF-binding to motifs within the E2f2 promoter by ChIP-PCR and analysis of the ChIP Seq data. We hypothesized that only a subset of the 3000 dysregulated genes would be direct EKLF targets. We limited the ChIP-seq library to display the top 5% most frequently represented fragments across the genome, and applied this criterion to the network of dysregulated mRNAs in the IPA cell cycle network. ChIP-seq identified peaks of EKLF association with 60% of the loci in this pathway. However, consistent with the role of EKLF as a transcriptional activator, 95% of the occupied genomic loci corresponded to mRNAs whose expression in EKLF-deficient FL cells was significantly decreased (p<0.05). The majority (59%) of these EKLF-bound sites were located at intragenic sites (i.e., introns), while a minority (15% and 26%) were found adjacent to the genes or in intergenic regions. We have shown that both the AHSP and E2f2 loci require EKLF to cause the locus to become activated and sensitive to DNase I digestion in erythroid cells. Based on the increased frequency of intragenic EKLF-binding sites, particularly in genes of the cell cycle network, we propose that the occupancy of intragenic sites by EKLF may facilitate chromatin modification.
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Perkins, Andrew C., Janelle R. Keys, Denise J. Hodge, and Michael R. Tallack. "Erythroid Kruppel-Like Factor Regulates E2F4 and the G1 Cdk Inhibitor, p18." Blood 106, no. 11 (November 16, 2005): 1357. http://dx.doi.org/10.1182/blood.v106.11.1357.1357.
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Abstract Erythroid Kruppel-Like Factor (EKLF) is a zinc finger transcription factor which is essential for β-globin gene expression. Knockout mice die from anemia at E15, but restoration of globin chain imbalance does not rescue anemia or increase survival. Cell lines derived from EKLF null mice undergo proliferation arrest upon reactivation of a conditional EKLF-ER fusion protein, suggesting a role in cell cycle control. A transcriptional profiling experiment comparing the global gene expression in EKLF null and wild type fetal liver identified many differentially expressed genes, a number of which function in G1 and at the G1/S checkpoint of the cell cycle. The Cyclin dependent kinase (Cdk) inhibitor, p18, and the S phase transcription factor E2F4 were both found to be significantly down regulated in EKLF null mice and this result was confirmed by real-time PCR. Interestingly, E2F4 knockout mice have a similar phenotype to EKLF knockout mice. Bioinformatic searches of the p18 and E2F4 genes shows that each contains phylogenetically conserved CACC box motifs capable of binding EKLF within longer regions of conservation in promoter and intron regions. The p18 gene contains two conserved CACCC sites upstream of the start of transcription, which are required for EKLF dependent promoter activity in luciferase reporter assays. The transcription factor E2F4 contains a conserved EKLF-binding CACC site within an intron that is closely associated with two conserved GATA1 binding sites. We show by a chromatin immunoprecipitation (ChIP) assays that the E2F4 intron and p18 promoter are occupied by EKLF in vivo. Together, these results suggest that EKLF is likely to directly regulate expression of key cell cycle genes in vivo to drive the switch from proliferation to differentiation of erythrocytes. The loss of EKLF is likely to result in aberrant proliferation and predisposition to leukemia.
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Drissen, Roy, Marieke von Lindern, Andrea Kolbus, Siska Driegen, Peter Steinlein, Hartmut Beug, Frank Grosveld, and Sjaak Philipsen. "The Erythroid Phenotype of EKLF-Null Mice: Defects in Hemoglobin Metabolism and Membrane Stability." Molecular and Cellular Biology 25, no. 12 (June 15, 2005): 5205–14. http://dx.doi.org/10.1128/mcb.25.12.5205-5214.2005.
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ABSTRACT Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Krüppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in β-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.
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Asano, Haruhiko, and George Stamatoyannopoulos. "Activation of β-Globin Promoter by Erythroid Krüppel-Like Factor." Molecular and Cellular Biology 18, no. 1 (January 1, 1998): 102–9. http://dx.doi.org/10.1128/mcb.18.1.102.
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ABSTRACT Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the β-globin gene CACCC box and is essential for β-globin gene expression. EKLF does not activate the γ gene, the CACCC sequence of which differs from that of the β gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the β gene by EKLF, the CACCC boxes of β and γ genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the β promoter carrying a γ CACCC box at a level comparable to that at which it activated the wild-type β promoter, whereas EKLF failed to activate a γ promoter carrying the β CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the β promoter carrying the γ CACCC box, or that EKLF activated the mutated β promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the β or γ promoter determined EKLF specificity, the proximal β CACCC box sequence was created at the position of the β promoter (−140) which corresponds to the position of the CACCC box on the γ promoter. Similarly, the β CACCC box was created in the position of the γ promoter (−90) corresponding to the position of the CACCC box in the β promoter. EKLF retained weak activation potential on the β−140CAC promoter, whereas EKLF failed to activate the γ−90βCAC promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the β promoter by EKLF is determined by the overall structure of the β promoter rather than solely by the sequence of the β gene CACCC box.
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Jansen, Valerie M., Shaji Ramachandran, Aurelie Desgardin, Jin He, Vishwas Parekh, Stephen M. Jane, and John M. Cunningham. "Context-Specific Roles for Erythroid Krüppel-Like Factor (EKLF) in Co-Ordinate High Level Expression of the Murine α- and β-Globin Genes." Blood 108, no. 11 (November 16, 2006): 365. http://dx.doi.org/10.1182/blood.v108.11.365.365.
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Abstract Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.
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Steiner, Laurie A., Vincent P. Schulz, Yelena Maksimova, Milind Mahajan, David M. Bodine, and Patrick G. Gallagher. "Dynamic CO-Localization of GATA1, NFE2, and EKLF and Changes in Gene Expression During Hematopoiesis." Blood 116, no. 21 (November 19, 2010): 741. http://dx.doi.org/10.1182/blood.v116.21.741.741.
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Abstract Abstract 741 Regulation of lineage choice during the development and differentiation of erythroid cells in hematopoiesis is a complex process. GATA1, NFE2, and EKLF are transcription factors critical for erythropoiesis. Focused studies, including detailed analyses of the human beta globin gene locus and a select group of erythrocyte membrane protein genes, have revealed that these three transcription factors may co-localize at common regulatory sites in erythroid-expressed genes. To address the hypothesis that GATA1, NFE2, and EKLF frequently co-localize on critical regulatory elements responsible for cell-type specific gene expression during erythropoiesis, chromatin immunoprecipitation coupled with ultrahigh throughput sequencing (ChIP-seq) was used to identify sites of GATA1, NFE2, and EKLF occupancy in human primary hematopoietic stem and progenitor cells (HSPCs) and human primary erythroid cells. ChIP was done using CD34+ HSPCs prepared by immunomagnetic bead selection and cultured CD71+/GPA+ erythroid cells (R3/R4 population) using antibodies against GATA1, NF-E2, and EKLF. The MACS algorithm (Zhang et al. Genome Biol, 2008) was used to identify regions of DNA-protein interaction, with a p-value ≤10e-5. Sites identified by MACS were ordered by p-value, and the 7000 sites with the most stringent p-values were selected for further analysis. Sites which occurred within 200bp of each other were treated as a single site. Unexpectedly, sites of GATA1, NFE2, and EKLF occupancy were common in HSPCs, with 6643 GATA1, 6657 NFE2, and 6579 EKLF sites identified, respectively. Sites identified in HSPCs were primarily in enhancers (>1kb from a RefSeq gene; 44% of GATA1, 49% of NFE2, and 51% of EKLF sites) and in introns (32% of GATA1, 34% of NFE2, and 34% of EKLF sites), with only a few sites at proximal promoters (within 1kb of a TSS; 7% of GATA1, 6% of NFE2, and 7% EKLF sites.) In erythroid cells, 6895 GATA1, 6907 NF-E2, and 6874 EKLF sites were identified. For all 3 factors, binding site occupancy varied greatly from that observed in HSPCs. Proximal promoter binding was much more common in erythroid cells than in HSPCs, with 19% of GATA1, 28% of NFE2 and 38% of EKLF sites found at promoters. Binding was frequently found at enhancers (41% of GATA, 38% NFE2, and 32% EKLF sites) and in introns (29% of GATA1, 26% of NFE2, and 21% of EKLF). To gain insight into three factor co-occupancy on a genome-wide scale, GATA1, EKLF, and NFE2 binding sites were compared using the Active Region Comparer (http://dart.gersteinlab. org/). Surprisingly, co-localization of all three factors was common in HSPCs, occurring at 2666 sites (40%, 40% and 45% of GATA1, NFE2, and EKLF sites). Sites of GATA1-NFE2-EKLF co-localization in HSPCs were located primarily at enhancers (51% of sites), in introns (32% of sites), and rarely at proximal promoters (6% of sites). In erythroid cells, co-localization of all three transcription factors was also common, occurring at 2445 sites (35%, 35%, and 36% of GATA1, NFE2, and EKLF sites, respectively). In contrast to HSPCs, sites of GATA1-NFE2-EKLF co-localization in erythroid cells were located primarily at proximal promoters (35% of sites) and enhancers (34% of sites), with co-localization in introns accounting for 20% of sites. A limited subset of sites, 1429 GATA1, 921 NFE2, and 1038 EKLF sites, were present in both HSPC and erythroid cells. Throughout the genome, there were only 233 sites of three factor co-localization in common in both HSPC and erythroid cells. Gene expression in HSPC and erythroid cell was analyzed via RNA hybridization to Illumina HumanHT-12 v3 Expression BeadChip arrays. In erythroid cells, genes with GATA1-NFE2-EKLF co-localization from 5kb upstream to 2kb downstream had significantly higher levels of mRNA expression than genes without GATA1-NFE2-EKLF co-localization (p<2.2e-16). The reverse was observed in HSPCs, where genes with GATA1-NFE2-EKLF co-localization had significantly lower levels of mRNA expression than genes without GATA1-NFE2-EKLF co-localization (p<7.3e-05). These data support the hypothesis that co-localization of GATA1, NFE2, and EKLF is a common finding in hematopoietic cells. Significant differences in factor co-localization and gene expression in HSPC and erythroid cells suggest that this coordinated binding orchestrates different patterns of gene expression during hematopoiesis. Disclosures: No relevant conflicts of interest to declare.
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Perkins, A. C., K. R. Peterson, G. Stamatoyannopoulos, H. E. Witkowska, and S. H. Orkin. "Fetal expression of a human Aγ globin transgene rescues globin chain imbalance but not hemolysis in EKLF null mouse embryos." Blood 95, no. 5 (March 1, 2000): 1827–33. http://dx.doi.org/10.1182/blood.v95.5.1827.004k10_1827_1833.
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Mice lacking the erythroid Kruppel-like factor (EKLF) die in utero at embryonic day 15 (E15) from severe anemia. EKLF−/− embryos display a marked deficit in β-globin gene expression. To test whether β-globin deficiency was solely responsible for the anemia and intrauterine death, we corrected the globin chain imbalance in EKLF−/− embryos by breeding with a strain of mice that express high levels of human γ-globin. Despite efficient production of hybrid m2-hγ2 hemoglobin in the fetal livers of EKLF−/− animals, hemolysis was not corrected and survival was not prolonged. We concluded that deficiency of nonglobin EKLF target genes is a major contributor to the definitive red blood cell abnormalities and prenatal death in EKLF−/−embryos. These results suggest that strategies designed to antagonize EKLF function in adults with hemoglobinopathy, in an attempt to reactivate γ-globin gene expression, may adversely affect other essential aspects of red blood cell physiology.
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Hung, Chun-Hao, Keh-Yang Wang, Yae-Huei Liou, Jing-Ping Wang, Anna Yu-Szu Huang, Tung-Liang Lee, Si-Tse Jiang, Nah-Shih Liao, Yu-Chiau Shyu, and Che-Kun James Shen. "Negative Regulation of the Differentiation of Flk2− CD34− LSK Hematopoietic Stem Cells by EKLF/KLF1." International Journal of Molecular Sciences 21, no. 22 (November 10, 2020): 8448. http://dx.doi.org/10.3390/ijms21228448.
Full textAbstract:
Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2− CD34− Lin− Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2− CD34− LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2− CD34− LSK-HSCs.
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Gregory, RC, DJ Taxman, D. Seshasayee, MH Kensinger, JJ Bieker, and DM Wojchowski. "Functional interaction of GATA1 with erythroid Kruppel-like factor and Sp1 at defined erythroid promoters." Blood 87, no. 5 (March 1, 1996): 1793–801. http://dx.doi.org/10.1182/blood.v87.5.1793.1793.
Full textAbstract:
Abstract GATA and CACC elements commonly are codistributed within the regulatory domains of a variety of erythroid genes. Using Drosophila S2 cells, the actions of GATA1, Sp1, and erythroid Kruppel-like factor (EKLF) at these elements within model erythroid promoters have been assessed. For each promoter studied (erythroid pyruvate kinase, glycophorin B, and a murine betamaj globin-derived construct, GCT) Sp1 and EKLF each activated transcription despite differences in CACC element sequence, orientation, and positioning. However, GATA1 acted in apparent cooperativity with Sp1 at the pyruvate kinase promoter; with EKLF at the betamaj globin-derived GCT promoter; and with either Sp1 or EKLF at the glycophorin B promoter. Thus, GATA1 may functionally interact with each of these Kruppel-like factors depending on promoter context; and at the GCT promoter, transcriptional activation by GATA1 and EKLF was > or = 10-fold higher than levels attributable to additive effects. The possibility that interactions between these activators may be direct was supported by the specific binding of baculoviral-expressed EKLF to GATA1. This report underlines the likelihood that discrete roles exist for Sp1 and EKLF in erythroid gene activation, and supports a mechanism of direct cooperativity for EKLF and GATA1 as coregulators.
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Gregory, RC, DJ Taxman, D. Seshasayee, MH Kensinger, JJ Bieker, and DM Wojchowski. "Functional interaction of GATA1 with erythroid Kruppel-like factor and Sp1 at defined erythroid promoters." Blood 87, no. 5 (March 1, 1996): 1793–801. http://dx.doi.org/10.1182/blood.v87.5.1793.bloodjournal8751793.
Full textAbstract:
GATA and CACC elements commonly are codistributed within the regulatory domains of a variety of erythroid genes. Using Drosophila S2 cells, the actions of GATA1, Sp1, and erythroid Kruppel-like factor (EKLF) at these elements within model erythroid promoters have been assessed. For each promoter studied (erythroid pyruvate kinase, glycophorin B, and a murine betamaj globin-derived construct, GCT) Sp1 and EKLF each activated transcription despite differences in CACC element sequence, orientation, and positioning. However, GATA1 acted in apparent cooperativity with Sp1 at the pyruvate kinase promoter; with EKLF at the betamaj globin-derived GCT promoter; and with either Sp1 or EKLF at the glycophorin B promoter. Thus, GATA1 may functionally interact with each of these Kruppel-like factors depending on promoter context; and at the GCT promoter, transcriptional activation by GATA1 and EKLF was > or = 10-fold higher than levels attributable to additive effects. The possibility that interactions between these activators may be direct was supported by the specific binding of baculoviral-expressed EKLF to GATA1. This report underlines the likelihood that discrete roles exist for Sp1 and EKLF in erythroid gene activation, and supports a mechanism of direct cooperativity for EKLF and GATA1 as coregulators.
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Sengupta, Tanushri, Ken Chen, Eric Milot, and James J. Bieker. "Acetylation of EKLF Is Essential for Epigenetic Modification and Transcriptional Activation of the β-Globin Locus." Molecular and Cellular Biology 28, no. 20 (August 18, 2008): 6160–70. http://dx.doi.org/10.1128/mcb.00919-08.
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ABSTRACT Posttranslational modifications of transcription factors provide alternate protein interaction platforms that lead to varied downstream effects. We have investigated how the acetylation of EKLF plays a role in its ability to alter the β-like globin locus chromatin structure and activate transcription of the adult β-globin gene. By establishing an EKLF-null erythroid line whose closed β-locus chromatin structure and silent β-globin gene status can be rescued by retroviral infection of EKLF, we demonstrate the importance of EKLF acetylation at lysine 288 in the recruitment of CBP to the locus, modification of histone H3, occupancy by EKLF, opening of the chromatin structure, and transcription of adult β-globin. We also find that EKLF helps to coordinate this process by the specific association of its zinc finger domain with the histone H3 amino terminus. Although EKLF interacts equally well with H3.1 and H3.3, we find that only H3.3 is enriched at the adult β-globin promoter. These data emphasize the critical nature of lysine acetylation in transcription factor activity and enable us to propose a model of how modified EKLF integrates coactivators, chromatin remodelers, and nucleosomal components to alter epigenetic chromatin structure and stimulate transcription.
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Miller, I. J., and J. J. Bieker. "A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins." Molecular and Cellular Biology 13, no. 5 (May 1993): 2776–86. http://dx.doi.org/10.1128/mcb.13.5.2776-2786.1993.
Full textAbstract:
We describe a novel erythroid cell-specific cDNA (EKLF [erythroid Krüppel-like factor]) isolated by enriching for genes expressed in a mouse erythroleukemia cell line but not expressed in a mouse monocyte-macrophage cell line. The complete cDNA sequence is predicted to encode a protein of approximately 38,000 Da that contains a proline-rich amino domain and three TFIIIA-like zinc fingers within the carboxy domain. Additional sequence analyses reveal that the EKLF zinc fingers are most homologous to the Krüppel family of transcription factors and also allow us to predict potential DNA-binding target sites for the EKLF protein. On the basis of this prediction, we show that EKLF is able to bind the sequence CCA CAC CCT, an essential element of the beta-globin promoter. Its tissue distribution establishes that the EKLF transcript is expressed only in bone marrow and spleen, the two hematopoietic organs of the mouse, and analysis of murine cell lines indicates that EKLF expression is limited to erythroid and mast cell lines. Cotransfection assays establish that EKLF transcriptionally activates a target promoter that contains its DNA-binding site. The tissue expression pattern of EKLF, in conjunction with its function as a transcriptional activator, strongly suggests that the EKLF protein may be intimately involved in establishment and/or maintenance of the erythroid cell phenotype.
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Miller, I. J., and J. J. Bieker. "A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins." Molecular and Cellular Biology 13, no. 5 (May 1993): 2776–86. http://dx.doi.org/10.1128/mcb.13.5.2776.
Full textAbstract:
We describe a novel erythroid cell-specific cDNA (EKLF [erythroid Krüppel-like factor]) isolated by enriching for genes expressed in a mouse erythroleukemia cell line but not expressed in a mouse monocyte-macrophage cell line. The complete cDNA sequence is predicted to encode a protein of approximately 38,000 Da that contains a proline-rich amino domain and three TFIIIA-like zinc fingers within the carboxy domain. Additional sequence analyses reveal that the EKLF zinc fingers are most homologous to the Krüppel family of transcription factors and also allow us to predict potential DNA-binding target sites for the EKLF protein. On the basis of this prediction, we show that EKLF is able to bind the sequence CCA CAC CCT, an essential element of the beta-globin promoter. Its tissue distribution establishes that the EKLF transcript is expressed only in bone marrow and spleen, the two hematopoietic organs of the mouse, and analysis of murine cell lines indicates that EKLF expression is limited to erythroid and mast cell lines. Cotransfection assays establish that EKLF transcriptionally activates a target promoter that contains its DNA-binding site. The tissue expression pattern of EKLF, in conjunction with its function as a transcriptional activator, strongly suggests that the EKLF protein may be intimately involved in establishment and/or maintenance of the erythroid cell phenotype.
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Mukherjee, Kaustav, James J. Bieker, and Venkata Srinivas Mohan Nimai Dangeti. "EKLF/Klf1 Regulates Erythroid Transcription By Its Pioneering Activity and Subsequent Control of RNA Pol II Pause-Release." Blood 138, Supplement 1 (November 5, 2021): 283. http://dx.doi.org/10.1182/blood-2021-151470.
Full textAbstract:
Abstract EKLF/Klf1 is a master transcriptional activator of critical genes that regulate both erythroid fate specification and terminal erythroid maturation. EKLF binds to DNA using three Zn-fingers at its C-terminus while the N-terminus constitutes a transcription activation domain (TAD) that interacts with various transcription co-factors including the protein acetylase CBP. An autosomal semi-dominant mutation at a single residue (E339D) in the mouse EKLF Zn-finger leads to Neonatal anemia (Nan). A mutation at the same residue in human EKLF (E325K) causes Congenital Dyserythropoietic Anemia type IV (CDA IV). Nan/Nan mice show lethality at embryonic day E10-11, in contrast to EKLF-/- homozygotes that survive until E15. Nan/+ heterozygotes survive to adulthood but are severely anemic, unlike EKLF+/- heterozygotes that display no aberrant phenotypes. The Nan-EKLF protein has an altered DNA binding specificity leading to a vastly altered transcriptome by two mechanisms. First, Nan-EKLF binding causes ectopic gene expression that significantly contributes to the severe anemia in Nan/+. Second, a subset of EKLF targets is downregulated in heterozygous Nan/+ mutants despite the presence of one copy of wild type EKLF, exacerbating the anemia. Thus, uncovering the mechanism by which gene expression is altered in Nan/+ may illuminate how EKLF normally activates transcription of its targets in vivo. To this end, we first examined the global occupancy of RNA Pol II phospho-Ser5 (as a paused mark) and phospho-Ser2 (as an elongation mark) in the mouse E13.5 fetal liver as a source of primary definitive erythroid cells. At promoters of ectopically expressed genes, where only Nan-EKLF (but not WT) binding is expected, we predominantly find increased levels of both paused and elongating RNA Pol II suggesting that Nan-EKLF binding activates transcription at ectopic genes by RNA Pol II recruitment and promoter proximal pausing. Further, we find increased levels of H3K27ac and CBP occupancy at these sites indicating that the mechanism of Pol II recruitment relies on CBP-mediated H3K27 acetylation and increased chromatin accessibility. Overall, this suggests robust pioneering activity of Nan-EKLF likely mediated by the interaction of its TAD with the CBP/p300 acetylase complex. At genes downregulated in Nan/+ we find two major patterns of Pol II occupancy. One is the converse of that seen at ectopic genes wherein there is a concomitant decrease in both Pol II p-Ser5 and p-Ser2 levels, along with lower H3K27ac and CBP levels suggesting EKLF gene activation has been lost at these sites in Nan/+. This includes cell cycle EKLF targets such as E2f2 and Rgcc. The second set of genes have comparable levels of p-Ser5 (paused) Pol II in Nan/+ and WT, but lower levels of p-Ser2 (elongating) Pol II in Nan/+. This suggests that although Pol II is being recruited to the TSS and pauses effectively, the pause-release step leading to effective transcription elongation is impaired. This subset includes important EKLF targets such as Bcl11a, Pax7, Xpo7, and several membrane transporters. As expected, CBP and H3K27ac levels are similar in WT and Nan/+ at these sites. To determine the cause of impaired RNA Pol II pause-release we examined the global occupancies of key transcription elongation factors such as P-TEFb and NELF. We find that levels of NELF, a negative elongation factor, remain unchanged in WT and Nan/+. However, levels of the P-TEFb subunit Cdk9, a positive elongation factor that facilitates release of paused RNA Pol II, is significantly lower at the TSS of these genes in Nan/+. This suggests that in Nan/+, possible reduction or loss of EKLF binding at some EKLF target promoters impairs effective recruitment of positive transcription elongation factors, resulting in a failure to efficiently release paused RNA Pol II. This causes downregulation of these EKLF target genes and contributes to the severe anemic phenotypes of the Nan mouse. We conclude that: EKLF exhibits expression control of its target genes at both the transcriptional initiation and elongation steps in vivo; EKLF can act as a pioneer transcription factor and increase chromatin accessibility through H3K27 acetylation by CBP leading to recruitment and pausing of RNA Pol II; and EKLF recruits the positive transcription elongation complex P-TEFb, enabling the controlled release of paused RNA Pol II at transcription start sites of a select group of its targets. Disclosures No relevant conflicts of interest to declare.
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Pilon, Andre M., Subramanian S. Ajay, Hatice Ozel Abaan, Elliott H. Margulies, Patrick G. Gallagher, and David M. Bodine. "Genome-Wide ChIP-Seq Reveals a Dramatic Shift in the EKLF Binding Profile Between Erythroid Progenitors and Erythroblasts." Blood 114, no. 22 (November 20, 2009): 565. http://dx.doi.org/10.1182/blood.v114.22.565.565.
Full textAbstract:
Abstract Abstract 565 Erythroid Kruppel-Like Factor (EKLF; KLF1) is the founding member of the Kruppel family of C2H2 zinc finger transcription factors. First identified as an activator of the beta-globin locus, EKLF facilitates chromatin remodeling and transcriptional activation of target genes, at least in part through recognition of a 9-base consensus motif (NCNCNCCCN). By comparing the transcriptional profiles of E13.5 wild type and Eklf-/- mice, we demonstrated that the lethal failure to complete definitive erythropoiesis in the fetal liver (FL) was due in part to dysregulation of an EKLF target gene, the cell cycle control factor, E2F2 (Pilon et al. 2008). To identify further direct targets of EKLF activation that affect erythropoiesis, we are coupling chromatin immunoprecipitation with ultra high-throughput massively parallel sequencing (ChIP-seq). ChIP-seq is increasingly being used to map protein-DNA interactions in vivo, allowing simultaneous genome-wide analysis of transcription factor occupancy, defining an ‘interactome‘. Using mice whose endogenous Eklf gene was replaced with a fully functional HA-tagged form of EKLF, chromatin was isolated at E13.5 from immature erythroid progenitors and maturing erythroblasts by ChIP. Using a highly specific high-affinity anti-HA antibody, libraries of HA-EKLF-bound chromatin were subjected to fluorescent in situ sequencing on a Solexa 1G platform, providing 36-base signature tags that were mapped to the mouse genome using the Eland software package. A control library was derived from E13.5 FL chromatin that was not enriched for HA-EKLF occupancy. For both progenitors and erythroblasts, >1.1×107tags were obtained. 72.5% and 78.7% of progenitor and erythroblast tags mapped to unique sites within the genome, respectively. The tags were highly enriched in the ∼10% of the genome within genes (genic; 42% of tags), sites ≤10 kb from the nearest gene (adjacent; 15%), as opposed to the ∼90% of the genome that is >10 kb from the nearest gene (intergenic; 22%) or in repetitive DNA (21%) p=2.2 ×10-16. Using the MACS software package clustered peaks of EKLF occupancy were identified throughout the genome, defining the EKLF ‘interactome‘. The vast majority of peaks were mapped to non-repetitive regions of the genome (98% in progenitors; 95% in erythroblasts). Progenitors contained 4,383 peaks of EKLF occupancy, while erythroblasts contained 15,396 peaks. Only 100 peaks were common between populations. This >3.5-fold increase in genomic EKLF occupancy between progenitors and erythroblasts (p=1×10-5) reflects the shift in the expression and activity of EKLF protein in erythropoiesis described previously (Bouilloux et al. 2008; Lohmann & Bieker 2008). To identify potential EKLF target genes, we partitioned the genome into 3 categories, relative to annotated RefSeq coordinates (genic) as well as adjacent and intergenic. In progenitors, the majority of EKLF binding (54%) occurred in intergenic regions, with a minority within (38%) or adjacent (7%) to genes. By contrast, the EKLF binding profile in erythroblasts was reversed, with 62% of the peaks in genic regions, and a minority at intergenic (26%) or adjacent (12%) sites.To assess the effect of this shift in EKLF binding on gene transcription, we used publicly availabel data from the inducible G1E model of erythroid maturation (GEO: GSE628) to correlate our ChIP-seq data with mRNA expression. Informatic analyses using MetaCore demonstrated that >2,200 EKLF-associated genes were differentially expressed during maturation (949 increasing expression; 1,298 decreasing expression, all p<0.05). Among progenitors, control of cell cycle S-phase entry and progression was a significantly represented network, highlighted by focal EKLF target genes like Cdk2, Cdk4, and p107, in agreement with our previous observations. Among erythroblasts, the erythropoietin (Epo) signaling pathway was most significantly represented, highlighted by focal EKLF target genes like Stat3 and Bcl-XL, reflecting the well-established importance of the Epo axis for erythroblast survival. These data indicate that shifts in EKLF occupancy during erythropoiesis correlate with distinct functional effects on gene expression. Further, these observations support a model in which transcriptional regulators (e.g., EKLF) may collect at intergenic locations when their activity is not required, but where they remain poised for rapid recruitment. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Wenjun, Shilpa Kadam, Beverly M. Emerson, and James J. Bieker. "Site-Specific Acetylation by p300 or CREB Binding Protein Regulates Erythroid Krüppel-Like Factor Transcriptional Activity via Its Interaction with the SWI-SNF Complex." Molecular and Cellular Biology 21, no. 7 (April 1, 2001): 2413–22. http://dx.doi.org/10.1128/mcb.21.7.2413-2422.2001.
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ABSTRACT Recruitment of modifiers and remodelers to specific DNA sites within chromatin plays a critical role in controlling gene expression. The study of globin gene regulation provides a convergence point within which to address these issues in the context of tissue-specific and developmentally regulated expression. In this regard, erythroid Krüppel-like factor (EKLF) is critical. EKLF is a red cell-specific activator whose presence is crucial for establishment of the correct chromatin structure and high-level transcriptional induction of adult β-globin. We now find, by metabolic labeling-immunoprecipitation experiments, that EKLF is acetylated in the erythroid cell. EKLF residues acetylated by CREB binding protein (CBP) in vitro map to Lys-288 in its transactivation domain and Lys-302 in its zinc finger domain. Although site-specific DNA binding by EKLF is unaffected by the acetylation status of either of these lysines, directed mutagenesis of Lys-288 (but not Lys-302) decreases the ability of EKLF to transactivate the β-globin promoter in vivo and renders it unable to be superactivated by coexpressed p300 or CBP. In addition, the acetyltransferase function of CBP or p300 is required for superactivation of wild-type EKLF. Finally, acetylated EKLF has a higher affinity for the SWI-SNF chromatin remodeling complex and is a more potent transcriptional activator of chromatin-assembled templates in vitro. These results demonstrate that the acetylation status of EKLF is critical for its optimal activity and suggest a mechanism by which EKLF acts as an integrator of remodeling and transcriptional components to alter chromatin structure and induce adult β-globin expression within the β-like globin cluster.
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Isern, Joan, Stuart T. Fraser, Zhiyong He, Hailan Zhang, and Margaret H. Baron. "Dose-dependent regulation of primitive erythroid maturation and identity by the transcription factor Eklf." Blood 116, no. 19 (November 11, 2010): 3972–80. http://dx.doi.org/10.1182/blood-2010-04-281196.
Full textAbstract:
Abstract The primitive erythroid (EryP) lineage is the first to differentiate during mammalian embryogenesis. Eklf/Klf1 is a transcriptional regulator that is essential for definitive erythropoiesis in the fetal liver. Dissection of the role(s) of Eklf within the EryP compartment has been confounded by the simultaneous presence of EryP and fetal liver–derived definitive erythroid (EryD) cells in the blood. To address this problem, we have distinguished EryP from their definitive counterparts by crossing Eklf+/− mutant and ϵ-globin::histone H2B-GFP transgenic mice. Eklf-deficient EryP exhibit membrane ruffling and a failure to acquire the typical discoidal erythroid shape but they can enucleate. Flow cytometric analyses of H2B-GFP+ EryP revealed that Eklf heterozygosity results in the loss of Ter119 surface expression on EryP but not on EryD. Null mutation of Eklf resulted in abnormal expression of a range of surface proteins by EryP. In particular, several megakaryocyte markers were ectopically expressed by maturing Eklf-null EryP. Unexpectedly, the platelet tetraspanin CD9 was detected on nucleated wild-type EryP but not on mature EryD and thus provides a useful marker for purifying circulating EryP. We conclude that Eklf gene dosage is crucial for regulating the surface phenotype and molecular identity of maturing primitive erythroid cells.
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Funnell, Alister P. W., Christopher A. Maloney, Lucinda J. Thompson, Janelle Keys, Michael Tallack, Andrew C. Perkins, and Merlin Crossley. "Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells." Molecular and Cellular Biology 27, no. 7 (February 5, 2007): 2777–90. http://dx.doi.org/10.1128/mcb.01658-06.
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ABSTRACT The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.
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Pilon, Andre M., Jacqueline Beaupre, James J. Bieker, Patrick G. Gallagher, and David M. Bodine. "Multiple Defects of Both Primitive and Definitive Erythrocytes in EKLF-Deficient Mice." Blood 110, no. 11 (November 16, 2007): 1234. http://dx.doi.org/10.1182/blood.v110.11.1234.1234.
Full textAbstract:
Abstract Erythroid Krüppel-Like Factor (EKLF) is the founding member of the mammalian Krüppel subfamily of transcription factors with 3 C2H2-type zinc fingers. In mice, loss of EKLF is lethal at day 14 of gestation (dE14), due to severe anemia. To study the physiological properties of the mixed population of circulating primitive and definitive erythrocytes in dE13.5 mice, we developed an osmotic fragility assay that compares the relative amount of embryonic (primitive) and adult (definitive) hemoglobin in the supernatant and pellet of cells exposed to increasing concentrations of NaCl. We found that both primitive and definitive EKLF-deficient cells were resistant to osmotic stress, consistent with the thalassemia-like phenotype of the cells. Flow cytometric analysis of WT dE13.5 fetal liver (FL) cells with TER119 and CD71 identified 5 previously described populations: R1+R2, composed of pro- and basophilic normoblasts, and R3+R4+R5, composed of more mature polychromatic and orthochromatic normoblasts and reticulocytes, respectively. Analysis of dE13.5 EKLF-deficient FL cells showed that R3+R4+R5 were absent, and morphological examination demonstrated that R1 and R2 were homogeneous populations of pro- and basophilic normoblasts. Colony-forming assays revealed an increase in the absolute number of BFU-E and CFU-E in EKLF-deficient R1 and R2 FL cells. Although the EKLF-deficient colonies required 24–72 more hours of culture than WT colonies to mature, they were composed of definitive erythroid cells as they expressed only adult β-globin. EKLF-deficient FL also contained 2-fold more CFU-Meg colonies (p<0.01). From these data we concluded that the lack of R3+R4+R5 cells in EKLF-deficient FL is due to a block in erythroid differentiation. We compared mRNA from sorted R1+R2 WT and EKLF-deficient FL cells by microarray and used Ingenuity Pathways Analysis (IPA) to demonstrate that the levels of mRNAs encoding proteins involved in the cell cycle and DNA replication were significantly altered. Cell cycle analyses showed that EKLF-deficient R1+R2 cells are significantly delayed exiting G0/G1 (p<0.002). A central node in the cell cycle network was E2F2, a transcription factor involved in cell cycle progression and differentiation. E2F2 mRNA was decreased to 7.6±1.6% of WT levels in EKLF-deficient FL, with a comparable reduction in protein. We hypothesized that E2F2 is a direct EKLF target gene, and examined the chromatin of the E2F2 locus in a high throughput DNase-I hypersensitive site (HS) assay. The E2F2 locus is sensitive to DNase I in WT FL cells and resistant to DNase I in EKLF-deficient FL cells. Three EKLF-dependent HS were identified: in the promoter, intron 2, and the 3′ untranslated region. We used chromatin immunoprecipitation to show that EKLF associates with consensus DNA-binding sites in the E2F2 promoter HS. IPA also demonstrated that apoptosis was abnormal in EKLF-deficient R1+R2 cells. Flow cytometry with annexin V staining demonstrated that EKLF-deficient R1+R2 cells were resistant to apoptosis (p<0.01), consistent with the severe anemia, indicating that apoptosis was not contributing to the differentiation block. Our results support a model in which EKLF-deficiency leads to a block in definitive erythroid maturation associated with decreased cell cycling, resulting in the limited production of dehydrated erythrocytes.
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Singleton, Belinda K., Victoria SS Fairweather, Winnie Lau, Stephen F. Parsons, Nicholas M. Burton, Jan Frayne, R. Leo Brady, and David J. Anstee. "A Novel EKLF Mutation in a Patient with Dyserythropoietic Anemia: The First Association of EKLF with Disease in Man." Blood 114, no. 22 (November 20, 2009): 162. http://dx.doi.org/10.1182/blood.v114.22.162.162.
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Abstract Abstract 162 We describe a single-point mutation in the transcription factor EKLF associated with dyserythropoietic anemia. The female Danish patient was extensively studied in the early 1990's (Wickramasinghe et al. Br J Haem 1991,79:322; Tang et al. Blood 1993,81:1636; Parsons et al. Blood 1994,83:860; Agre et al. J Clin Invest 1994,94:1050). The patient was severely anemic at birth and required repeated transfusions during childhood. Notable features included persistent expression of epsilon and zeta embryonic globins, an HbF level of 40%, novel intra-erythroblastic and intra-erythrocytic inclusions and deficiency of erythroid proteins CD44 and Aquaporin 1. Evidence that EKLF plays a major role in globin gene regulation in particular and erythroid gene expression in general led us to examine EKLF in this patient, her unaffected sister, and her parents. The coding sequence of EKLF was normal wild-type in both healthy parents and sister, but the patient had a mutation on one allele of EKLF in the second zinc finger domain (Glu325Lys). This mutation is at a site that is central to the expected site of interaction of EKLF with DNA. However, modelling revealed that reversal of the charge at this position in a critical DNA-binding domain was likely to create a novel direct contact between Lys325 and a phosphate on the DNA backbone, hence maintaining rather than disrupting binding of EKLF to promoter regions. To test this, we created recombinant forms of the EKLF zinc finger domain, corresponding to the wild-type, Glu325Lys and 3 presumed loss-of-function mutations previously associated with the rare blood group In(Lu) phenotype (Singleton et al. Blood 2008,112:2081). Using fluorescence-based binding assays, mutant EKLF proteins Arg328Leu, Arg328His, and Arg331Gly showed virtually no binding to the beta globin (HBB) promoter sequence, as expected. In contrast, there was no significant difference in the binding of EKLF Glu325Lys and wild-type EKLF to the promoter sequence. We then transfected full-length wild-type and mutated EKLF into K562 cells and measured the effect on expression of several genes by quantitative real-time PCR. Transfectants with wild-type EKLF showed an average 13.7 fold increase in EKLF mRNA expression (SD 4.8, n=5) compared with a clone transfected with the empty vector. This was associated with an elevation in HBB and CD44H mRNA expression (average 12.8 (SD 10.9, n=5) and 27.0 (SD 21.7, n=5) fold respectively, compared with the empty vector clone). In contrast, transfectants with EKLF Glu325Lys, although expressing slightly lower levels of EKLF mRNA than the wild-type clones (average 8.0 fold compared with the empty vector clone, SD 5.2, n=11), showed much reduced HBB and CD44H expression (average 1.9 (SD 1.6, n=11) and 1.4 (SD 1.4, n=11) fold respectively, compared with the empty vector clone). Our findings indicate that EKLF Glu325Lys has a reduced ability to activate HBB and CD44H expression, thus establishing a link between the mutation and the patient's phenotype. This reduction, however, does not appear to be explained by differences in the ability of the mutant EKLF to bind to the HBB promoter, implying that other mechanisms of gene regulation must be affected in the patient. Disclosures: No relevant conflicts of interest to declare.
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Zhu, Jianqiong, Kyung Chin, Wulin Aerbajinai, Chutima Kumkhaek, Hongzhen Li, Matthew M. Hsieh, Courtney D. Fitzhugh, and Griffin P. Rodgers. "A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease." Blood 128, no. 22 (December 2, 2016): 3506. http://dx.doi.org/10.1182/blood.v128.22.3506.3506.
Full textAbstract:
Abstract Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor's blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.
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Starck, Joëlle, Nathalie Cohet, Colette Gonnet, Sandrine Sarrazin, Zina Doubeikovskaia, Alexandre Doubeikovski, Alexis Verger, Martine Duterque-Coquillaud, and François Morle. "Functional Cross-Antagonism between Transcription Factors FLI-1 and EKLF." Molecular and Cellular Biology 23, no. 4 (February 15, 2003): 1390–402. http://dx.doi.org/10.1128/mcb.23.4.1390-1402.2003.
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ABSTRACT FLI-1 is an ETS family transcription factor which is overexpressed in Friend erythroleukemia and contributes to the blockage of differentiation of erythroleukemic cells. We show here that FLI-1 represses the transcriptional activity of the β-globin gene promoter in MEL cells and interacts with two of its critical transactivators, GATA-1 and EKLF. Unexpectedly, FLI-1 enhances the stimulating activity of GATA-1 on a GATA-1-responsive promoter but represses that of EKLF on β-globin and an EKLF-responsive artificial promoters. This repressive effect of FLI-1 requires the ETS DNA binding domain and its association with either the N- or C-terminal domain, which themselves interact with EKLF but not with GATA-1. Furthermore, the FLI-1 ETS domain alone behaves as an autonomous repression domain when linked to the Gal4 DNA binding domain. Taken together, these data indicate that FLI-1 represses EKLF-dependent transcription due to the repression activity of its ETS domain and its indirect recruitment to erythroid promoters by protein-protein interaction with EKLF. Reciprocally, we also show that EKLF itself represses the FLI-1-dependent megakaryocytic GPIX gene promoter, thus further suggesting that functional cross-antagonism between FLI-1 and EKLF might be involved in the control of the erythrocytic versus megakaryocytic differentiation of bipotential progenitors.
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Hung, Chun-Hao, Tung-Liang Lee, Anna Yu-Szu Huang, Kang-Chung Yang, Yu-Chiau Shyu, Shau-Ching Wen, Mu-Jie Lu, Shinsheng Yuan, and Che-Kun James Shen. "A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis." International Journal of Molecular Sciences 22, no. 15 (July 27, 2021): 8024. http://dx.doi.org/10.3390/ijms22158024.
Full textAbstract:
The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf−/−) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.
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Pilon, Andre M., Murat O. Arcasoy, Serena E. Vayda, Holly K. Dressman, James J. Bieker, David M. Bodine, and Patrick G. Gallagher. "Defects in E2F1/2 Expression Are Associated with Abnormalities in Cell Cycle and Differentiation in EKLF-Deficient Erythroid Cells." Blood 108, no. 11 (November 16, 2006): 84. http://dx.doi.org/10.1182/blood.v108.11.84.84.
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Abstract Mice deficient in the erythroid transcription factor EKLF die ~dE14 from severe anemia, attributed to decreased β-globin expression. Recent reports using microarray analyses indicate that expression of numerous genes is perturbed in erythroid cells lacking EKLF. We performed flow cytometry of WT dE14 fetal liver (FL) cells with TER119 and CD71 and identified 5 previously described populations: R1+R2 composed of BFU-E and CFU-E, respectively, and R3+R4+R5 composed of more mature erythroblasts. The same analysis of dE14 EKLF-deficient FL cells showed R3+R4+R5 were absent, indicating a block in erythroid differentiation. This differentiation block introduced a bias into the previous microarray data, as EKLF-deficient FL contain only immature erythroid cells (R1+R2), while WT FL cells are predominantly more differentiated (R3+R4+R5). Thus, expression of many genes was decreased due to a loss of more mature erythroblasts, rather than due to the action of EKLF or an intermediary. To obtain more rigorous comparative data, we performed microarray analyses with EKLF-deficient FL cells and R1+R2 populations from WT FL cells. Expression of numerous genes was deregulated. Of note, many genes significantly down regulated in previous microarray analyses were not down regulated when the 2 similar populations were compared. Ingenuity Pathway Analysis of the microarray data identified a biologic network involved in cell cycle and DNA replication. At the central nodes of the network were E2F1 and E2F2, transcription factors involved in cell cycle control and differentiation. In quantitative RT-PCR, E2F1 and E2F2 expression in EKLF-deficient mRNA was decreased to 40.5±1.6 and 7.6±1.6% of WT, respectively. Western blot analysis demonstrated comparable reductions in both E2F proteins. Cell cycle analyses showed that EKLF-deficient R1 cells exhibit a significant delay exiting G0+G1 and entering S phase (p<0.001). Both R1 and R2 cells exhibited a defect exiting S and entering G2+M (p<0.004). Colony forming assays revealed that EKLF-deficient FL cells had decreased frequency of BFU-E in R1 (p<0.005), with a defect in ability to generate CFU-E, and decreased frequency of CFU-E in R2 (p<0.001) with a defect in ability to differentiate into more mature erythroblasts. Flow cytometry with annexin V staining revealed that EKLF-deficient cells were resistant to apoptosis, indicating apoptosis was not contributing to the block. Based on these data, we hypothesized that E2F1/2 were EKLF target genes. Thus we examined the chromatin at the E2F2 locus in WT and EKLF-deficient FL cells using a high throughput assay to identify DNase-I hypersensitive sites (HS). Three HS were identified in WT that were absent in EKLF-deficient chromatin, one in the promoter region, one in intron 2, and one in the 3′ region. The E2F2 promoter HS region contains 2 EKLF consensus binding sites, which were examined in gel mobility shift assays for their ability to bind EKLF. One of these probes yielded a complex that co-migrated with a control β-globin probe complex. This complex was competed by an excess of E2F2 or β-globin probe and supershifted with an anti-EKLF MoAb. These results support the hypothesis that defects in E2F1/2 expression are associated with abnormalities in cell cycle and differentiation, contributing to a failure of definitive erythropoiesis in EKLF-deficient mice.
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Zhu, Jianqiong, Hongzhen Li, Wulin Aerbajinai, Chutima Kumkhaek, Kyung Chin, and Griffin Rodgers. "A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Cultured Mouse Erythrocytes." Blood 132, Supplement 1 (November 29, 2018): 3479. http://dx.doi.org/10.1182/blood-2018-99-115209.
Full textAbstract:
Abstract β-hemoglobinopathies are inherited disorders caused by mutations/deletions in the β-globin chain that lead to structurally defective β-globin chains or reduced (or absent) β-globin chain production. These diseases affect multiple organs and are associated with considerable morbidity and mortality, representing a major public health challenge. Current gene therapy approaches for the treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in human CD34+ cells. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from mouse hematopoietic progenitor cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 1.8-fold, δ-globin gene expression 3.3-fold, and γ-globin gene expression 1.7-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.6-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2 was increased from 2.1 % in vector-transduced cells to 8.9% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 6.3% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 30.6% in medium-form Eklf-GATA-transduced cells as compared with 40.7% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. ChIP-sequencing analysis has demonstrated that Eklf-GATA1 fusion proteins and GATA1 having a similar protein-DNA binding pattern at a global level. Our results have found that the long form Eklf-GATA1 fusion protein has a major effect on δ-globin induction than β-globin; the medium form Eklf-GATA1 is able to elevate δ-globin expression without having an effect on β-globin expression. The above findings indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.
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Jansen, Valerie M., Tatiana Abramova, Eun-Hee Shim, Shaji Ramachandran, Aurelie Desgardin, Vishwas Parekh, Stephen Jane, and John M. Cunningham. "Altered Erythroid and Megakaryocytic Differentiation in Mice Expressing a Unique Chromatin Remodeling Domain of Erythroid Krüppel-Like Factor (EKLF)." Blood 112, no. 11 (November 16, 2008): 132. http://dx.doi.org/10.1182/blood.v112.11.132.132.
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Abstract The zinc finger-encoding transacting factor EKLF binds key regulatory elements of many erythroid-specific genes, and is essential for definitive erythropoiesis. Mice lacking this factor (EKLF−/−) die of anemia by E15.5 of gestation, failing to activate β-globin gene transcription, and demonstrating a block in the erythroid differentiation program at the primitive erythroblast stage. In contrast, megakaryocytic progenitors are amplified in EKLFnull embryos, with increased Fli-1 gene expression (a marker of early megakaryocytic differentiation), consistent with the idea that EKLF modulates the megakaryocyticerythroid (M-E) differentiation switch. We have demonstrated that an amino terminal mutant of EKLF (Δ221EKLF), is required to induce chromatin remodeling at the β-globin promoter in an EKLF-null erythroid cell line, but additional amino terminal sequences are required for initiation of β-globin gene transcription (Brown et al., 2002). To evaluate the role of this chromatin remodeling (CR) domain in erythroid and megakaryocytic differentiation in vivo, we have generated a knock-in allele of EKLFCR allele. Similar to EKLF-null embryos, mice homozygous for this mutant allele die of anemia by E15.5 of gestation. In contrast to erythroid cells lacking EKLF, EKLFCR/CR progenitors demonstrate appropriate binding of the CR encoding domain to all EKLF-regulatory sequences; a block in erythropoiesis at a more a mature stage in differentiation a chromatin architecture and histone modification pattern at erythroid-specific genes that recapitulates the events observed in EKLF+/+ erythroblasts at a similar stage of erythroid ontogeny; a failure of terminal erythroid gene transcription. Examining the role of EKLFCR in megakaryopoiesis, we observed inhibition of megakaryocytic progenitor amplification in EKLFCR/CR fetal hematopoietic cell populations when compared to EKLF-null embryos; loss of Fli-1 gene expression in EKLFCR expressing cells; binding of the EKLFCR mutant protein to the Fli-1 promoter with inhibition of gene transcription; a repressed chromatin architecture at megakaryocytic gene loci. In contrast to these results, mice homozygous for a knockin allele encoding the zinc finger DNA binding domain alone (Δ253EKLF), a region shown previously to be sufficient for chromatin remodeling in vitro, demonstrate erythroid and megakaryocytic phenotypes that resemble those observed in EKLF-null hematopoietic progenitors. Taken together, our results suggest strongly that the unique EKLFCR domain is necessary and sufficient to modulate the chromatin-specific roles of EKLF at erythroid- and megakaryocytic-specific loci in definitive hematopoietic cells in vivo.
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Bieker, J. J., and C. M. Southwood. "The erythroid Krüppel-like factor transactivation domain is a critical component for cell-specific inducibility of a beta-globin promoter." Molecular and Cellular Biology 15, no. 2 (February 1995): 852–60. http://dx.doi.org/10.1128/mcb.15.2.852.
Full textAbstract:
Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.
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Singleton, Belinda K., Winnie Lau, Victoria S. S. Fairweather, Nicholas M. Burton, Marieangela C. Wilson, Steve F. Parsons, Ben M. Richardson, et al. "Mutations in the second zinc finger of human EKLF reduce promoter affinity but give rise to benign and disease phenotypes." Blood 118, no. 11 (September 15, 2011): 3137–45. http://dx.doi.org/10.1182/blood-2011-04-349985.
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Abstract Mutations in the human erythroid Krüppel-like factor (EKLF) can lead to either anemia or the benign InLu phenotype. To elucidate the relationship between these mutations and the differing phenotypes, we prepared recombinant forms of wild-type and 5 mutant EKLF proteins and quantitated their binding affinity to a range of EKLF-regulated genes. Missense mutants (R328H, R328L, and R331G) from persons with InLu phenotype did not bind DNA. Hence, as with the heterozygous loss of function nonsense (L127X, S270X, and K292X) and frameshift (P190Lfs and R319Efs) EKLF mutations, monoallelic loss of EKLF does not result in haploinsufficiency at all loci. In contrast, K332Q has a slightly reduced DNA binding affinity (∼ 2-fold) for all promoters examined but exhibits a phenotype only in a compound heterozygote with a nonfunctional allele. E325K also has a reduced, but significant, binding affinity, particularly for the β-globin gene but results in a disease phenotype even with the wild-type allele expressed, although not as a classic dominant-negative mutant. E325K protein may therefore actively interfere with EKLF-dependent processes by destabilizing transcription complexes, providing a rational explanation for the severity of the disease phenotype. Our study highlights the critical role of residues within the second EKLF zinc finger domain.
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Shyu, Yu-Chiau, Tung-Liang Lee, Shau-Ching Wen, Hsin Chen, Wei-Yuan Hsiao, Xin Chen, JauLang Hwang, and Che-Kun James Shen. "Subcellular Transport of EKLF and Switch-On of Murine Adult βmaj Globin Gene Transcription." Molecular and Cellular Biology 27, no. 6 (January 22, 2007): 2309–23. http://dx.doi.org/10.1128/mcb.01875-06.
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ABSTRACT Erythroid Krüppel-like factor (EKLF) is an essential transcription factor for mammalian β-like globin gene switching, and it specifically activates transcription of the adult β globin gene through binding of its zinc fingers to the promoter. It has been a puzzle that in the mouse, despite its expression throughout the erythroid development, EKLF activates the adult βmaj globin promoter only in erythroid cells beyond the stage of embryonic day 10.5 (E10.5) but not before. We show here that expression of the mouse βmaj globin gene in the aorta-gonad-mesonephros region of E10.5 embryos and in the E14.5 fetal liver is accompanied by predominantly nuclear localization of EKLF. In contrast, EKLF is mainly cytoplasmic in the erythroid cells of E9.5 blood islands in which βmaj is silenced. Remarkably, in a cultured mouse adult erythroleukemic (MEL) cell line, the activation of the βmaj globin gene by dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) induction is also paralleled by a shift of the subcellular location of EKLF from the cytoplasm to the nucleus. Blockage of the nuclear import of EKLF in DMSO-induced MEL cells with a nuclear export inhibitor repressed the transcription of the βmaj globin gene. Transient transfection experiments further indicated that the full-sequence context of EKLF was required for the regulation of its subcellular locations in MEL cells during DMSO induction. Finally, in both the E14.5 fetal liver cells and induced MEL cells, the β-like globin locus is colocalized the PML oncogene domain nuclear body, and concentrated with EKLF, RNA polymerase II, and the splicing factor SC35. These data together provide the first evidence that developmental stage- and differentiation state-specific regulation of the nuclear transport of EKLF might be one of the steps necessary for the switch-on of the mammalian adult β globin gene transcription.
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Zhu, Jianqiong, Kyung Chin, Wulin Aerbajinai, Cecelia Trainor, Peter Gao, and Griffin P. Rodgers. "Recombinant erythroid Kruppel-like factor fused to GATA1 up-regulates delta- and gamma-globin expression in erythroid cells." Blood 117, no. 11 (March 17, 2011): 3045–52. http://dx.doi.org/10.1182/blood-2010-07-294751.
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Abstract The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, α2δ2) and fetal hemoglobin (HbF, α2γ2) both inhibit the polymerization of hemoglobin S, which results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, α2β2) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to up-regulate δ-globin to increase HbA2 expression, we created a series of EKLF-GATA1 fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATA1, and then tested their effects on hemoglobin expression. EKLF-GATA1 fusion proteins activated δ-, γ-, and β-globin promoters in K562 cells, and significantly up-regulated δ- and γ-globin RNA transcript and protein expression in K562 and/or CD34+ cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter, and may represent a new genetic therapeutic approach to β-hemoglobinopathies.
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Chen, Xiaoyong, and James J. Bieker. "Unanticipated Repression Function Linked to Erythroid Krüppel-Like Factor." Molecular and Cellular Biology 21, no. 9 (May 1, 2001): 3118–25. http://dx.doi.org/10.1128/mcb.21.9.3118-3125.2001.
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ABSTRACT The erythroid cell-specific transcription factor erythroid Krüppel-like factor (EKLF) is an important activator of β-globin gene expression. It achieves this by binding to the CACCC element at the β-globin promoter via its zinc finger domain. The coactivators CBP and P300 interact with, acetylate, and enhance its activity, helping to explain its role as a transcription activator. Here we show that EKLF can also interact with the corepressors mSin3A and HDAC1 (histone deacetylase 1) through its zinc finger domain. When linked to a GAL4 DNA binding domain, full-length EKLF or its zinc finger domain alone can repress transcription in vivo. This repressive activity can be relieved by the HDAC inhibitor trichostatin A. Although recruitment of EKLF to a promoter is required to show repression, its zinc finger domain cannot bind directly to DNA and repress transcription simultaneously. In addition, the target promoter configuration is important for enabling EKLF to exhibit any repressive activity. These results suggest that EKLF may function in vivo as a transcription repressor and play a previously unsuspected additional role in regulating erythroid gene expression and differentiation.
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Ma, Wen-Bing, Xiao-Han Wang, Chang-Yan Li, Huan-Huan Tian, Jie Zhang, Jun-Jie Bi, Guang-Ming Ren, et al. "GPS2 promotes erythroid differentiation by control of the stability of EKLF protein." Blood 135, no. 25 (June 18, 2020): 2302–15. http://dx.doi.org/10.1182/blood.2019003867.
Full textAbstract:
Abstract Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2−/− embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.
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Zhu, Jianqiongz, Kyung Chin, Wulin Aerbajinai, Cecelia D. Trainor, Gao Perter, and Griffin P. Rodgers. "Recombinant Erythroid Kruppel-Like Factor Fused to GATA1 up-Regulates δ-Globin Expression In Erythroid Cells." Blood 116, no. 21 (November 19, 2010): 3752. http://dx.doi.org/10.1182/blood.v116.21.3752.3752.
Full textAbstract:
Abstract Abstract 3752 The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, α2δ2) and fetal hemoglobin (HbF, a2γ2) both inhibit the polymerization of hemoglobin S that results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, α2β2) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to upregulate δ-globin to increase HbA2 expression, we created a series of EKLF-GATAl fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATAl and then tested their effects on hemoglobin expression. EKLF-GATAl fusion proteins activated δ-, γ-, and β-globin promoters in K562 cells, and significantly upregulated δ- and γ-globin RNA transcripts and proteins expression in K562 and CD34+ cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter, and may represent a potentially new genetic therapeutic approach to β-hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.
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Pilon, Andre M., Dewang Zhou, Mitchell J. Weiss, Timothy M. Townes, David M. Bodine, and Patrick G. Gallagher. "The Human Alpha Hemoglobin Stabilizing Protein (AHSP) Gene Locus in EKLF-Deficient Erythroid Cells." Blood 106, no. 11 (November 16, 2005): 1740. http://dx.doi.org/10.1182/blood.v106.11.1740.1740.
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Abstract AHSP is an erythroid-specific protein that complexes with free α-hemoglobin, protecting it from precipitation. AHSP has been proposed as a modifier gene in β thalassemia and as a candidate gene for unexplained Heinz body anemias, thus understanding its regulation may lead to novel therapies for these disorders. Identified as an erythroid-specific, GATA-1 inducible gene, decreased AHSP mRNA has been found in the fetal livers of mice deficient in the erythroid transcription factor EKLF by both microarray and RNA subtraction analysis. In fetal livers from d13.5 EKLF-deficient mice, AHSP/α-globin mRNA ratios were decreased to 11–16% of wild type by RT-PCR and RPA. In the same fetal livers, no AHSP protein was detected on Western blots with a MoAB against AHSP. EKLF interacts with the proximal CACCC box of the β-globin gene promoter, establishing local chromatin structure and directing high-level β-globin transcription. We hypothesized that chromatin across the AHSP locus would be perturbed in erythroid cells from EKLF-deficient mice. We performed DNase I hypersensitive site (HS) mapping and chromatin immunoprecipitation (ChIP) analysis using wild type and EKLF deficient fetal liver cells. A strong HS was identified in the AHSP 5′ flanking DNA in the core promoter region, that was absent in day 13.5 fetal liver DNA from EKLF-deficient mice. Fine mapping placed this 5′ HS over a CACCC site in the core AHSP promoter. ChIP across the entire AHSP locus with d13.5 fetal liver chromatin identified 2 regions of hyperacetylation of histones H3 and H4 in wild type mice, one corresponding to the 5′ HS and the other 3′ to the AHSP coding sequence. Both of these hyperacetylated regions were hypoacetylated in EKLF-deficient fetal liver cells. ChIP across the AHSP locus with chromatin obtained from mice with an HA tag knocked into the 3′ end of the EKLF gene identified a peak of EKLF binding extending from the 5′HS to intron one, peaking over the core promoter CACCC site. The sequence of this region (ACCCACCCT) has a single mismatch compared to the EKLF consensus site (CCNCNCCCN). Using the AHSP CACCC site as probe in mobility shift assays with rEKLF protein yielded a complex that migrated at the same mobility as a complex obtained with a control β-globin promoter CACCC site probe. Both AHSP and control β-globin complexes were effectively competed by an excess of unlabeled AHSP probe, unlabeled β-globin probe, or ELKF antiserum. Mutant AHSP CACCC probes did not form DNA-protein complexes nor did they effectively displace wild type AHSP CACCC or β-globin CACCC probes in competition assays. Probes with the AHSP CACCC site mutated to the β-globin sequence (A to C) or the other 2 possibilities (A to G, A to T) yielded complexes comparable to wild type AHSP and control β-globin CACCC probes. In transfection assays in K562 cells, an AHSP promoter-luciferase reporter plasmid was transactivated by an EKLF expression plasmid to a degree comparable to a β-globin promoter-luciferase plasmid. These results support the hypotheses that the hemolytic anemia in EKLF-deficient mice is exacerbated by decreased AHSP expression and that EKLF acts as a transcription factor and a chromatin modulator for genes other than β-globin. Our data also support the hypothesis that AHSP and EKLF may be modifier genes for the β-thalassemia syndromes.
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Siatecka, Miroslawa, Li Xue, and James J. Bieker. "Sumoylation of EKLF Promotes Transcriptional Repression and Is Involved in Inhibition of Megakaryopoiesis." Molecular and Cellular Biology 27, no. 24 (October 15, 2007): 8547–60. http://dx.doi.org/10.1128/mcb.00589-07.
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ABSTRACT Erythroid Krüppel-like factor (EKLF [KLF1]) is a transcriptional regulator that plays a critical role within a specific subset of hematopoietic cells, particularly in the erythroid lineage and its immediate precursor, the megakaryocyte-erythroid progenitor (MEP). We find that EKLF is posttranslationally modified by sumoylation at a single site near its amino terminus and that PIAS1 plays a critical role in this process. Mutation of this site has little effect on EKLF's ability to function as a transcriptional activator; however, it has a dramatic effect on its repressive abilities. The mechanism of repression likely involves a novel small ubiquitin-related modifier (SUMO)-dependent EKLF interaction with the Mi-2β component of the NuRD repression complex. Mutated EKLF is attenuated in its ability to repress megakaryocyte differentiation, implicating EKLF sumoylation status in differentiative decisions emanating from the MEP. These studies demonstrate a novel mechanism by which transcription factor sumoylation can alter protein-protein interactions and bipotential lineage decisions.
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Gallagher, Patrick G., Murat O. Arcasoy, Serena E. Vayda, Holly K. Dressman, James J. Bieker, and David M. Bodine. "A Differentiation Block in Erythroid Cells Lacking Erythroid Krupple-Like Factor (EKLF)." Blood 106, no. 11 (November 16, 2005): 526. http://dx.doi.org/10.1182/blood.v106.11.526.526.
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Abstract Mice deficient in the erythroid specific zinc-finger transcription factor EKLF die ~d14-15 of gestation of severe anemia, attributed to decreased expression of β-globin. The morphology of fetal-liver derived erythroid cells in EKLF-deficient mice does not mimic that seen in thalassemia, but instead shows hemolysis with uniform, nucleated erythroid progenitor cells. This has led to the hypothesis that a block in erythroid differentiation contributes to the anemia in EKLF-deficient mice. To address this, we performed microarray analyses with Affymetrix GeneChip Mouse Genome 430 2.0 arrays and RNA from d13.5 fetal livers of wild type (WT) and EKLF-deficient mice. Three independent EKLF +/+ and −/− RNA samples were analyzed. Numerous genes were down regulated including AHSP, pyruvate kinase, ankyrin, β spectrin and band 3. Verification of reduced expression of selected genes demonstrated that expression levels of many genes identified as down regulated via microarray analyses were minimally reduced in EKLF −/− RNA (<20%) compared to normal (Rh 30, protein 4.2, protein 4.9, p55, AQP1, and ALAS-E). Flow cytometry of WT d14.5 fetal liver cells using TER 119 and CD71 was performed. In WT fetal livers, this identifies 5 populations, designated R1-R5, with R1/R2 composed of primitive progenitors and proerythroblasts and R3, R4, and R5 composed of more mature erythroblasts (Blood102:3938, 2003). In EKLF −/− fetal livers, R3, R4, and R5, populations involved in terminal erythroid differentiation, were completely absent, suggesting many of the genes identified by microarray analyses were differentially expressed because of a bias introduced by a differentiation block to more mature erythroid cells. Confirming this hypothesis, we demonstrated that genes with <20% difference in expression between WT and EKLF-deficient fetal liver mRNA had 4-fold or higher levels in wild type R3+R4+R5 RNA compared to R1+R2 RNA. To better understand how differentially expressed genes were integrated into specific regulatory and signaling pathway networks, we used Ingenuity Pathway Analysis. A subset of focus genes incorporated into a biological network with highly a significant scores (>40) was generated containing 35 focus genes. The biological function of this network involved cell cycle and DNA replication. At the central nodes of this network were E2F1 and E2F2, transcription factors involved in cell cycle control. Cell cycle analysis demonstrated that EKLF-deficient R1 cells exhibited a significant delay exiting G0+G1 and entering S phase and both R1 and R2 cells exhibited a defect in exiting S and entering G2+M. Colony assays with R1 and R2 cells revealed that EKLF-deficient fetal liver cells had decreased frequency of CFU-E, but similar absolute numbers of CFU-E as WT. As predicted by the cell cycle defect, EKLF−/− FL cells were severely (~10 fold) deficient in their ability to generate BFU-E. Flow cytometry with annexin V revealed no difference between WT and EKLF-deficient cells indicated that apoptosis was not contributing to the differentiation block. These results support the hypothesis that the failure of definitive erythropoiesis in EKLF deficient mice is due to decreased expression of many erythroid genes involved in erythroid differentiation, stabilization of α-globin protein, membrane stability, and glycolysis, not simply decreased transcription of the β-globin gene.
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Guy, Louis-Georges, Qi Mei, Andrew C. Perkins, Stuart H. Orkin, and Lee Wall. "Erythroid Krüppel-Like Factor Is Essential for β-Globin Gene Expression Even in Absence of Gene Competition, But Is Not Sufficient to Induce the Switch From γ-Globin to β-Globin Gene Expression." Blood 91, no. 7 (April 1, 1998): 2259–63. http://dx.doi.org/10.1182/blood.v91.7.2259.
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Abstract Different genes in the β-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage–specific β-globin gene. In transgenic mice carrying the complete human β-globin locus, EKLF ablation not only impairs human β-globin–gene expression but also results in increased expression of the human γ-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from γ-globin to β-globin transcription. However, we show here that the function of EKLF for β-globin–gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from γ-globin to β-globin–gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of γ-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.
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Guy, Louis-Georges, Qi Mei, Andrew C. Perkins, Stuart H. Orkin, and Lee Wall. "Erythroid Krüppel-Like Factor Is Essential for β-Globin Gene Expression Even in Absence of Gene Competition, But Is Not Sufficient to Induce the Switch From γ-Globin to β-Globin Gene Expression." Blood 91, no. 7 (April 1, 1998): 2259–63. http://dx.doi.org/10.1182/blood.v91.7.2259.2259_2259_2263.
Full textAbstract:
Different genes in the β-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage–specific β-globin gene. In transgenic mice carrying the complete human β-globin locus, EKLF ablation not only impairs human β-globin–gene expression but also results in increased expression of the human γ-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from γ-globin to β-globin transcription. However, we show here that the function of EKLF for β-globin–gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from γ-globin to β-globin–gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of γ-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.
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Singleton, Belinda K., Nicholas M. Burton, Carole Green, R. Leo Brady, and David J. Anstee. "Mutations in EKLF/KLF1 form the molecular basis of the rare blood group In(Lu) phenotype." Blood 112, no. 5 (September 1, 2008): 2081–88. http://dx.doi.org/10.1182/blood-2008-03-145672.
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Abstract Comparison of normal erythroblasts and erythroblasts from persons with the rare In(Lu) type of Lu(a-b-) blood group phenotype showed increased transcription levels for 314 genes and reduced levels for 354 genes in In(Lu) cells. Many erythroid-specific genes (including ALAS2, SLC4A1) had reduced transcript levels, suggesting the phenotype resulted from a transcription factor abnormality. A search for mutations in erythroid transcription factors showed mutations in the promoter or coding sequence of EKLF in 21 of 24 persons with the In(Lu) phenotype. In all cases the mutant EKLF allele occurred in the presence of a normal EKLF allele. Nine different loss-of-function mutations were identified. One mutation abolished a GATA1 binding site in the EKLF promoter (−124T>C). Two mutations (Leu127X; Lys292X) resulted in premature termination codons, 2 (Pro190LeufsX47; Arg319GlufsX34) in frameshifts, and 4 in amino acid substitution of conserved residues in zinc finger domain 1 (His299Tyr) or domain 2 (Arg328Leu; Arg328His; Arg331Gly). Persons with the In(Lu) phenotype have no reported pathology, indicating that one functional EKLF allele is sufficient to sustain human erythropoiesis. These data provide the first description of inactivating mutations in human EKLF and the first demonstration of a blood group phenotype resulting from mutations in a transcription factor.
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Varricchio, Lilian, Carmela Dell'Aversana, Angela Nebbioso, Giovanni Migliaccio, Lucia Altucci, James J. Bieker, and Anna Rita F. Migliaccio. "Identification of a New Functional HDAC Complex Composed by HDAC5, GATA1 and EKLF in Human Erythroid Cells." Blood 120, no. 21 (November 16, 2012): 979. http://dx.doi.org/10.1182/blood.v120.21.979.979.
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Abstract Abstract 979 Histone deacetylation, the reaction that maintains chromatin in a condensed configuration preventing gene expression, is catalyzed by the histone deacetylase (HDAC) superfamily. The human HDAC family includes 18 different isoforms classified on the basis of their sequence homology to HDACs from Saccharomyces Cerevisiae into class I (HDAC1, −2, −3, and −8), IIa (HDAC4, −5, −7, and −9), IIb (HDAC6 and −10) and IV (HDAC11). Class I HDACs bind the DNA directly while class IIa HDACs shuffles other proteins between nucleus and cytoplasm. While the role of individual class I HDACs in erythropoiesis is starting to emerge, that of class IIa and b HDACs is still largely unknown. To clarify the role played by class IIa HDACs in the control of human erythropoiesis, an extensive analysis of expression, activity, and function of different classes of HDACs during the maturation of erythroblasts derived in vitro from adult blood or cord blood was performed. HDACs expression/activity. Erythroid maturation was associated with increased expression of class I HDACs (both mRNA and protein) which, in the case of HDAC1, was also associated with increased enzymatic activity and association with its NuRD partner GATA1. By contrast, reductions either in expression (HDAC4) or activity (HDAC5) of class IIa HDACs were observed with maturation. In addition, GATA1 and EKLF were consistently found associated in human erythroblasts but EKLF was not found associated with HDAC1. The extent of nuclear-cytoplasmic trafficking of class I (HDAC1 and 2) and IIa (HDAC4 and 5) and of the transcription factors EKLF and GATA1 in response to EPO was determined. HDAC2/EKLF/GATA1 and HDAC4 were found constitutively present in the nucleus and in the cytoplasm, respectively. By contrast, the nuclear concentration of HDAC1 increased while that of HDAC5 and of GATA1fl decreased upon stimulation with EPO. The last two observations suggested that HDAC5, GATA1 and EKLF might be associated in a complex. Identification of the HDAC5/EKLF/GATA1 complex. A series of IPs followed by WB experiments showed that HDAC5 was consistently associated with EKLF and GATA1 and conversely, both GATA1 (preferentially GATA1fl over GATA1s) and EKLF were consistently associated with HDAC5 (Fig 1A and not shown). Interestingly also pERK was detected in IPs with HDAC5, EKLF and GATA1 antibodies. These results indicate that in erythroid cells HDAC5 forms a complex with GATA1, EKLF and pERK. Identification of the biological activity of the HDAC5/GATA1/EKLF/pERK complex. The association between GATA1/EKLF was greater in cells generated with cord blood (which express high HbF levels) than in those derived from adult blood and their association decreased with maturation, suggesting that the complex may regulate HbF expression. To confirm this hypothesis, HDAC5/GATA1 association and γ/(γ+ β) mRNA ratios were determined in erythroid cells induced to mature in the presence of a pan-class II-specific (APHA9, ID50=20 μM for HDAC4) HDAC inhibitor (HDACi) (Fig 1B)1. Cells exposed in parallel to the class I/IIa-specific (UBHA24, ID50 =0.2 and 0.6 μM for HDAC1 and HDAC4, respectively) HDACi, were used as control. Exposure to APHA9 reduced the association between GATA1 and HDAC5 and increased γ/(γ + β) mRNA expression ratio, while this association was not affected by exposure to the class I/II HDACi which, as expected, also increased γ/(γ+ β) mRNA ratio. Conclusions. These data identify a new HDAC complex formed by HDAC5, EKLF and GATA1 that regulates γ/(γ + β) ratio. We hypothesize that the biological role of this new complex is to shuffle GATA1 and EKLF from the cytoplasm to the nucleus, making them able to engage into the NuRD and Sin3A complex respectively, and that inhibition of the activity of this complex affects γ-globin expression indirectly by limiting the amount of GATA1and EKLF available to associate with NuRD and Sin3A. Disclosures: No relevant conflicts of interest to declare.
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Pilon, Andre M., Clara Wong, Lisa J. Garrett-Beal, Mitchell Weiss, Patrick G. Gallagher, and David M. Bodine. "Chromatin Remodeling of the Mouse AHSP Gene Requires EKLF." Blood 104, no. 11 (November 16, 2004): 375. http://dx.doi.org/10.1182/blood.v104.11.375.375.
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Abstract Alpha-Hemoglobin Stabilizing Protein (AHSP) is an erythroid-specific protein that binds to α-globin, preventing precipitation of α-hemoglobin tetramers. Our interest has focused on how the AHSP gene is specifically expressed in erythroid cells, and we have investigated the roles of cis acting DNA sequences, the transcription factor EKLF and chromatin structure on AHSP gene expression. We have previously shown that the AHSP gene has a single mRNA initiation site followed by a non-coding exon. Putative promoter sequences from −904, −479 or −170 were active in luciferase reporter assays only when the constructs contained the downstream 269 bp containing exon 1 and intron 1. The −904/+269, −479/+269 and −170/+269 constructs gave 53.3+2.0 to 122.1+8.8 -fold increased levels of luciferase expression in K562 cells compared to plasmids without exon 1 and intron 1 (p<0.001; Gallagher et al., BLOOD 102, 267a, 2003). In vitro DNase I footprinting and EMSA assays revealed two regions (−75 to −67 and +153 to +164) that bound GATA-1. Analysis of 5 lines of transgenic mice with between 1 and 11 copies of the −170/+269 promoter fused to the human γ-globin gene demonstrated position independent expression (5/5 lines express) of γ-globin mRNA, at levels that were 4.6% the level of mouse α-globin mRNA per transgene copy. There was no correlation between transgene copy number and the level of γ-globin mRNA and 3/5 lines exhibited variegated expression. We concluded that sequences upstream of −170 or downstream of +269 are required for authentic expression from the AHSP promoter. To examine the role of EKLF in AHSP expression, we used subtractive hybridization, microarray and RNase protection analysis to compare AHSP mRNA levels in fetal liver cells from wild type and EKLF−/− mice. EKLF −/− fetal liver cells had 9 -fold less AHSP mRNA than wild type fetal liver cells, which we propose would exacerbate the moderate β- thalassemia described in EKLF −/− mice. Based on the observation that EKLF associates with an erythroid chromatin remodeling complex (Armstrong et al. Cell 95, 93–104, 1998), we hypothesized that EKLF was involved in chromatin remodeling at the AHSP locus. We assayed for DNase I Hypersensitive sites (HS) in chromatin from 13.5 day wild type and EKLF−/− mouse fetal liver nuclei. We demonstrated a strong DNase I HS between −200 and −400 of the AHSP gene, just upstream of the minimal −170 promoter, that was not present in chromatin from EKLF−/− fetal liver cells. To examine histone acetylation across the 3.5 kb AHSP locus we performed Chromatin Immune Precipitation analysis on wild type and EKLF −/− fetal liver chromatin using 13 different primer pairs (~300 bp intervals). In wild type chromatin there are two regions where histones H3 and H4 were hyperacetylated relative to a control region from the mouse α-globin gene promoter. The 5′ region corresponded to the DNase I HS at −400 to −200, while the second region maps 3′ to the AATAAA signal in the AHSP gene. Histones H3 and H4 were also acetylated in the interval between the hyperacetylated regions, while the chromatin upstream and downstream (~1 kb in each direction) of these regions was hypoacetylated. In contrast, all sites were hypoacetylated in chromatin from EKLF−/− fetal liver cells, correlating with the severe reduction in AHSP gene expression. We conclude that EKLF is required for remodeling the chromatin of the AHSP locus and that EKLF could be a modifier gene for the thalassemia syndromes.
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Neuwirtova, Radana, Ota Fuchs, Dana Provaznikova, Jaroslav Cermak, Magda Siskova, Anna Jonasova, and Kyra Michalova. "Fli-1 and EKLF Gene Expression in Patients with MDS 5q- Syndrome." Blood 114, no. 22 (November 20, 2009): 2788. http://dx.doi.org/10.1182/blood.v114.22.2788.2788.
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Abstract Abstract 2788 Poster Board II-764 Introduction. Patients with MDS-5q- syndrome have macrocytic anemia often with hypoplastic erythropoiesis and on the contrary thrombocythemia with effective though dysplastic megakaryopoiesis. Megakaryocytes and erythroid cells are thought to share a common progenitor MEP (T.P.McDonald et al., Exp. Hematology 1993). There are two key transcription factors which together with other transcription factors and relevant cytokines and receptors determine the hemopoietic differentiation of the common stem cell: erythroid Krüppel-like factor (EKLF) for erythroid lineage and Friend leukemia virus integration 1 (FLi-1) for megakaryopoiesis (Pilar Frontelo et al., Blood 2007; G.A.Blobel, Blood 2007; F.Bouilloux et al., Blood 2008). There is functional cross antagonism between FLi-1 and EKLF (J.Starck et al., Mol. Cel. Biology 2003). FLi-1 is active only if dephosphorylated (H.Huang et al., ASH Abstracts 2008). The question is whether both factors play any role in 5q- syndrome. Methods. FLi-1 and EKLF gene expressions were determined in mononuclear cells isolated from the whole blood or bone marrow using Ficoll-Paque PLUS. Expression of both factors was measured by quantitative real-time PCR. RT-PCR products were verified by electrophoresis and direct sequencing. The assays were performed for sample in duplicate. Glyceraldehyd-3-phosphate dehydrogenase (GAPDH), FLi-1 and EKLF were amplified in 25 μl reaction mixture containing 12.5 μl SYBR Green JumpStart Taq Ready Mix, 2.5 μl 2 μM FLi-1 or EKLF forward and reverse primers, 0,25 μl internal reference dye and 1 μl cDNA. Relative levels of FLi-1 and EKLF mRNAs were calculated to the level of housekeeping GAPDH mRNA. Results. FLi-1 and EKLF were measured in blood mononuclear cells of 8 patients fulfilling all criterias of 5q- syndrome. FLi1mRNA/GAPDHmRNA was higher in all samples, average value was 0.0930 (0.0242-0.4274) compared to control value 0.0194. FLi1mRNA/GAPDHmRNA in bone marrow mononuclear cells of 7 patients with 5q- syndrome was higher in all samples but one. The average value was 0.0827 (0.0070-0.2554) compared to healthy controls 0.0044. EKLF gives very low values in the majority of patients′ blood and bone marrow samples as well as in healthy controls. The evaluation is therefore less reliable then FLi-1 assessment. EKLFmRNA/GAPDHmRNA in blood was 0.0004 (0.0-0.0023) compared to the control 0.0222. The results of EKLF in 5 bone marrow samples are inconsistent. Three are lower than the control (0.0068), 1 of remaining 2 samples is extremely high (0.3491). It is interesting that this patient is the only one who responded to erythropoietin and is transfusion independent. Summary. Our preliminary results with FLi-1 and EKLF gene expression measurement are in agreement with expected findings: increased FLi-1 expression corresponds to thrombocytemia in 5q- syndrome patients and expression of EKLF, lower than in controls would correspond to anemia in these patients. However, EKLF values are less reliable because of very low values in patients as well as in controls and because of inconsistent results in bone marrow samples. We prepare to follow both factors in 5q- patients after the treatment with lenalidomide. Lenalidomide improves anemia in 5q- syndrome patients and temporarily causes decrease of thrombocytes (A.List et al., N.Engl.J.Med. 2005, 2006). Inhibition of phosphatases by lenalidomide (S.Wei et al., Proc.Natl.Acad.Sci.USA 2009) can stop FLi-1 dephosphorylation which leads to FLi-1 inactivation. Hypotetically inactive FLi-1 would enable EKLF to induce MEP into erythroid lineage. Supported by MSM 0021620808 Disclosures: No relevant conflicts of interest to declare.
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Siatecka, Miroslawa, Felix Lohmann, Sujin Bao, and James J. Bieker. "EKLF Directly Activates the p21WAF1/CIP1 Gene by Proximal Promoter and Novel Intronic Regulatory Regions during Erythroid Differentiation." Molecular and Cellular Biology 30, no. 11 (April 5, 2010): 2811–22. http://dx.doi.org/10.1128/mcb.01016-09.
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ABSTRACT The switch from proliferation to differentiation during the terminal stages of erythropoiesis is a tightly controlled process that relies in part on transcription factor-mediated activation of cell cycle components. EKLF is a key transcription factor that is necessary for the initial establishment of the red cell phenotype. Here, we find that EKLF also plays a role during the subsequent differentiation process, as it induces p21WAF1/CIP1 expression independent of p53 to regulate the changes in the cell cycle underlying erythroid maturation. EKLF activates p21 not only by directly binding to an EKLF site within a previously characterized GC-rich region in the p21 proximal promoter but also by occupancy at a novel, phylogenetically conserved region that contains consensus CACCC core motifs located downstream from the p21 TATA box. Our findings demonstrate that EKLF, likely in coordination with other transcription factors, directly contributes to the complex set of events that occur at the final erythroid cell divisions and accentuates terminal differentiation directly by activation of CDK inhibitors such as p21.