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Andrew C Perkins - One of the best experts on this subject based on the ideXlab platform.
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KLF1 acts as a pioneer transcription factor to open chromatin and facilitate recruitment of gata1
Blood, 2018Co-Authors: Kevin R Gillinder, Graham Magor, Charles C Bell, Melissa Ilsley, Stephen Huang, Andrew C PerkinsAbstract:Only a small subset of transcription factors (TFs) can act as pioneer factors; i.e. those that can 9open9 otherwise 9closed9 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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KLF1 null neonates display hydrops fetalis and a deranged erythroid transcriptome
Blood, 2014Co-Authors: Graham Magor, Michael R Tallack, Kevin R Gillinder, Charles C Bell, Naomi Mccallum, Andrew C Perkins, Bronwyn WilliamsAbstract:We describe a case of severe neonatal anemia with kernicterus caused by compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1-null human. The phenotype of severe nonspherocytic hemolytic anemia, jaundice, hepatosplenomegaly, and marked erythroblastosis is more severe than that present in congenital dyserythropoietic anemia type IV as a result of dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found that human KLF1 acts like mouse KLF1 to coordinate expression of many genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell-cycle regulators, and blood group antigens. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. We also identify new roles for KLF1 in autophagy, global transcriptional control, and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.
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new insights into the mechanism of dominant anemia caused by zinc finger mutations in KLF1
Blood, 2014Co-Authors: Andrew C Perkins, Graham Magor, Michael R Tallack, Kevin R Gillinder, Mathieu Lajoie, Melissa Ilsley, Michael J Landsberg, Timothy L Bailey, Joel P Mackay, James J BiekerAbstract:Kruppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor [1, 2]. A number of studies have shown up to ~700 genes are poorly expressed when KLF1 is absent [3-6]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF1 [7]. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF1 [8]. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (= E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation [9]. To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1 nan into an erythroid cell line derived from KLF1 -/- fetal liver cells [10]. We performed ChIP-seq to determine differences in genome occupancy in vivo , and identified novel sites occupied by EKLF-E339D but not by wild type KLF1. Using de novo motif discovery [11], we find KLF1 nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to any known TFs, so we think it represents a sequence specificity not normally encoded in mammals. Ectopic binding to non-erythroid gene promoters is accompanied by aberrant gene expression as determined by 4sU labelling and deep sequencing of tamoxifen-induced primary nuclear RNAs. We find a 4-fold greater number of genes induced by KLF1-nan compared with wild type KLF1 which is consistent with degenerate genome occupancy. We compared the KLF1-nan dependent genes with RNA-seq performed in primary fetal liver for KLF1 +/nan versus KLF1 +/- mice. We confirmed aberrant binding using EMSA and surface plasmon resonance (SPR) using recombinant GST-KLF1 zinc finger domains expressed in E.coli. The degenerate motif is consistent with structural models of how the second zinc finger of KLF1 specifically interacts with its binding site [12, 13]. We are undertaking structural studies to confirm this modelling. Together RNA-seq, ChIP-seq and SPR studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. To our knowledge this mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel. References: 1.Perkins, A.C., A.H. Sharpe, and S.H. Orkin. Nature, 1995. 375 (6529): p. 318-22. 2.Nuez, B., et al., Nature, 1995. 375 (6529): p. 316-8. 3.Pilon, A.M., et al., Mol Cell Biol, 2006. 26 (11): p. 4368-77. 4.Drissen, R., et al., Mol Cell Biol, 2005. 25 (12): p. 5205-14. 5.Hodge, D., et al., Blood, 2006. 107 (8): p. 3359-70. 6.Tallack, M.R., et al., Genome Res, 2012. 22 (12):2385-98 7.Arnaud, L., et al., Am J Hum Genet. 87 (5): p. 721-7. 8.Siatecka, M., et al., Proc Natl Acad Sci U S A. 2010. 107 (34):15151-6 9.Heruth, D.P., et al., Genomics, 2010. 96 (5): p. 303-7. 10.Coghill, E., et al., Blood, 2001. 97 (6): p. 1861-1868. 11.Bailey, T.L., et al., Nucleic Acids Res, 2009. 37 (Web Server issue): p. W202-8. 12.Schuetz, A., et al., Cell Mol Life Sci, 2011. 68 (18): p. 3121-31. 13.Oka, S., et al., Biochemistry, 2004. 43 (51): p. 16027-35. Disclosures No relevant conflicts of interest to declare.
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mutations in the zinc finger domain of human and mouse KLF1 cause congenital dyserythropoietic anemia cda via promiscuous dna binding and ectopic target gene expression
Blood, 2013Co-Authors: Kevin R Gillinder, Graham Magor, Michael R Tallack, Mathieu Lajoie, Melissa Ilsley, Michael J Landsberg, Timothy L Bailey, Andrew C PerkinsAbstract:Kruppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor1, 2. A number of studies have shown up to ∼700 genes are poorly expressed when KLF1 is absent3-6. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF17. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF18. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (i.e. E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation9. To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1nan into an erythroid cell line derived from KLF1 -/- fetal liver cells10. We performed ChIP-seq to determine genome occupancy site preferences for KLF1 and KLF1nan. We identified about 4-fold the number of binding sites within the genome for KLF1nan versus KLF1; many of these are ectopic or promiscuous. Using de novo motif discovery11, we find KLF1nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to known TFs, so we think it represents specificity not normally present in mammals. The degenerate motif is consistent with models of how the second zinc finger of KLF1 specifically interacts with the 9bp consensus binding site12,13. We also isolated nascent RNA from wild type and mutant cells, to identify primary transcriptional targets of KLF1 and aberrant targets of the KLF1nanmutation. We performed primary transcript RNA-seq and validation using RT-PCR of pre-processed nuclear transcripts. Together the RNA-seq and ChIP-seq studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. This mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel. References: 1. Perkins, A.C., A.H. Sharpe, and S.H. Orkin. Nature, 1995. 375 (6529): p. 318-22. 2. Nuez, B., et al., Nature, 1995. 375 (6529): p. 316-8. 3. Pilon, A.M., et al., Mol Cell Biol, 2006. 26 (11): p. 4368-77. 4. Drissen, R., et al., Mol Cell Biol, 2005. 25 (12): p. 5205-14. 5. Hodge, D., et al., Blood, 2006. 107 (8): p. 3359-70. 6. Tallack, M.R., et al., Genome Res, 2012. 22 (12):2385-98 7. Arnaud, L., et al., Am J Hum Genet. 87 (5): p. 721-7. 8. Siatecka, M., et al., Proc Natl Acad Sci U S A. 2010. 107 (34):15151-6 9. Heruth, D.P., et al., Genomics, 2010. 96 (5): p. 303-7. 10. Coghill, E., et al., Blood, 2001. 97 (6): p. 1861-1868. 11. Bailey, T.L., et al., Nucleic Acids Res, 2009. 37 (Web Server issue): p. W202-8. 12. Schuetz, A., et al., Cell Mol Life Sci, 2011. 68 (18): p. 3121-31. 13. Oka, S., et al., Biochemistry, 2004. 43 (51): p. 16027-35. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.
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dynamics and mechanics of KLF1 regulation in erythropoiesis
Blood, 2013Co-Authors: Kevin R Gillinder, Graham Magor, Michael R Tallack, Melissa Ilsley, Jim R Hughes, James O J Davies, Douglas R Higgs, Andrew C PerkinsAbstract:Kruppel-like factor-1 (KLF1) is a C2H2 zinc finger transcription factor which is essential for broad erythroid gene expression and erythropoiesis in vivo . A number of studies have shown ∼700 genes are poorly expressed when KLF1 is absent [1-8]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice [9,10], and partly responsible for congenital anemia in humans and mice with dominant mutations in KLF1 [11,12]. To determine whether KLF1-dependent genes are direct or indirect targets of KLF1, we have previously performed global ChIP-seq experiments identifying 945-1350 regions of KLF1 occupancy in the mouse genome [7]. About 15% of these regions fall within the promoters of KLF1 target genes but surprisingly, most are thousands of kilobases distant from any known gene. Many of these distant sites exhibit co-occupancy with other transcriptional regulators involved in erythropoiesis, including GATA1. Approximately half of the KLF1 occupied sites are found within regions of mono-methylation of lysine 4 on histone 3 (H3K4me1). These regions are devoid of histones tri-methylated at the same residue (H3K4me3). This methylation signature is commonly associated with regions of the genome that act as transcriptional enhancers [13,14] and many are also bound by the co-activator, p300. The nature and function of these distant sites, particularly those without enhancer marks, is interesting as they may shed light on novel mechanisms of action of KLF1 and associated transcription factors. The transcriptional machinery of the cell, including many transcription factors is found in large sub-nuclear compartments called transcriptional factories [15]. KLF1 has been found localized to a subset of these in erythroid cells. KLF1 is also required for long-range looping of the β-globin gene into these transcription factories [16]. Other erythroid genes involved in the production of a functional haemoglobin molecule such as α-globin and haem synthesis enzymes are often found in the same transcription factory. This strongly suggests KLF1 can employ this sub-nuclear machine to co-ordinate the transcriptional output from many genes and thereby direct erythroid cell differentiation. To explore the function of KLF1-bound loci, we have performed multiplexed chromosome conformation capture (3C) coupled with sequencing (Capture-seq) using a tamoxifen responsive, KLF1 inducible cell line to investigate the role of KLF1 in chromosomal looping. In addition, we have analysed primary transcriptional output of KLF1 target genes by nascent RNA-seq. As expected β-globin and a-globin transcription is rapidly induced, becoming detectable within 5 minutes. However, the transcriptional response of dematin and a set direct KLF1 target genes is much slower. Thus, the mechanism of KLF1 transcriptional activation differs between target gene loci. We find a dynamic role of KLF1-dependent chromosomal looping and transcriptional co-factor recruitment required to effect gene transcription during erythropoiesis. We will discuss models of differentiation transcription regulation by KLF1. References: 1. Drissen R, et al. (2005). Molecular and Cellular Biology 25: 5205–5214. 2. Funnell APW, et al. (2007). Molecular and Cellular Biology 27: 2777–2790. 3. Hodge D, et al. (2006). Blood 107: 3359–3370. 4. Pilon AM, et al. (2008). Molecular and Cellular Biology 28: 7394–7401. 5. Siatecka M, et al. (2010). PNAS 107: 15151–15156. 6. Siatecka M, Bieker JJ (2011). Blood 118: 2044–2054. 7. Tallack MR, et al. (2010). Genome Res 20: 1052–1063. 8. Tallack MR, Perkins AC (2010). IUBMB Life 62: 886–890. 9. Perkins AC, Sharpe AH, Orkin SH (1995). Nature 375: 318–322. 10. Nuez B, et al. (1995). Nature 375: 316–318. 11. Arnaud L, S et al. (2010). Am J Hum Genet 87: 721–727. 12. Borg J, et al. (2011). Haematologica 96: 635–638. 13. Zentner GE, et al. (2011). Genome Res 21: 1273–1283. 14. Pekowska A, et al. (2011). EMBO J 30: 4198–4210. 15. Osborne CS, et al. (2004). Nat Genet 36: 1065–1071. 16. Schoenfelder S, et al. (2010). Nat Genet 42: 53–61. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.
Mukesh K. Jain - One of the best experts on this subject based on the ideXlab platform.
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the kruppel like factor klf2 inhibits peroxisome proliferator activated receptor gamma expression and adipogenesis
Journal of Biological Chemistry, 2003Co-Authors: Sucharita Sen Banerjee, Susan Gray, Mark W Feinberg, Masafumi Watanabe, Richard L Haspel, Diane J Denkinger, Rodney S Kawahara, Hans Hauner, Mukesh K. JainAbstract:Abstract Obesity is an important public health problem associated with a number of disease states such as diabetes and arteriosclerosis. As such, an understanding of the mechanisms governing adipose tissue differentiation and function is of considerable importance. We recently reported that the Kruppel-like zinc finger transcription factor KLF15 can induce adipocyte maturation and GLUT4 expression. In this study, we identify that a second family member, KLF2/Lung Kruppel-like factor (LKLF), as a negative regulator of adipocyte differentiation. KLF2 is highly expressed in adipose tissue, and studies in cell lines and primary cells demonstrate that KLF2 is expressed in preadipocytes but not mature adipocytes. Constitutive overexpression of KLF2 but not KLF15 potently inhibits peroxisome proliferator-activated receptor-γ (PPARγ) expression with no effect on the upstream regulators C/EBPβ and C/EBPδ. However, the expression of C/EBPα and SREBP1c/ADD1 (adipocyte determination and differentiation factor-1/sterol regulatory element-binding protein-1), two factors that feedback in a positive manner to enhance PPARγ function, was also markedly reduced. In addition, transient transfection studies show that KLF2 directly inhibits PPARγ2 promoter activity (70% inhibition;p < 0.001). Using a combination of promoter mutational analysis and gel mobility shift assays, we have identified a binding site within the PPARγ2 promoter, which mediates this inhibitory effect. These data identify a novel role for KLF2 as a negative regulator of adipogenesis.
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the kruppel like factor klf2 inhibits peroxisome proliferator activated receptor γ expression and adipogenesis
Journal of Biological Chemistry, 2003Co-Authors: Sucharita Sen Banerjee, Susan Gray, Mark W Feinberg, Masafumi Watanabe, Richard L Haspel, Diane J Denkinger, Rodney S Kawahara, Hans Hauner, Mukesh K. JainAbstract:Obesity is an important public health problem associated with a number of disease states such as diabetes and arteriosclerosis. As such, an understanding of the mechanisms governing adipose tissue differentiation and function is of considerable importance. We recently reported that the Kruppel-like zinc finger transcription factor KLF15 can induce adipocyte maturation and GLUT4 expression. In this study, we identify that a second family member, KLF2/Lung Kruppel-like factor (LKLF), as a negative regulator of adipocyte differentiation. KLF2 is highly expressed in adipose tissue, and studies in cell lines and primary cells demonstrate that KLF2 is expressed in preadipocytes but not mature adipocytes. Constitutive overexpression of KLF2 but not KLF15 potently inhibits peroxisome proliferator-activated receptor-γ (PPARγ) expression with no effect on the upstream regulators C/EBPβ and C/EBPδ. However, the expression of C/EBPα and SREBP1c/ADD1 (adipocyte determination and differentiation factor-1/sterol regulatory element-binding protein-1), two factors that feedback in a positive manner to enhance PPARγ function, was also markedly reduced. In addition, transient transfection studies show that KLF2 directly inhibits PPARγ2 promoter activity (70% inhibition;p
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the kruppel like factor KLF15 regulates the insulin sensitive glucose transporter glut4
Journal of Biological Chemistry, 2002Co-Authors: Susan Gray, Sucharita Sen Banerjee, Mark W Feinberg, Masafumi Watanabe, Richard L Haspel, Sarah C Hull, Chay T Kuo, Ana S Depina, Mukesh K. JainAbstract:Resistance to the stimulatory effects of insulin on glucose utilization is a key feature of type 2 diabetes, obesity, and the metabolic syndrome. Recent studies suggest that insulin resistance is primarily caused by a defect in glucose transport. GLUT4 is the main insulin-responsive glucose transporter and is expressed predominantly in muscle and adipose tissues. Whereas GLUT4 has been shown to play a critical role in maintaining systemic glucose homeostasis, the mechanisms regulating its expression are incompletely understood. We have cloned the murine homologue of KLF15, a member of the Kru¨ppel-like family of transcription factors. KLF15 is highly expressed in adipocytes and myocytes in vivo and is induced when 3T3-L1 preadipocytes are differentiated into adipocytes. Overexpression of KLF15 in adipose and muscle cell lines potently induces GLUT4 expression. This effect is specific to KLF15 as overexpression of two other Kru¨ppel-like factors, KLF2/LKLF and KLF4/GKLF, did not induce GLUT4 expression. Both basal (3.3-fold, p
Clinton Jones - One of the best experts on this subject based on the ideXlab platform.
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pioneer transcription factors progesterone receptor and kruppel like transcription factor 4 cooperatively stimulate the bovine herpesvirus 1 icp0 early promoter and productive late protein expression
Virus Research, 2020Co-Authors: Laximan Sawant, Nishani Wijesekera, Clinton JonesAbstract:Bovine herpesvirus 1 (BoHV-1), including commercially available modified live vaccines, readily infect the fetus and ovaries, which can cause reproductive failure. The BoHV-1 latency-reactivation cycle in sensory neurons further complicates reproductive failure because progesterone sporadically induces reactivation from latency. The progesterone receptor (PR) and Kruppel-like transcription factor 15 (KLF15) cooperatively stimulate productive infection and the immediate early transcription unit 1 (IEtu1) promoter. In addition to the IEtu1 promoter, the bICP0 gene also contains a separate early (E) promoter. In this study, we tested the hypothesis that PR and KLF family members transactivate the bICP0 E promoter. PR and KLF4 stimulated bICP0 E promoter activity and expression of late productive viral protein expression in a cooperative manner. Additional studies revealed three enhancer domains within the bICP0 E promoter were responsive to PR and KLF4. Chromatin immunoprecipitation studies demonstrated PR and KLF4 occupy bICP0 E promoter sequences in transfected Neuro-2A cells and at late times following infection of bovine kidney cells. Co-immunoprecipitation studies indicated PR and KLF4 stably interact with each other. These studies suggest cooperative activation of the bICP0 E promoter by PR and KLF4 correlate with interactions between these pioneer transcription factors.
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two pioneer transcription factors kruppel like transcription factor 4 and glucocorticoid receptor cooperatively transactivate the bovine herpesvirus 1 icp0 early promoter and stimulate productive infection
Journal of Virology, 2019Co-Authors: Fouad S Elmayet, Laximan Sawant, Prasanth Thunuguntla, Jing Zhao, Clinton JonesAbstract:An important site for bovine herpesvirus 1 (BoHV-1) latency is sensory neurons within trigeminal ganglia (TG). The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. Expression of four Kruppel-like transcription factors (KLF), i.e., KLF4, KLF6, PLZF (promyelocytic leukemia zinc finger), and KLF15, are induced in TG neurons early during dexamethasone-induced reactivation. The glucocorticoid receptor (GR) and KLF15 form a feed-forward transcription loop that cooperatively transactivates the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter that drives bovine infected cell protein 0 (bICP0) and bICP4 expression. Since the bICP0 gene also contains a separate early (E) promoter, we tested the hypothesis that GR and KLF family members transactivate the bICP0 E promoter. GR and KLF4, both pioneer transcription factors, cooperated to stimulate bICP0 E promoter activity in a ligand-independent manner in mouse neuroblastoma cells (Neuro-2A). Furthermore, GR and KLF4 stimulated productive infection. Mutating both half GR binding sites did not significantly reduce GR- and KLF4-mediated transactivation of the bICP0 E promoter, suggesting that a novel mechanism exists for transactivation. GR and KLF15 cooperatively stimulated bICP0 activity less efficiently than GR and KL4: however, KLF6, PLZF, and GR had little effect on the bICP0 E promoter. GR, KLF4, and KLF15 occupied bICP0 E promoter sequences in transfected Neuro-2A cells. GR and KLF15, but not KLF4, occupied the bICP0 E promoter at late times during productive infection of bovine cells. Collectively, these studies suggest that cooperative transactivation of the bICP0 E promoter by two pioneer transcription factors (GR and KLF4) correlates with stimulating lytic cycle viral gene expression following stressful stimuli.IMPORTANCE Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes lifelong latency in sensory neurons. Reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone. We predict that increased corticosteroid levels activate the glucocorticoid receptor (GR). Consequently, viral gene expression is stimulated by the activated GR. The immediate early transcription unit 1 promoter (IEtu1) drives expression of two viral transcriptional regulatory proteins, bovine infected cell protein 0 (bICP0) and bICP4. Interestingly, a separate early promoter also drives bICP0 expression. Two pioneer transcription factors, GR and Kruppel-like transcription factor 4 (KLF4), cooperatively transactivate the bICP0 early (E) promoter. GR and KLF15 cooperate to stimulate bICP0 E promoter activity but significantly less than GR and KLF4. The bICP0 E promoter contains enhancer-like domains necessary for GR- and KLF4-mediated transactivation that are distinct from those for GR and KLF15. Stress-induced pioneer transcription factors are proposed to activate key viral promoters, including the bICP0 E promoter, during early stages of reactivation from latency.
James J Bieker - One of the best experts on this subject based on the ideXlab platform.
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KLF1 eklf expression in acute leukemia is correlated with chromosomal abnormalities
Blood Cells Molecules and Diseases, 2020Co-Authors: Adnan Mansoor, Mohammad Omer Mansoor, Jay Patel, Shuchun Zhao, Yasodha Natkunam, James J BiekerAbstract:KLF1 (EKLF) is a master regulator of erythropoiesis and controls expression of a wide array of target genes. We interrogated human tissue microarray samples via immunohistological analysis to address whether levels of KLF1 protein are associated with leukemia. We have made the unexpected findings that higher KLF1 levels are correlated with cells containing abnormal chromosomes, and that high KLF1 expression is not limited to acute myeloid leukemia (AML) associated with erythroid/megakaryoblastic differentiation. Expression of KLF1 is associated with poor survival. Further analyses reveal that KLF1 directly regulates a number of genes that play a role in chromosomal integrity. Together these results suggest that monitoring KLF1 levels may provide a new marker for risk stratification and prognosis in patients with AML.
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a kruppel like factor 1 KLF1 mutation associated with severe congenital dyserythropoietic anemia alters its dna binding specificity
Molecular and Cellular Biology, 2020Co-Authors: Klaudia Kulczynska, James J Bieker, Miroslawa SiateckaAbstract:Kruppel-like factor 1 (KLF1/EKLF) is a transcription factor that globally activates genes involved in erythroid cell development. Various mutations are identified in the human KLF1 gene. The E325K mutation causes congenital dyserythropoietic anemia (CDA) type IV, characterized by severe anemia and non-erythroid-cell-related symptoms. The CDA mutation is in the second zinc finger of KLF1 at a position functionally involved in its interactions with DNA. The molecular parameters of how CDA-KLF1 exerts its biological effects have not been addressed. Here, using an in vitro selection strategy, we determined the preferred DNA-binding site for CDA-KLF1. Binding to the deduced consensus sequence is supported by in vitro gel shifts and by in vivo functional reporter gene studies. Two significant changes compared to wild-type (WT) binding are observed: G is selected as the middle nucleotide, and the 3' portion of the consensus sequence is more degenerate. As a consequence, CDA-KLF1 did not bind the WT consensus sequence. However, activation of ectopic sites is promoted. Continuous activation of WT target genes occurs if they fortuitously contain the novel CDA site nearby. Our findings provide a molecular understanding of how a single mutation in the KLF1 zinc finger exerts effects on erythroid physiology in CDA type IV.
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a kruppel like factor 1 KLF1 mutation associated with severe congenital dyserythropoietic anemia alters its dna binding specificity
bioRxiv, 2019Co-Authors: Klaudia Kulczynska, James J Bieker, Miroslawa SiateckaAbstract:Abstract Kruppel-like factor 1 (KLF1/EKLF) is a transcription factor that globally activates genes involved in erythroid cell development. Various mutations are identified in the human KLF1 gene. The E325K mutation causes congenital dyserythropoietic anemia (CDA) type IV, characterized by severe anemia and non-erythroid-related symptoms. The CDA mutation is in the second zinc finger of KLF1 at a position functionally involved in its interactions with DNA. The molecular parameters of how CDA-KLF1 exerts its biological effects have not been addressed. Here, using an in vitro selection strategy we determined the preferred DNA-binding site for CDA-KLF1. Binding to the deduced consensus sequence is supported by in vitro gel shifts and by in vivo functional reporter gene studies. Two significant changes compared to WT binding are observed: G is selected as the middle nucleotide and the 3’-portion of the consensus sequence is more degenerate. As a consequence CDA-KLF1 did not bind the WT consensus sequence. However, activation of ectopic sites is promoted. Continuous activation of WT target genes occurs if they fortuitously contain the novel CDA site nearby. Our findings provide a molecular understanding of how a single mutation in the KLF1 zinc finger exerts an effects on erythroid physiology in CDA type IV.
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new insights into the mechanism of dominant anemia caused by zinc finger mutations in KLF1
Blood, 2014Co-Authors: Andrew C Perkins, Graham Magor, Michael R Tallack, Kevin R Gillinder, Mathieu Lajoie, Melissa Ilsley, Michael J Landsberg, Timothy L Bailey, Joel P Mackay, James J BiekerAbstract:Kruppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor [1, 2]. A number of studies have shown up to ~700 genes are poorly expressed when KLF1 is absent [3-6]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF1 [7]. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF1 [8]. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (= E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation [9]. To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1 nan into an erythroid cell line derived from KLF1 -/- fetal liver cells [10]. We performed ChIP-seq to determine differences in genome occupancy in vivo , and identified novel sites occupied by EKLF-E339D but not by wild type KLF1. Using de novo motif discovery [11], we find KLF1 nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to any known TFs, so we think it represents a sequence specificity not normally encoded in mammals. Ectopic binding to non-erythroid gene promoters is accompanied by aberrant gene expression as determined by 4sU labelling and deep sequencing of tamoxifen-induced primary nuclear RNAs. We find a 4-fold greater number of genes induced by KLF1-nan compared with wild type KLF1 which is consistent with degenerate genome occupancy. We compared the KLF1-nan dependent genes with RNA-seq performed in primary fetal liver for KLF1 +/nan versus KLF1 +/- mice. We confirmed aberrant binding using EMSA and surface plasmon resonance (SPR) using recombinant GST-KLF1 zinc finger domains expressed in E.coli. The degenerate motif is consistent with structural models of how the second zinc finger of KLF1 specifically interacts with its binding site [12, 13]. We are undertaking structural studies to confirm this modelling. Together RNA-seq, ChIP-seq and SPR studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. To our knowledge this mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel. References: 1.Perkins, A.C., A.H. Sharpe, and S.H. Orkin. Nature, 1995. 375 (6529): p. 318-22. 2.Nuez, B., et al., Nature, 1995. 375 (6529): p. 316-8. 3.Pilon, A.M., et al., Mol Cell Biol, 2006. 26 (11): p. 4368-77. 4.Drissen, R., et al., Mol Cell Biol, 2005. 25 (12): p. 5205-14. 5.Hodge, D., et al., Blood, 2006. 107 (8): p. 3359-70. 6.Tallack, M.R., et al., Genome Res, 2012. 22 (12):2385-98 7.Arnaud, L., et al., Am J Hum Genet. 87 (5): p. 721-7. 8.Siatecka, M., et al., Proc Natl Acad Sci U S A. 2010. 107 (34):15151-6 9.Heruth, D.P., et al., Genomics, 2010. 96 (5): p. 303-7. 10.Coghill, E., et al., Blood, 2001. 97 (6): p. 1861-1868. 11.Bailey, T.L., et al., Nucleic Acids Res, 2009. 37 (Web Server issue): p. W202-8. 12.Schuetz, A., et al., Cell Mol Life Sci, 2011. 68 (18): p. 3121-31. 13.Oka, S., et al., Biochemistry, 2004. 43 (51): p. 16027-35. Disclosures No relevant conflicts of interest to declare.
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eklf KLF1 a tissue restricted integrator of transcriptional control chromatin remodeling and lineage determination
Molecular and Cellular Biology, 2013Co-Authors: Yvette Y Yien, James J BiekerAbstract:Erythroid Kruppel-like factor (EKLF or KLF1) is a transcriptional regulator that plays a critical role in lineage-restricted control of gene expression. KLF1 expression and activity are tightly controlled in a temporal and differentiation stage-specific manner. The mechanisms by which KLF1 is regulated encompass a range of biological processes, including control of KLF1 RNA transcription, protein stability, localization, and posttranslational modifications. Intact KLF1 regulation is essential to correctly regulate erythroid function by gene transcription and to maintain hematopoietic lineage homeostasis by ensuring a proper balance of erythroid/megakaryocytic differentiation. In turn, KLF1 regulates erythroid biology by a wide variety of mechanisms, including gene activation and repression by regulation of chromatin configuration, transcriptional initiation and elongation, and localization of gene loci to transcription factories in the nucleus. An extensive series of biochemical, molecular, and genetic analyses has uncovered some of the secrets of its success, and recent studies are highlighted here. These reveal a multilayered set of control mechanisms that enable efficient and specific integration of transcriptional and epigenetic controls and that pave the way for proper lineage commitment and differentiation.
Joyce A Lloyd - One of the best experts on this subject based on the ideXlab platform.
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kruppel like transcription factor KLF1 is required for optimal γ and β globin expression in human fetal erythroblasts
PLOS ONE, 2016Co-Authors: Divya S Vinjamur, Yousef N Alhashem, David C Williams, Safa F Mohamad, Parth B Amin, Joyce A LloydAbstract:In human adult erythroid cells, lower than normal levels of Kruppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies.
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KLF1 regulates β-globin, total globin and BCL11A expression in UCB erythroblasts.
2016Co-Authors: Divya S Vinjamur, Yousef N Alhashem, David C Williams, Safa F Mohamad, Parth Amin, Joyce A LloydAbstract:The amount of KLF1, β-globin, γ-globin and BCL11A mRNA was measured by qRT-PCR and normalized to Cyclophilin A mRNA. The fold change in expression of these genes in KLF1 shRNA-treated samples was calculated by setting the value in Scr shRNA controls to 100. Each point on the Pearson’s correlation plots represents a biological replicate, i.e. cells obtained from a different umbilical cord blood sample. Three of the samples were infected with the K1V1 shRNA, and the remaining samples (the majority) were infected with the K1V2 shRNA. (A) A strong, positive correlation was observed between the amounts of KLF1 and β-globin mRNA (N = 19, r2 = 0.65, Prob>F = 0.0001); (B) The amount of total globin (γ+β) expression positively correlates with residual KLF1 mRNA (N = 19, r2 = 0.54, Prob>F = 0.0003) (C) There is a modest positive correlation between the amounts of KLF1 and BCL11A mRNA (N = 17, r2 = 0.27, Prob>F = 0.032). (D) There is no linear relationship between the amounts of KLF1 and γ-globin mRNA (N = 19, r2 = 0.16, Prob>F = 0.095).
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kruppel like transcription factors KLF1 and klf2 have unique and coordinate roles in regulating embryonic erythroid precursor maturation
Haematologica, 2014Co-Authors: Divya S Vinjamur, Jack L Haar, Kristen J Wade, Safa F Mohamad, Stephen T Sawyer, Joyce A LloydAbstract:The Kruppel-like transcription factors KLF1 and KLF2 are essential for embryonic erythropoiesis. They can partially compensate for each other during mouse development, and coordinately regulate numerous erythroid genes, including the β-like globins. Simultaneous ablation of KLF1 and KLF2 results in earlier embryonic lethality and severe anemia. In this study, we determine that this anemia is caused by a paucity of blood cells, and exacerbated by diminished β-like globin gene expression. The anemia phenotype is dose-dependent, and, interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. Mechanistic studies led to the discovery that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous loss of KLF1 and KLF2 than with loss of a single factor. KLF1 and KLF2 coordinately regulate several proliferation-associated genes, including Foxm1. Differential expression of FoxM1, in particular, correlates with the observed KLF1 and KLF2 gene dosage effects on anemia. Furthermore, KLF1 binds to the FoxM1 gene promoter in blood cells. Thus KLF1 and KLF2 coordinately regulate embryonic erythroid precursor maturation through the regulation of multiple homeostasis-associated genes, and KLF2 has a novel and essential role in this process.
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transcription factors KLF1 and klf2 positively regulate embryonic and fetal β globin genes through direct promoter binding
Journal of Biological Chemistry, 2011Co-Authors: Yousef N Alhashem, Divya S Vinjamur, Mohua Basu, Ursula Klingmuller, Karin M L Gaensler, Joyce A LloydAbstract:Kruppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Kruppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ∈- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ∈- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ∈- and γ-globin genes and locus control region in KLF1−/− embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos.
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eklf and klf2 have compensatory roles in embryonic β globin gene expression and primitive erythropoiesis
Blood, 2007Co-Authors: Priyadarshi Basu, Jerry B. Lingrel, Wafaa Lemsaddek, Latasha C Redmond, Jack L Haar, Mohua Basu, Tina K. Lung, Thanh Giang Sargent, David C Williams, Joyce A LloydAbstract:The Kruppel-like C2/H2 zinc finger transcription factors (KLFs) control development and differentiation. Erythroid Kruppel-like factor (EKLF or KLF1) regulates adult β-globin gene expression and is necessary for normal definitive erythropoiesis. KLF2 is required for normal embryonic Ey- and βh1-, but not adult βglobin, gene expression in mice. Both EKLF and KLF2 play roles in primitive erythroid cell development. To investigate potential interactions between these genes, EKLF/KLF2 double-mutant embryos were analyzed. EKLF−/−KLF2−/− mice appear anemic at embryonic day 10.5 (E10.5) and die before E11.5, whereas single-knockout EKLF−/− or KLF2−/− embryos are grossly normal at E10.5 and die later than EKLF−/−KLF2−/− embryos. At E10.5, Ey- and βh1-globin mRNA is greatly reduced in EKLF−/−KLF2−/−, compared with EKLF−/− or KLF2−/− embryos, consistent with the observed anemia. Light and electron microscopic analyses of E9.5 EKLF−/−KLF2−/− yolk sacs, and cytospins, indicate that erythroid and endothelial cells are morphologically more abnormal than in either single knockout. EKLF−/−KLF2−/− erythroid cells are markedly irregularly shaped, suggesting membrane abnormalities. EKLF and KLF2 may have coordinate roles in a common progenitor to erythroid and endothelial cells. The data indicate that EKLF and KLF2 have redundant functions in embryonic β-like globin gene expression, primitive erythropoiesis, and endothelial development.