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Pandya, Kumar, David Donze, and Tim M. Townes. "Novel Transactivation Domain in Erythroid Kruppel-like Factor (EKLF)." Journal of Biological Chemistry 276, no. 11 (November 22, 2000): 8239–43. http://dx.doi.org/10.1074/jbc.m008457200.
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Perkins, Andrew. "Erythroid Kruppel like factor: from fishing expedition to gourmet meal." International Journal of Biochemistry & Cell Biology 31, no. 10 (October 1999): 1175–92. http://dx.doi.org/10.1016/s1357-2725(99)00083-7.
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Tallack, Michael R., Janelle R. Keys, and Andrew C. Perkins. "Erythroid Kruppel-like factor regulates the G1 Cdk inhibitor p18." Blood Cells, Molecules, and Diseases 38, no. 2 (March 2007): 168. http://dx.doi.org/10.1016/j.bcmd.2006.10.111.
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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.
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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.
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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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Crossley, M., A. P. Tsang, J. J. Bieker, and S. H. Orkin. "Regulation of the erythroid Kruppel-like factor (EKLF) gene promoter by the erythroid transcription factor GATA-1." Journal of Biological Chemistry 269, no. 22 (June 1994): 15440–44. http://dx.doi.org/10.1016/s0021-9258(17)40698-3.
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Ilsley, Melissa, Kevin R. Gillinder, Graham Magor, Merlin Crossley, and Andrew C. Perkins. "Fine-Tuning Erythropoiesis By Competition Between Krüppel-like Factors for Promoters and Enhancers." Blood 128, no. 22 (December 2, 2016): 1036. http://dx.doi.org/10.1182/blood.v128.22.1036.1036.
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Abstract Krüppel-like factors (KLF) are a group of 17 transcription factors with highly conserved DNA-binding domains that contain three C-terminal C2H2-type zinc fingers and a variable N-terminal domain responsible for recruiting cofactors 1. KLFs participate in diverse roles in stem cell renewal, early patterning, organogenesis and tissue homeostasis. Krüppel-like factor 1 (KLF1) is an erythroid-specific KLF responsible for coordinating many aspects of terminal erythroid differentiation 2. It functions as a transcriptional activator by recruiting cofactors such as p300 and chromatin modifiers such as Brg1 via N-terminal transactivation domains 3. Krüppel-like factor 3 (KLF3) acts as a transcriptional repressor via recruitment of C-terminal binding proteins 4. In erythropoiesis, KLF1 directly activates KLF3 via an erythroid-specific promoter 5. Some KLF1 target genes are upregulated in Klf3-/- fetal liver suggesting possible competition between the two factors for promoter/enhancer occupancy. We generated three independent clones of the erythroid cell line, J2E, by retroviral transduction of a tamoxifen-inducible version of Klf3 (Klf3-ERTM) as previously described 6. Using next-generation sequencing of newly synthesised RNA (4sU-labeling), we show KLF3 induction leads to immediate repression of a set of ~580 genes; a subset of these (54) are also directly induced by KLF1 in K1-ER cells, suggesting antagonistic regulation. Indeed, ChIP-seq revealed KLF1 and KLF3 bind many of the same regulatory sites within the erythroid cell genome. KLF3 also binds an independent set of promoters which are not bound by KLF1, suggesting it also plays a KLF1-independent role in maintenance of gene repression. By de novo motif discovery we confirm KLF3 binds preferably to a extended CACCC motif, R-CCM-CRC-CCN, so the DNA-binding specificity in vivo is indistinguishable from the KLF1 binding specificity 7, and is independent of co-operating DNA-binding proteins or cofactors. Using Q-PCR of KLF1 ChIPed DNA in J2E-Klf3ER cells, we show that overexpression of KLF3 directly displaces KLF1 from many key target sites such as the E2f2 enhancer and this leads to down regulation of gene expression. This is the first proof that KLF1 and KLF3 directly compete for key promoters and enhancers which drive erythroid cell proliferation and differentiation. We propose KLF3 acts to 'fine-tune' transcription in erythropoiesis by repressing genes activated by KLF1 and that this negative feedback system is necessary for precise control over the generation of erythrocytes. It also works independently of KLF1 perhaps via competition for binding with other KLF/SP factors. References: 1. van Vliet J, Crofts LA, Quinlan KG, Czolij R, Perkins AC, Crossley M. Human KLF17 is a new member of the Sp/KLF family of transcription factors. Genomics. 2006;87(4):474-482. 2. Tallack MR, Magor GW, Dartigues B, et al. Novel roles for KLF1 in erythropoiesis revealed by mRNA-seq. Genome Res. 2012. 3. Perkins A, Xu X, Higgs DR, et al. "Kruppeling" erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants unveiled by genomic sequencing. Blood. 2016. 4. Dewi V, Kwok A, Lee S, et al. Phosphorylation of Kruppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity. J Biol Chem. 2015;290(13):8591-8605. 5. Funnell AP, Maloney CA, Thompson LJ, et al. Erythroid Kruppel-like factor directly activates the basic Kruppel-like factor gene in erythroid cells. Mol Cell Biol. 2007;27(7):2777-2790. 6. Coghill E, Eccleston S, Fox V, et al. Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood. 2001;97(6):1861-1868. 7. Tallack MR, Whitington T, Yuen WS, et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome Res. 2010;20(8):1052-1063. Disclosures Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria.
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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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Donze, David, Tim M. Townes, and James J. Bieker. "Role of Erythroid Kruppel-like Factor in Human - to -Globin Gene Switching." Journal of Biological Chemistry 270, no. 4 (January 27, 1995): 1955–59. http://dx.doi.org/10.1074/jbc.270.4.1955.
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Tallack, Michael R., Janelle R. Keys, and Andrew C. Perkins. "Erythroid Kruppel-like Factor Regulates the G1 Cyclin Dependent Kinase Inhibitor p18INK4c." Journal of Molecular Biology 369, no. 2 (June 2007): 313–21. http://dx.doi.org/10.1016/j.jmb.2007.02.109.
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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.
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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., 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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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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Yien, Yvette Y., and James J. Bieker. "Functional Interactions between Erythroid Kruppel-like Factor (EKLF/KLF1) and Protein Phosphatase PPM1B/PP2Cβ." Journal of Biological Chemistry 287, no. 19 (March 5, 2012): 15193–204. http://dx.doi.org/10.1074/jbc.m112.350496.
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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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Mas, C., M. Lussier-Price, S. Soni, T. Morse, G. Arseneault, P. Di Lello, J. Lafrance-Vanasse, J. J. Bieker, and J. G. Omichinski. "Structural and functional characterization of an atypical activation domain in erythroid Kruppel-like factor (EKLF)." Proceedings of the National Academy of Sciences 108, no. 26 (June 13, 2011): 10484–89. http://dx.doi.org/10.1073/pnas.1017029108.
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Zhang, W., and J. J. Bieker. "Acetylation and modulation of erythroid Kruppel-like factor (EKLF) activity by interaction with histone acetyltransferases." Proceedings of the National Academy of Sciences 95, no. 17 (August 18, 1998): 9855–60. http://dx.doi.org/10.1073/pnas.95.17.9855.
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Keys, Janelle R., Michael R. Tallack, Denise J. Hodge, Simon O. Cridland, Rakesh David, and Andrew C. Perkins. "Genomic organisation and regulation of murine alpha haemoglobin stabilising protein by erythroid Kruppel-like factor." British Journal of Haematology 136, no. 1 (January 2007): 150–57. http://dx.doi.org/10.1111/j.1365-2141.2006.06381.x.
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Luo, Qi, Xiaojing Ma, Sharon M. Wahl, James J. Bieker, Merlin Crossley, and Luis J. Montaner. "Activation and Repression of Interleukin-12 p40 Transcription by Erythroid Kruppel-like Factor in Macrophages." Journal of Biological Chemistry 279, no. 18 (February 19, 2004): 18451–56. http://dx.doi.org/10.1074/jbc.m400320200.
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Manwani, Deepa, Mariann Galdass, and James J. Bieker. "Altered Regulation of β like Globin Genes by a Redesigned Erythroid Transcription Factor." Blood 104, no. 11 (November 16, 2004): 1212. http://dx.doi.org/10.1182/blood.v104.11.1212.1212.
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Abstract The well characterized switch during ontogeny of globin gene expression from embryonic/ fetal to adult type is a result of a complex interplay between cis and trans acting regulatory elements at the beta globin locus. Trans acting elements include tissue specific transcription factors that bind specific motifs within the beta globin gene cluster with high specificity. Erythroid Kruppel like factor (EKLF) is one such erythroid specific, zinc finger transcription factor that is critical for the activation of the beta globin promoter and for consolidating the switch from gamma to beta globin during development. The ability to willfully regulate the expression of endogenous genes using redesigned zinc finger transcription factors is an emerging field. There is tremendous appeal in utilizing the understanding of transcriptional control pathways to design tools that will elucidate molecular mechanisms and provide potential therapeutic tools. To this end we redesigned Erythroid Kruppel Like Factor (EKLF) as a transcriptional repressor. The zinc finger DNA binding domain was linked to the repressor domain from the Drosophila Engrailed protein with the prediction that this construct (ENG/ZNF) would bind the beta globin promoter and repress it. It was hypothesized that embryonic/fetal globin activation would result by a competitive mechanism. When introduced transiently into cells these transcription factors are effective in repressing the adult beta globin promoter CACCC element, the natural target for EKLF. In stable MEL clones, repression of the adult beta globin gene is accompanied by a reactivation of the endogenous embryonic globin gene. In order to study this effect in the context of a whole animal we generated transgenic mice expressing ENG/ZNF. A 271 bp region 5′of the ANK-1 gene was chosen to drive expression in transgenic mice as it provides erythroid specific expression with copy number dependence and minimal position dependence. D13.5 fetal livers were subject to RT-PCR analysis in the linear range to quantitate the ratios of BH1 to alpha globin transcripts. The 9 ENG/ZNF transgenic embryos express BH1 mRNa in a range of values that is statistically higher than in 9 control littermates (Mann Whitney U test, p value 0.02) and beta major globin mRNA at lower levels. We further studied ENG/ZNF in the developmentally plastic environment of differentiating murine embryonic stem cells. The construct was stably integrated into a targeting site upstream of the HPRT locus under the control of a tetracycline inducible promoter. The Doxycycline induction of ENG/ZNF transgene expression results in a 4 fold activation of embryonic globin at day 6 of embryoid body development; however there is no evidence of beta globin repression. Since at this stage of embryoid body development, primitive erythroid cells are 100–500 fold more abundant than definitive erythroid cells, this may reflect a differential effect of EKLF in primitive erythroid cells. To evaluate this further, we are currently performing analyses in primitive versus definitive erythroid colonies. In conclusion, our studies support the competitive model of globin switching and may contribute to the delineation of a stage specific role of EKLF. In addition, transcriptional reagents that augment gamma globin expression hold promise as novel therapeutic agents for sickle cell disease and other hemoglobinopathies.
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Shyu, Yu-Chiau, Shau-Ching Wen, Tung-Liang Lee, Xin Chen, Chia-Tse Hsu, Hsin Chen, Ruei-Lin Chen, Jau-Lang Hwang, and Che-Kun James Shen. "Chromatin-binding in vivo of the erythroid kruppel-like factor, EKLF, in the murine globin loci." Cell Research 16, no. 4 (April 2006): 347–55. http://dx.doi.org/10.1038/sj.cr.7310045.
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Lee, J. S., C. H. Lee, and J. H. Chung. "The beta -globin promoter is important for recruitment of erythroid Kruppel-like factor to the locus control region in erythroid cells." Proceedings of the National Academy of Sciences 96, no. 18 (August 31, 1999): 10051–55. http://dx.doi.org/10.1073/pnas.96.18.10051.
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Siatecka, M., K. E. Sahr, S. G. Andersen, M. Mezei, J. J. Bieker, and L. L. Peters. "Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Kruppel-like factor." Proceedings of the National Academy of Sciences 107, no. 34 (August 9, 2010): 15151–56. http://dx.doi.org/10.1073/pnas.1004996107.
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Siatecka, Miroslawa, Shefali Soni, Antanas Planutis, and James J. Bieker. "Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3)." Journal of Biological Chemistry 290, no. 15 (February 24, 2015): 9929–40. http://dx.doi.org/10.1074/jbc.m114.610246.
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Gillinder, Kevin R., Graham Magor, Charles Bell, Melissa D. Ilsley, Stephen Huang, and Andrew Perkins. "KLF1 Acts As a Pioneer Transcription Factor to Open Chromatin and Facilitate Recruitment of GATA1." Blood 132, Supplement 1 (November 29, 2018): 501. http://dx.doi.org/10.1182/blood-2018-99-119608.
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Abstract Only a small subset of transcription factors (TFs) can act as pioneer factors; i.e. those that can 'open' otherwise 'closed' chromatin to facilitate assembly of TF complexes and co-factors to enable transcription. The KLF/SP family of TFs bind to a 9-10 bp consensus motif in DNA to activate or repress target gene expression. We have studied the potential for KLF1, which is essential for erythropoiesis, to provide a pioneering function in erythroid progentior cells. Previous ChIP-seq studies have shown KLF1 binds a few thousand enhancers and promoters to activate erythroid cell gene expression 1. It often binds near to other key erythroid TFs such as GATA1 and SCL/TAL1, so is likely to work in concert with them in some contexts. We have employed an inducible stable KLF1-ERTM construct to rescue gene expression and differentiation of Klf1-/- erythroid cell lines 2. We employed ChIP-seq, ATAC-seq and DNAse1 HS to show KLF1 can bind to closed sites in chromatin and induce an open state. We show this is essential for recruitment of the settler transcription, GATA1, at certain co-bound sites but not others. This pioneering function occurs at ~300 key erythroid enhancers and super-enhancers such the one at -26kb in the a-globin LCR and one within the body of the E2f2 gene 3 but rarely at promoters. We further show that two different neomorphic mutations in the KLF1 DNA-binding domain lead to ectopic pioneering (opening of closed chromatin) and aberrant gene activation 4. We generated a series of N-terminal deletions in KLF1 and employed ATAC-seq to map the domain/s within KLF1 responsible for the pioneering activity and show it is distinct from DNA-binding activity. The domain is responsible for bromodomain protein recruitment, the likely effector of chromatin remodelling. We have also examined whether KLF3, which acts as a transcription repressor via recruitment of the co-repressor, CtBP2, can force the closure of otherwise open chromatin 5. We find it cannot. Rather, KLF3 (and likely other members of this subclade) works via active recruitment of co-repressors rather than rendering chromatin inaccessible. This likely enables rapid reactivation of pioneered enhancers without the need to reprogram chromatin. This work has broad implications for how the KLF/SP family of TFs work in vivo to reprogram cells and direct differentiation. We will present data for such activity in non-erythroid cell systems. References:Tallack MR, Whitington T, Yuen WS, et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome Res. 2010;20(8):1052-1063.Coghill E, Eccleston S, Fox V, et al. Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood. 2001;97(6):1861-1868.Tallack MR, Keys JR, Humbert PO, Perkins AC. EKLF/KLF1 controls cell cycle entry via direct regulation of E2f2. J Biol Chem. 2009;284(31):20966-20974.Gillinder KR, Ilsley MD, Nebor D, et al. Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability. Nucleic Acids Res. 2017;45(3):1130-1143.Turner J, Crossley M. Cloning and characterization of mCtBP2, a co-repressor that associates with basic Kruppel-like factor and other mammalian transcriptional regulators. Embo J. 1998;17(17):5129-5140. Disclosures Perkins: Novartis Oncology: Honoraria.
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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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Yokomizo, Tomomasa, Kazuteru Hasegawa, Hiroyuki Ishitobi, Motomi Osato, Masatsugu Ema, Yoshiaki Ito, Masayuki Yamamoto, and Satoru Takahashi. "Runx1 is involved in primitive erythropoiesis in the mouse." Blood 111, no. 8 (April 15, 2008): 4075–80. http://dx.doi.org/10.1182/blood-2007-05-091637.
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Abstract Targeted disruption of the Runx1/ AML1 gene in mice has demonstrated that it is required for the emergence of definitive hematopoietic cells but that it is not essential for the formation of primitive erythrocytes. These findings led to the conclusion that Runx1 is a stage-specific transcription factor acting only during definitive hematopoiesis. However, the zebrafish and Xenopus homologs of Runx1 have been shown to play roles in primitive hematopoiesis, suggesting that mouse Runx1 might also be involved in the development of primitive lineages. In this study, we show that primitive erythrocytes in Runx1−/− mice display abnormal morphology and reduced expression of Ter119, Erythroid Kruppel-like factor (EKLF, KLF1), and GATA-1. These results suggest that mouse Runx1 plays a role in the development of both primitive and definitive hematopoietic cells.
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Parga, Juan A., Ana I. Rodriguez-Perez, Maria Garcia-Garrote, Jannette Rodriguez-Pallares, and Jose L. Labandeira-Garcia. "NRF2 Activation and Downstream Effects: Focus on Parkinson’s Disease and Brain Angiotensin." Antioxidants 10, no. 11 (October 20, 2021): 1649. http://dx.doi.org/10.3390/antiox10111649.
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Reactive oxygen species (ROS) are signalling molecules used to regulate cellular metabolism and homeostasis. However, excessive ROS production causes oxidative stress, one of the main mechanisms associated with the origin and progression of neurodegenerative disorders such as Parkinson’s disease. NRF2 (Nuclear Factor-Erythroid 2 Like 2) is a transcription factor that orchestrates the cellular response to oxidative stress. The regulation of NRF2 signalling has been shown to be a promising strategy to modulate the progression of the neurodegeneration associated to Parkinson’s disease. The NRF2 pathway has been shown to be affected in patients with this disease, and activation of NRF2 has neuroprotective effects in preclinical models, demonstrating the therapeutic potential of this pathway. In this review, we highlight recent advances regarding the regulation of NRF2, including the effect of Angiotensin II as an endogenous signalling molecule able to regulate ROS production and oxidative stress in dopaminergic neurons. The genes regulated and the downstream effects of activation, with special focus on Kruppel Like Factor 9 (KLF9) transcription factor, provide clues about the mechanisms involved in the neurodegenerative process as well as future therapeutic approaches.
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Sengupta, Ananya, Ghanshyam Upadhyay, Asif Chowdhury, and Shireen Saleque. "Regulation Of Erythro-Megakaryocytic Lineage Bifurcation By The Gfi1b Gene Target Rgs18." Blood 122, no. 21 (November 15, 2013): 1191. http://dx.doi.org/10.1182/blood.v122.21.1191.1191.
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Abstract The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis. Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively. Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures: No relevant conflicts of interest to declare.
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Bianchi, Elisa, Roberta Zini, Simona Salati, Elena Tenedini, Ruggiero Norfo, Enrico Tagliafico, Rossella Manfredini, and Sergio Ferrari. "c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression." Blood 116, no. 22 (November 25, 2010): e99-e110. http://dx.doi.org/10.1182/blood-2009-08-238311.
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The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34+ hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb–silenced CD34+ cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34+ cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.
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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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Perrine, Susan P., Rishikesh Mankidy, Michael S. Boosalis, James J. Bieker, and Douglas V. Faller. "EKLF Is Recruited to the γ-Globin Gene Promoter as a Co-Activator and Is Required for γ-Globin Gene Induction by Short-Chain Fatty Acids." Blood 110, no. 11 (November 16, 2007): 1771. http://dx.doi.org/10.1182/blood.v110.11.1771.1771.
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Abstract The erythroid Kruppel-like factor, EKLF, is an essential transcription factor for mammalian β-type globin gene switching, and specifically activates transcription of the adult β-globin gene through binding of its zinc finger domain to the β-globin promoter. We report now that EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives. We found that specific knockdown of EKLF levels by siRNA prevents SCFA induced-expression of an integrated γ-globin promoter in a stably-expressed mLCRβprRluc AγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. In chromatin immunoprecipitation (ChIP) assays, EKLF was found to be actively recruited to the endogenous γ-globin gene promoter with exposure of human erythroid progenitors, and hematopoietic cell lines, to SCFA derivatives. The human SWI/WNF complex is a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure in an ATP-dependent manner. We found that the SWI/SNF complex chromatin-modifying core ATPase BRG1 is also required for γ-globin gene induction by SCFA derivatives. Furthermore, BRG1 is actively recruited to the endogenous γ-globin promoter of human erythroid progenitors with exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings all demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. Recently. we also identified a γ-globin-specific repressor complex, consisting of NCoR and HDAC3, which is displaced from the proximal γ-globin promoter by exposure to SCFA derivatives prior to activation of transcription (Blood, 108:3179–86, 2006). Collectively, these studies identify critical activating and repressing cofactors regulating γ-globin gene expression, and provide new targets for therapeutic interventions.
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Tewari, R. "Erythroid Kruppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta -globin locus control region." EMBO Journal 17, no. 8 (April 15, 1998): 2334–41. http://dx.doi.org/10.1093/emboj/17.8.2334.
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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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Miki, T., N. Kawamata, S. Hirosawa, and N. Aoki. "Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel-like zinc-finger protein." Blood 83, no. 1 (January 1, 1994): 26–32. http://dx.doi.org/10.1182/blood.v83.1.26.26.
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Abstract Chromosomal translocations involving band 3q27 are the recently described nonrandom cytogenetic abnormalities in B-cell malignancies. We have previously cloned the breakpoint region of 3q27, designated as the BCL5 locus, from the B-cell line carrying the t(3;22). The cDNA for the BCL5 gene was cloned from the human liver cDNA library. The nucleotide sequencing analysis showed that the BCL5 gene encodes a potential transcription factor containing six repeats of the Cys2-His2 zinc-finger motif resembling the Drosophila segmentation gene Kruppel. The calculated molecular weight was 78.8 kD, which was supported by an in vitro transcription and translation experiment. A part of the sequence was essentially identical to that of a genomic fragment, ZNF51, previously reported to be located at 3qter. The translocation occurred in the 5′ region of the BCL5 gene, and the protein-coding exons were fused to the Ig-lambda gene in a head-to-head configuration in the cell line carrying t(3;22). The BCL5 cDNA probe detected a major transcript of 3.8 kb in Burkitt's lymphoma cell lines and an aberrant transcript in the t(3;22) cell line, whereas no transcript was detected in myeloid, monocytoid, erythroid, T-lymphoid, and Epstein-Barr virus- immortalized B-lymphoblastoid cell lines.
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Miki, T., N. Kawamata, S. Hirosawa, and N. Aoki. "Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel-like zinc-finger protein." Blood 83, no. 1 (January 1, 1994): 26–32. http://dx.doi.org/10.1182/blood.v83.1.26.bloodjournal83126.
Full textAbstract:
Chromosomal translocations involving band 3q27 are the recently described nonrandom cytogenetic abnormalities in B-cell malignancies. We have previously cloned the breakpoint region of 3q27, designated as the BCL5 locus, from the B-cell line carrying the t(3;22). The cDNA for the BCL5 gene was cloned from the human liver cDNA library. The nucleotide sequencing analysis showed that the BCL5 gene encodes a potential transcription factor containing six repeats of the Cys2-His2 zinc-finger motif resembling the Drosophila segmentation gene Kruppel. The calculated molecular weight was 78.8 kD, which was supported by an in vitro transcription and translation experiment. A part of the sequence was essentially identical to that of a genomic fragment, ZNF51, previously reported to be located at 3qter. The translocation occurred in the 5′ region of the BCL5 gene, and the protein-coding exons were fused to the Ig-lambda gene in a head-to-head configuration in the cell line carrying t(3;22). The BCL5 cDNA probe detected a major transcript of 3.8 kb in Burkitt's lymphoma cell lines and an aberrant transcript in the t(3;22) cell line, whereas no transcript was detected in myeloid, monocytoid, erythroid, T-lymphoid, and Epstein-Barr virus- immortalized B-lymphoblastoid cell lines.
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Xu, Zhixiong, Xianzhang Meng, Ying Cai, and Stephen J. Brandt. "Recruitment of mSin3A and Histone Deacetylase 2 (HDAC2) by the Chromatin Remodeling Protein BRG1 Mediates Transcriptional Repression in Erythroid Progenitors." Blood 104, no. 11 (November 16, 2004): 1601. http://dx.doi.org/10.1182/blood.v104.11.1601.1601.
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Abstract The Swi/Snf chromatin remodeling complexes are critical for activating or repressing transcription of specific genes and require one of two homologous ATPases, Brg1 and Brm. Although Brg1 is also known to be involved in ß-globin gene transcription and a recent report showed that Brg1-containing complexes could be recruited to the ß-globin promoter and its locus control region (LCR), the exact roles of Brg1 in erythroid gene expression and differentiation have not been directly investigated. We previously showed that Brg1, but not Brm, is recruited to the Protein 4.2 (P4.2) promoter by a multi-protein DNA-binding complex containing TAL1/SCL, E47, GATA-1, LMO2, and Ldb1. This complex transactivates P4.2 gene expression through tandem E box-GATA elements in its proximal promoter. We report here that overexpression of Brg1 in murine erythroleukemia (MEL) cells significantly reduced the proportion of benzidine-stained cells and dramatically inhibited expression of mRNA for P4.2 and ß-globin but not glycophorin A and erythroid Kruppel-like factor in MEL cells induced to differentiate with dimethyl sulfoxide (DMSO). Further, overexpression of Brg1 repressed P4.2 promoter activity in a dose-dependent manner in DMSO-treated MEL cells, while treatment of cells with trichostatin A, a specific HDAC inhibitor, attenuated Brg1-directed inhibition of reporter activity in transient transfection analysis. In addition, coimmunoprecipitation analysis showed that Brg1 interacted stably with the corepressor mSin3A and HDAC2 in erythroid cells, suggesting that HDAC activity is involved in Brg1-mediated transcriptional repression. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that Brg1 occupancy of the P4.2 and ß-globin promoters and hypersensitive site 2 (HS2) of the ß-globin LCR decreased upon erythroid differentiation. Importantly, the selective transcriptional repression of P4.2 and ß-globin gene expression by Brg1 correlated with increased loading of mSin3A and HDAC2 on the P4.2 promoter and ß-globin HS2, respectively. Together, these results suggest that Brg1-mediated transcriptional repression of select erythroid genes is characterized by the localized recruitment of a corepressor complex containing mSin3A and HDAC2 in a process of active repression.
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Kapetanaki, Maria G., Deva Sharma, Oluwabukola T. Gbotosho, Valerie Schrott, Frances Weidart, Solomon Fiifi Ofori-Acquah, Grant C. Bullock, and Gregory J. Kato. "The Oxidant Response Transcription Factor NRF2 Mediates Heme Activation of Placenta Growth Factor Expression in Erythroid Cells, a Contributor to Pulmonary Hypertension in Sickle Cell Disease." Blood 126, no. 23 (December 3, 2015): 403. http://dx.doi.org/10.1182/blood.v126.23.403.403.
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Abstract Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PlGF) which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PlGF levels, leading us to hypothesize a mechanistic link between excessive iron and the induction of the PlGF protein. We have published evidence that heme-bound iron stimulates the PlGF promoter up to 400-fold in human K562 cells and in primary human erythroid cells. Gene transfer, knockout and reconstitution experiments have documented the requirement for erythroid Kruppel-like factor (EKLF; KLF1), confirmed by chromatin immunoprecipitation. We are currently investigating the role of other transcription factors known to sense intracellular iron. Using cultured human erythroid cells (K562 cells) treated with heme-bound iron (hemin) and quantitative real-time PCR, we have assessed the time course of heme regulation of the transcripts of several members of the Nrf2-small Maf family of transcription factors. The transcripts for the antioxidant NRF2, NFE2, MafF, MafG and BACH1 are dynamically regulated following exposure of the cells to heme. Gene expression knockdown and small molecule inhibitor and activator experiments have revealed a central role of NRF2 in activating the PlGF promoter in response to heme, further supported by chromatin immunoprecipitation experiments. Specifically, the PlGF promoter is robustly activated by the well characterized non-oxidative NRF2 agonist sulforaphane, and also activated by the nonspecific oxidant hydrogen peroxide. Furthermore, heme activation of the PlGF promoter is inhibited by the NRF2 inhibitor brusatol, by siRNA directed against NRF2, and by the nonspecific antioxidant N-acetyl cysteine. Although the small Maf proteins are often considered to be nonspecific, interchangeable heterodimeric partners of NRF2, our knockdown data suggest a specific role for MafG cooperating with NRF2 in transducing the heme signal on the PlGF promoter. Our knockdown and sulforaphane results also support a role for NRF2 in regulating the transcript levels of all the known NRF2 family members. We have found similarities and differences in the regulation of the PlGF promoter with another well described heme-responsive promoter, heme oxygenase-1. We are currently investigating these regulatory effects on heme on PlGF expression in vivo and in primary erythroid cells from Nrf2 knockout mice. Our results to date support a mechanism in which accelerated heme turnover in sickle cell disease promotes robust expression of PlGF in erythroblasts during erythroid differentiation, through a pathway that involves EKLF, NRF2 and MafG. Our prior published data in collaboration with the Malik lab has documented a causal role for PlGF in promoting expression of the potent vasoconstrictor endothelin-1 accompanied by the development of pulmonary hypertension, corroborated by data from humans with SCD. This mechanism helps to explain the clinical observation that heavily transfused, iron overloaded adults with SCD are more likely to develop pulmonary hypertension, as a potential consequence of excess heme trafficking from turnover of transfused red cells. These results might inspire greater adherence to existing approved therapies to chelate iron in SCD. Disclosures No relevant conflicts of interest to declare.
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Wang, Xunde, Gregory J. Kato, and Laurel Mendelsohn. "Iron Containing Compound Stimulates Expression of Pulmonary Hypertension Promoting Factor PlGF." Blood 118, no. 21 (November 18, 2011): 900. http://dx.doi.org/10.1182/blood.v118.21.900.900.
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Abstract Abstract 900 We have previously reported elevated plasma levels of placental growth factor (PlGF) and endothelin-1 (ET-1) in patients with sickle cell disease with high estimated pulmonary artery systolic pressure, a marker of pulmonary hypertension, and gene transfer experiments in mice document that PlGF stimulates ET-1 expression and pulmonary hypertension. Among other markers, PlGF in SCD subjects is correlated with markers of excessive iron burden, including serum ferritin and transferrin, markers that have been previously associated with pulmonary hypertension in SCD in many studies. We therefore hypothesized that iron stimulates expression of PlGF. Because tissue postnatal expression of PlGF in vivo is restricted primarily to early erythroid cells, we chose K562 erythroleukemia cells as a cell culture model. We find that heme-bound iron (hemin) induces PlGF mRNA robustly in a dose-dependent and time-dependent fashion. The PlGF transcript rises robustly within hours of hemin stimulation, and reaches levels of 40 to 80-fold over baseline in a real time PCR assay. Heme analog compound, mesoporphyrin without iron fails to induce PlGF, but as a control induces hemeoxygenase-1 mRNA. Iron plus mesoporphyrin induces PlGF strongly, indicating a critical requirement for iron in inducing PlGF transcript. In promoter-reporter luciferase constructs transfected into K562 cells, hemin induces the human PlGF promoter significantly, and a minimal promoter construct contains binding sites for erythroid Kruppel-like factor (KLF-1). KLF-1 mRNA levels rise in hemin-stimulated K562 cells in parallel with PlGF, and in peripheral blood mRNA from SCD subjects and healthy controls, the level of KLF-1 transcript correlates closely with PlGF transcript (r=0.82, p<0.0001). Additional studies of KLF-1 are under way. Our results indicate for the first time a specific mechanistic pathway induced by iron that is linked in humans with SCD and in mice to vasculopathy and pulmonary hypertension. Rather a simple epiphenomenon of frequent transfusion or source of nonspecific oxidative stress, iron may trigger a KLF-1/PlGF/ET-1 linear mechanistic pathway to stimulate pulmonary hypertension. This suggests that PlGF levels are linked to the iron overload in patients with SCD. Further details in this pathway are under investigation. 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.
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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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Goodwin, Andrew J., Jane M. McInerney, Michelle A. Glander, Oded Pomerantz, and Christopher H. Lowrey. "In VivoFormation of a Human β-Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1." Journal of Biological Chemistry 276, no. 29 (April 13, 2001): 26883–92. http://dx.doi.org/10.1074/jbc.m008410200.
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Gao, Xiang, Shuangpeng Jiang, Zhangzhen Du, Angtin Ke, Qingwei Liang, and Xu Li. "KLF2 Protects against Osteoarthritis by Repressing Oxidative Response through Activation of Nrf2/ARE Signaling In Vitro and In Vivo." Oxidative Medicine and Cellular Longevity 2019 (November 19, 2019): 1–18. http://dx.doi.org/10.1155/2019/8564681.
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Osteoarthritis (OA) is a multifactorial and inflammatory disease characterized by cartilage destruction that can cause disability among aging patients. There is currently no effective treatment that can arrest or reverse OA progression. Kruppel-like factor 2 (KLF2), a member of the zinc finger family, has emerged as a transcription factor involved in a wide variety of inflammatory diseases. Here, we identified that KLF2 expression is downregulated in IL-1β-treated human chondrocytes and OA cartilage. Genetic and pharmacological overexpression of KLF2 suppressed IL-1β-induced apoptosis and matrix degradation through the suppression of reactive oxygen species (ROS) production. In addition, KLF2 overexpression resulted in increased expression of heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) through the enhanced nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Further, Nrf2 inhibition abrogated the chondroprotective effects of KLF2. Safranin O/fast green and TUNEL staining demonstrated that adenovirus-mediated overexpression of KLF2 in joint cartilage protects rats against experimental OA by inhibiting cartilage degradation and chondrocyte apoptosis. Immunohistochemical staining revealed that KLF2 overexpression significantly decreases MMP13 expression caused by OA progression in vivo. This in vitro and in vivo study is the first to investigate the antioxidative effect and mechanisms of KLF2 in OA pathogenesis. Our results collectively provide new insights into OA pathogenesis regulated by KLF2 and a rationale for the development of effective OA intervention strategies.
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Perrine, Susan P., Rishikesh Mankidy, Michael S. Boosalis, James J. Bieker, and Douglas V. Faller. "Erythroid Kruppel-like factor (EKLF) is recruited to theγ-globingene promoter as a co-activator and is required forγ-globingene induction by short-chain fatty acid derivatives." European Journal of Haematology 82, no. 6 (June 2009): 466–76. http://dx.doi.org/10.1111/j.1600-0609.2009.01234.x.
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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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Bruns, Ingmar, Ulrich Steidl, Guido Kobbe, Roland Fenk, Slawomir Kliszewski, Sabrina Pechtel, Rainer Haas, and Ralf Kronenwett. "Distinct Gene Expression Pattern of CD34+ Stem and Progenitor Cells Mobilized by Pegfilgrastim after Cytotoxic Therapy in Multiple Myeloma in Comparison to G-CSF." Blood 106, no. 11 (November 16, 2005): 5191. http://dx.doi.org/10.1182/blood.v106.11.5191.5191.
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Abstract Background: Current regimens for peripheral blood stem cell (PBSC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of G-CSF starting shortly after cytotoxic therapy. Recently a polyethylenglycole (PEG)-conjugated G-CSF (pegfilgrastim) has been introduced which has a substantially longer half-life than the original formula. Here, we compared the molecular phenotypes of CD34+ stem and progenitor cells mobilized by G-CSF with those mobilized by pegfilgrastim. Study design and Methods: We examined immunomagnetically enriched CD34+ cells from leukapheresis products of 8 patients who received G-CSF and of 8 patients who were given pegfilgrastim using Affymetrix HG Focus GeneChips covering 8793 genes. The statistical scripting language ‘R’ was used for data analysis. Significantly differentially expressed genes were identified with the Significance Analysis of Microarrays (SAM) algorithm. Results: Comparing CD34+ cells mobilized by G-CSF with pegfilgrastim-mobilized CD34+ cells 108 genes were differentially expressed (fold change 1.25 – 14.0, q- value 2.45–14.44%). 38 genes had a higher and 70 genes had a lower expression in CD34+ cells mobilized by G-CSF. We found upregulation of genes characteristic for erythropoietic differentiation including haemoglobin chains and Erythroid Kruppel-like factor in G-CSF-mobilized CD34+ cells. Utilizing clonogenic assays we were able to functionally corroborate this finding as G-CSF-mobilized cells gave rise to a significantly higher number of burst-forming units erythroid (BFU-E) as compared to colony forming units granulocyte-macrophage (CFU-GM) (p=0.016). Cell cycle regulating genes were differentially expressed as well. Genes encoding for proteins that cause cell cycle arrest including human HTm4 were upregulated in G-CSF-mobilized cells, as opposed to an upregulation of cell cycle-promoting genes including Cyclin D2 and Hepatocyte Leukemia Factor (HLF) in pegfilgrastim-mobilized cells. Moreover in pegfilgrastim-mobilized CD34+ cells we saw an upregulation of multiple genes involved in cellular immunogenicity like MHC class I and II antigens and genes encoding for proteins playing a role in antigen presentation. Conclusion: Unconjugated G-CSF seems to be associated with an increased mobilisation of erythroid progenitors or an induction of erythropoiesis. Pegfilgrastim might result in mobilization of more immunogenic CD34+ cells. Unconjugated G-CSF and pegfilgrastim both seem to have an effect on cell cycle. Unconjugated G-CSF might rather induce cell cycle arrest and pegfilgrastim seems to lead to an increase of the cell cycle activity. This may be due to potentially different effects of continuously high serum levels of G-CSF maintained by pegfilgrastim and the pulsatile daily G-CSF injections on CD34+ cells.
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Macari, Elizabeth R., and Christopher H. Lowrey. "Simvastatin and tBHQ Act Synergistically to Increase γ-Globin Gene Expression Through the Transcription Factors KLF2 and NRF2." Blood 118, no. 21 (November 18, 2011): 2149. http://dx.doi.org/10.1182/blood.v118.21.2149.2149.
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Abstract Abstract 2149 While increased fetal hemoglobin (HbF) levels have proven therapeutic benefit for people with sickle cell disease and β-thalassemia, none of the current HbF inducing agents have the optimal combination of safety, efficacy and ease of use that would make them applicable to most hemoglobinopathy patients. In an effort to develop new strategies for HbF induction, we have recently shown that drugs that activate the NF-E2 related factor 2 (NRF2)/antioxidant response signaling pathway stimulate HbF production in primary human erythroid cells. This discovery prompted us to investigate ways to further enhance HbF levels achieved with NRF2 activators alone. Recent reports from the cardiovascular literature have uncovered a synergy between Kruppel-like factor 2 (KLF2) and NRF2. In vascular endothelial cells, shear stress induces a battery of genes that protect against atherosclerotic cardiovascular disease and this induction is mediated by the transcription factors KLF2 and NRF2 and includes synergistic activation of NRF2 target genes by the two factors. Interestingly, HMG-CoA reductase inhibitors (statins), are strong activators of the transcription factor, KLF2. These findings suggested to us that combining statins with drugs that activate NRF2 signaling might synergistically activate γ-globin gene expression and HbF production. An additional rationale for this approach is that several NRF2 activating drugs are either already approved or undergoing clinical testing and that statins are among the most widely used drugs. To test this hypothesis, we first treated K562 cells with various concentrations of simvastatin and observed a dose dependent increase in KLF2 mRNA and protein expression, with 5μM statin resulting in more than 200-fold increase in steady state mRNA levels but no change in γ-globin mRNA. When combined with tBHQ, 5μM statin synergistically increased γ-globin levels compared to either drug alone at 24 and 48hrs. To investigate the specificity of this synergy, we created a stable K562 cell line that overexpressed murine klf2. Treating these cells with tBHQ enhanced γ-globin expression compared to tBHQ treated WT K562 cells, reproducing the effect we saw with statin and tBHQ combination treatment in WT K562 cells. This suggests that KLF2 is responsible for the synergistic effects of statin when combined with tBHQ. To further investigate the mechanism of statin action we performed KLF2 and NRF2 ChIP studies. Statin treatment strongly increased KLF2 binding to HS2 of the β-globin LCR (30-fold over IgG) while tBHQ induced NRF2 binding to the NF-E2 region of LCR HS2 30-fold and 10-fold over IgG in K562 and in primary human erythroid cells, respectively. Binding at HS1, HS3 or HS4 was not increased for either factor. In a single experiment performed so far, combined tBHQ and statin treatment of differentiating primary human erythroid cells increased KLF2 and NRF2 target gene NQO1 mRNA. γ-globin mRNA was induced to levels equivalent to those seen with 5-azacytidine. These data provide preliminary evidence suggesting that combining NRF2 activators with widely used statins may be a safe and effective way to achieve therapeutic HbF levels in β-thalassmia and sickle cell disease patients. Disclosures: No relevant conflicts of interest to declare.
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Peralta, Raechel, Audrey Low, Aneeza Kim, Sue Murray, Shuling Guo, Sue Freier, Tim M. Townes, and Gene Hung. "Targeting BCL11A and KLF1 For The Treatment Of Sickle Cell Disease and β-Thalassemia In Vitro using Antisense Oligonucleotides." Blood 122, no. 21 (November 15, 2013): 1022. http://dx.doi.org/10.1182/blood.v122.21.1022.1022.
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Abstract Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis. B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression. To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures: Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.
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Kalra, Inderdeep S., Md M. Alam, and Betty S. Pace. "Transcriptional Regulation of γ-Globin Gene Expression by KLF4." Blood 116, no. 21 (November 19, 2010): 645. http://dx.doi.org/10.1182/blood.v116.21.645.645.
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Abstract Abstract 645 Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in various gene promoters. The CACCC element is also critical for developmental regulation of the human γ-globin and β-globin genes; therefore studies to identify transcription factors that bind the CACCC element to alter gene expression are desirable. By microarray-based gene profiling, we identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased simultaneously with γ-globin silencing during in vitro erythroid maturation. Subsequent reverse transcription quantitative PCR (RT-qPCR) analysis confirmed KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively in erythroid progenitors from day 7 to day 28 with over 90% γ-globin gene silencing. The effects of known fetal hemoglobin inducers hemin (50μM) and sodium butyrate (2mM) on KLF factor expression was tested in K562 cells. Hemin and sodium butyrate increased KLF4 3-fold (p<0.05) and 13-fold (p<0.01) respectively while KLF12 was only induced by butyrate. Likewise, hemin treatment of KU812 leukemia cells, which actively express γ-globin and β-globin, produced a 7-fold increase in KLF4 (p<0.05) while KLF12 levels were not changed suggesting KLF4 may be directly involved in γ-globin gene regulation. To characterize its role further siRNA-mediated loss of function studies were performed in K562 cells. A 60% knockdown of KLF4 expression produced 40% attenuation of γ-globin transcription (p<0.05). To confirm this effect, rescue experiments were performed as follows: K562 cells were treated with 100nM siKLF4 alone or in combination with the pMT3-KLF4 expression vector (10 and 20μg) for 48 hrs. The 40% knockdown of γ-globin expression produced by siKLF4 was rescued to baseline levels after enforced pMT3-KLF4 expression (p<0.05). To establish whether KLF4 directly stimulates γ-globin promoter activity, we performed co-transfection of pMT3-KLF4 and the Gγ-promoter (-1500 to +36) cloned into the pGL4.17 Luc2/neo vector; a dose-dependent increase in luciferase activity (2- to 5-fold; p<0.001) was observed. Furthermore, enforced expression of pMT3-KLF4 augmented endogenous γ-globin expression 2-fold (p<0.01). Collectively, these studies suggest that KLF4 acts as a trans-activator of γ-globin gene transcription. To address the physiological relevance of these findings, studies were extended to human primary erythroid cells grown in a two-phase liquid culture system. At day 11 when γ-globin gene expression was maximal, siKLF4 treatment produced a 60% decrease in γ/β-globin mRNA levels (p<0.001). By contrast, enforced pMT3-KLF4 expression enhanced γ/β-globin 1.5-fold at day 11 and day 28 (after γ-globin silencing); HbF levels were induced 1.5-fold (p<0.05) which was demonstrated by enzyme-linked immunosorbent assay. To gain insights into the molecular mechanism of KLF4-mediated γ-globin regulation, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) were completed. Since CREB binding protein (CBP) is known to function as a co-activator for KLF1, 4 and 13, we also tested its role in γ-globin gene regulation. EMSA performed with K562 nuclear extract and a [γ-32P] labeled γ-CACC probe (-155 to -132 relative to the γ-globin cap site) produced three DNA-protein complexes; the addition of KLF4 or CBP antibody resulted in a marked decrease in intensity of all complexes suggesting these factors bind the γ-CACC element. ChIP assay demonstrated 10-fold and 20-fold chromatin enrichment with KLF4 and CBP antibody respectively (p<0.001) confirming in vivo binding at the γ-CACC region. Lastly, co-immunoprecipitation established protein-protein interaction between KLF4 and CBP in K562 cells. Future studies will investigate the role of CBP in KLF4-mediated γ-globin regulation which will provide molecular targets for fetal hemoglobin induction and treatment of sickle cell anemia and β-thalassemia. Disclosures: No relevant conflicts of interest to declare.
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Lee, J. S., H. Ngo, D. Kim, and J. H. Chung. "Erythroid Kruppel-like factor is recruited to the CACCC box in the beta -globin promoter but not to the CACCC box in the gamma -globin promoter: The role of the neighboring promoter elements." Proceedings of the National Academy of Sciences 97, no. 6 (March 7, 2000): 2468–73. http://dx.doi.org/10.1073/pnas.040476297.
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Mitchell, W. Beau, Merlin Nithya Gnanapragasam, Julie A. Jaffray, James J. Bieker, and Deepa Manwani. "Case Report of Erythroid Transcription Factor EKLF Mutation Causing a Rare Form of Congenital Dyserythropoetic Anemia in a Patient of Taiwanese Origin." Blood 118, no. 21 (November 18, 2011): 2154. http://dx.doi.org/10.1182/blood.v118.21.2154.2154.
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Abstract Abstract 2154 Congenital dyserythropoietic anemias (CDA) are a rare, inherited form of blood disorders characterized by dyserythropoiesis in the bone marrow, anemia, jaundice and splenomegaly. There are three major types of CDAs, although rarer variants have been identified. We describe a patient with an unusual type of CDA, of which only four other cases have been reported. Our patient had severe hemolytic anemia, increased fetal hemoglobin and abnormal bone marrow pathology inconsistent with previously described forms of CDA. On further study he was also found to have a mutation in KLF1, the gene encoding Erythroid-Kruppel like growth factor (EKLF). Here we describe the full clinical characteristics of our patient and define the diagnostic clinical features of this new variant of CDA by comparing with one of the previously reported patients designated as ME in the table. EKLF is an erythroid specific transcription factor that is essential for b-globin expression, the switch from fetal to adult globin and definitive erythropoiesis (Siatecka, M and Bieker, J. Blood prepublished May 2011). Various mutations in KLF1 have been identified, some causing the benign In(Lu) type of Lu blood group phenotype. Recently, a missense, dominant-negative KLF1 mutation was reported, c.973G>A, which resulted in a previously unidentified type of CDA (Singleton et al. ASH Abstract 162, 2009; Arnaud et al. Am J Hum Genet 2010,87:721-727). The G-to-A transition in exon 3 of KLF1 results in the substitution of glutamate 325 by a lysine (E325K) in the second zinc finger. The mutated area of the zinc finger was found to be essential for binding of EKLF to DNA motifs causing a profound dysregulation of globin gene expression. The mutation was found to have a dominant-negative effect on the transcriptional activity of EKLF, thus making the heterozygous patients symptomatic. Our patient, JL, is an 8 year old male Taiwanese immigrant found to have hyperbilirubinemia and anemia at birth. He is a developmentally normal child with height 10th centile, weight in the 25th centile and spleen palpable to his suprapubic area. He has chronic hemolytic anemia, with baseline hemoglobin 7–9 g/dL, MCV 83 fL and RDW 22% and reticulocyte count 16%. Peripheral blood smear shows marked anisopoikilocytosis, schistocytes, mild polychromasia and nucleated RBCs, many with double nuclei. Bone marrow aspirate revealed a hypercellular marrow with erythroid hyperplasia and dyserythropoiesis. Electron microscopy analysis of the bone marrow showed rare immature erythroid cells with marked heterochromatin. Several cells showed a peripheral double membrane of the cytoplasm and there was rare invagination of nuclear membrane with intranuclear precipitated material. JL received blood transfusions every 2 months for the first 3 years of his life while living in Taiwan. Since moving to the U.S. in 2003, he has only received 2 transfusions secondary to aplastic crisis. Osmotic fragility testing showed mildly increased increased fragility. Hemoglobin electrophoresis revealed an elevated fetal hemoglobin level of 42%. Gene analysis for alpha or beta globin mutations was negative. RBC enzyme testing revealed an ADA of 6.1 and decreased FFK. Due to the unique combination of anemia, elevated fetal hemoglobin, and bone marrow morphology suggestive of, but not fully diagnostic for CDA I, II or III, we tested his EKLF gene and identified the heterozygous E325K mutation. JL's two siblings, mother and father have normal hemoglobin levels and peripheral blood smears. They also have normal In(Lu) blood group phenotype. Since the E325K mutation is dominant-negative, his phenotypically normal family members were not tested for the mutation. Four other patients have also been identified as having an E325K mutation in their EKLF gene. The patients had severe hemolytic anemia, elevated fetal hemoglobin, and bone marrow morphology showing dyserythropoiesis. Patients' erythrocytes also had low CD44 and water channel AQP1 expression, which is known to be regulated by EKLF. One patient was described with multiple congenital anomalies: hepatomegaly, micropenis, hypospadias, enlarged fontanel and hypertelorism. Taken together, the key clinical characteristics of this rare CDA are: severe normocytic anemia, highly elevated Hb F, presence of nucleated RBCs in the peripheral blood, erythroid hyperplasia with limited dyserythropoiesis in the bone marrow, splenomegaly, and growth delay. Disclosures: No relevant conflicts of interest to declare.