Abstract:Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opp… Show more
“…83 Defect in enucleation could then be related to decreased GPX4 expression because (i) the GPX4 enhancer/promoter region carries an KLF1-binding consensus site, 84 (ii) GPX4 downregulation is observed in fetal liver cells of Klf1 2/2 mice, 85 and in polyE and orthoE of the E339D KLF1-mutant Nan mice. 86 Moreover, when studied in vitro, Eklf 2/2 erythroid cells undergo normal nuclear condensation and polarization but are blocked at the last step of enucleation. 87,88 Considering these data, studying GPX4 expression, lipid raft clustering, and CAR formation in erythroid cells from CDA IV patients to identify their potential involvement in the phenotype in addition to the already described cell cycle defect would be of great interest.…”
The selenoprotein glutathione peroxidase 4 (GPX4), the only member of the glutathione peroxidase family able to directly reduce cell membrane–oxidized fatty acids and cholesterol, was recently identified as the central regulator of ferroptosis. GPX4 knockdown in mouse hematopoietic cells leads to hemolytic anemia and to increased spleen erythroid progenitor death. The role of GPX4 during human erythropoiesis is unknown. Using in vitro erythroid differentiation, we show here that GPX4-irreversible inhibition by 1S,3R-RSL3 (RSL3) and its short hairpin RNA–mediated knockdown strongly impaired enucleation in a ferroptosis-independent manner not restored by tocopherol or iron chelators. During enucleation, GPX4 localized with lipid rafts at the cleavage furrows between reticulocytes and pyrenocytes. Its inhibition impacted enucleation after nuclear condensation and polarization and was associated with a defect in lipid raft clustering (cholera toxin staining) and myosin-regulatory light-chain phosphorylation. Because selenoprotein translation and cholesterol synthesis share a common precursor, we investigated whether the enucleation defect could represent a compensatory mechanism favoring GPX4 synthesis at the expense of cholesterol, known to be abundant in lipid rafts. Lipidomics and filipin staining failed to show any quantitative difference in cholesterol content after RSL3 exposure. However, addition of cholesterol increased cholera toxin staining and myosin-regulatory light-chain phosphorylation, and improved enucleation despite GPX4 knockdown. In summary, we identified GPX4 as a new actor of human erythroid enucleation, independent of its function in ferroptosis control. We described its involvement in lipid raft organization required for contractile ring assembly and cytokinesis, leading in fine to nucleus extrusion.
“…83 Defect in enucleation could then be related to decreased GPX4 expression because (i) the GPX4 enhancer/promoter region carries an KLF1-binding consensus site, 84 (ii) GPX4 downregulation is observed in fetal liver cells of Klf1 2/2 mice, 85 and in polyE and orthoE of the E339D KLF1-mutant Nan mice. 86 Moreover, when studied in vitro, Eklf 2/2 erythroid cells undergo normal nuclear condensation and polarization but are blocked at the last step of enucleation. 87,88 Considering these data, studying GPX4 expression, lipid raft clustering, and CAR formation in erythroid cells from CDA IV patients to identify their potential involvement in the phenotype in addition to the already described cell cycle defect would be of great interest.…”
The selenoprotein glutathione peroxidase 4 (GPX4), the only member of the glutathione peroxidase family able to directly reduce cell membrane–oxidized fatty acids and cholesterol, was recently identified as the central regulator of ferroptosis. GPX4 knockdown in mouse hematopoietic cells leads to hemolytic anemia and to increased spleen erythroid progenitor death. The role of GPX4 during human erythropoiesis is unknown. Using in vitro erythroid differentiation, we show here that GPX4-irreversible inhibition by 1S,3R-RSL3 (RSL3) and its short hairpin RNA–mediated knockdown strongly impaired enucleation in a ferroptosis-independent manner not restored by tocopherol or iron chelators. During enucleation, GPX4 localized with lipid rafts at the cleavage furrows between reticulocytes and pyrenocytes. Its inhibition impacted enucleation after nuclear condensation and polarization and was associated with a defect in lipid raft clustering (cholera toxin staining) and myosin-regulatory light-chain phosphorylation. Because selenoprotein translation and cholesterol synthesis share a common precursor, we investigated whether the enucleation defect could represent a compensatory mechanism favoring GPX4 synthesis at the expense of cholesterol, known to be abundant in lipid rafts. Lipidomics and filipin staining failed to show any quantitative difference in cholesterol content after RSL3 exposure. However, addition of cholesterol increased cholera toxin staining and myosin-regulatory light-chain phosphorylation, and improved enucleation despite GPX4 knockdown. In summary, we identified GPX4 as a new actor of human erythroid enucleation, independent of its function in ferroptosis control. We described its involvement in lipid raft organization required for contractile ring assembly and cytokinesis, leading in fine to nucleus extrusion.
“…This phenotype is more severe than a complete loss of function of Klf1 [5, 6]. We previously showed the KLF1-E339D protein binds to a degenerate DNA motif in vitro and in vivo, and this corrupts the erythroid transcriptome leading to hemolysis [24–26]. That is, KLF1-E339D has a neomorphic biochemical function which results in red blood cell destruction.Fig.…”
Background
Mutations in the transcription factor,
KLF1
, are common within certain populations of the world. Heterozygous missense mutations in
KLF1
mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis.
Results
To investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a
Klf1
−/−
genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding.
Conclusions
Since all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.
Electronic supplementary material
The online version of this article (10.1186/s12864-019-5805-z) contains supplementary material, which is available to authorized users.
“…A similar reasoning could be applied to Xpo7 , one of the most downregulated genes in Nan E12.5 fetal liver, since it is expressed at lower levels in primitive erythroid cells [51]. However, Xpo7 was previously identified as a common deregulated gene between Nan fetal liver and adult spleen [22], supporting the notion that its expression is directly affected in Nan erythroid cells. Xpo7 caught our attention since a recent paper described that it is required for nuclear condensation and enucleation during terminal erythroid differentiation in vitro [25].…”
Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by
Klf1
knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1
Nan
variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1
Nan
variant during fetal development. We show that
Nan
embryos have defects in erythroid maturation. RNA-sequencing of the KLF1
Nan
fetal liver cells revealed that Exportin 7 (
Xpo7
) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1
Nan
fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1
Nan
erythroid cells.
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