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Mutations in linker-2 of KLF1 impair expression of membrane transporters and cytoskeletal proteins causing hemolysis

ABSTRACT: The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotypes. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generated a mouse model of this disease by crossing Klf1+/- mice with Klf1H350R/+ mice that harbour a missense mutation in linker-2. Klf1H350R/- mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrated loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq showed no change in DNA-binding specificity, but a global reduction in affinity, which was confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.

ORGANISM(S): Mus musculus

PROVIDER: GSE94351 | GEO | 2024/07/03

REPOSITORIES: GEO

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Missense mutations in linker-2 of KLF1 impair expression of membrane transporters and cytoskeletal proteins to cause hemolysis

Project description:The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotypes. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generated a mouse model of this disease by crossing Klf1+/- mice with Klf1H350R/+ mice that harbour a missense mutation in linker-2. Klf1H350R/- mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrated loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq showed no change in DNA-binding specificity, but a global reduction in affinity, which was confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.

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KLF1 null neonates display hydrops fetalis and a deranged erythroid transcriptome

Project description:We describe a case of severe neonatal anemia with kernicterus due to 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 DAT-negative non-spherocytic hemolytic anaemia (NSHA), jaundice, hepato-splenomegaly, and marked erythroblastosis is more severe than that present in CDA type IV due to 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 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. mRNA sequencing on peripheral blood from a family trio (mother, father and proband) where parents were asymptomatic and proband had severe neonatal anemia.

2015-01-01 | E-GEOD-60514 | biostudies-arrayexpress

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Project description:The onset of erythropoiesis is under strict developmental control, with direct and indirect inputs influencing its derivation from the hematopoietic stem cell. A major regulator of this transition is KLF1/EKLF, a zinc finger transcription factor that plays a global role in all aspects of erythropoiesis. Here, we have identified a short, conserved enhancer element in KLF1 intron 1 that is important for establishing optimal levels of KLF1 in mouse and human cells. Chromatin accessibility of this site exhibits cell-type specificity and is under developmental control during the differentiation of human CD34+ cells towards the erythroid lineage. This site binds GATA1, SMAD1, TAL1, and ETV6. In vivo editing of this region in cell lines and primary cells reduces KLF1 expression quantitatively. However, we find that, similar to observations seen in pedigrees of families with KLF1 mutations, downstream effects are variable, suggesting that the global architecture of the site is buffered towards keeping the KLF1 genetic region in an active state. We propose that modification of intron 1 in both alleles is not equivalent to complete loss of function of one allele.

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Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability

Project description:The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo. Here we demonstrate how a missense point mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and semi-dominant neonatal anemia. We employed RNA-seq, ChIP-seq and 4sU-RNA-seq to identify the direct transcriptional consequences of aberrant DNA-binding events genome wide. Funding: National Health and Medical Research Council [APP1082429 to A.C.P.]; United States Public Health Service [R01 GM103544 to T.B., R01 DK100692 to L.L.P. and R01 DK46865 to J.J.B.]; Victorian Government Operational Infrastructure Support Scheme [St Vincent's Institute of Medical Research to M.W.P]. Funding for open access charge: Mater Research - UQ [APP1082429].

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