Corrupted DNA-binding specificity and ectopic transcription underpin dominant neomorphic mutations in KLF/SP transcription factors.

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.

Krüppel-like factors compete for promoters and enhancers to fine-tune transcription.

Krüppel-like factors (KLFs) are a family of 17 transcription factors characterized by a conserved DNA-binding domain of three zinc fingers and a variable N-terminal domain responsible for recruiting cofactors. KLFs have diverse functions in stem cell biology, embryo patterning, and tissue homoeostasis. KLF1 and related family members function as transcriptional activators via recruitment of co-activators such as EP300, whereas KLF3 and related members act as transcriptional repressors via recruitment of C-terminal Binding Proteins. KLF1 directly activates the Klf3 gene via an erythroid-specific promoter. Herein, we show KLF1 and KLF3 bind common as well as unique sites within the erythroid cell genome by ChIP-seq. We show KLF3 can displace KLF1 from key erythroid gene promoters and enhancers in vivo. Using 4sU RNA labelling and RNA-seq, we show this competition results in reciprocal transcriptional outputs for >50 important genes. Furthermore, Klf3-/- mice displayed exaggerated recovery from anemic stress and persistent cell cycling consistent with a role for KLF3 in dampening KLF1-driven proliferation. We suggest this study provides a paradigm for how KLFs work in incoherent feed-forward loops or networks to fine-tune transcription and thereby control diverse biological processes such as cell proliferation.

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

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 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 anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.

Identification of novel hypomorphic and null mutations in Klf1 derived from a genetic screen for modifiers of α-globin transgene variegation.

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated α-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for modifiers of murine metastable epialleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation. Both strains harbour point mutations in the erythroid transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain and a homozygous null phenotype. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have chronic microcytic anaemia which models the phenotype in a recently described family. This is the first genetic evidence that the linkers between the zinc fingers of transcription factors have a function beyond that of a simple spacer.

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

The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10 bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotype. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generate 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 demonstrate loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq show no change in DNA-binding specificity, but a global reduction in affinity, which is 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.

Author Correction: EPO does not promote interaction between the erythropoietin and beta-common receptors.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

EPO does not promote interaction between the erythropoietin and beta-common receptors.

A direct interaction between the erythropoietin (EPOR) and the beta-common (ßc) receptors to form an Innate Repair Receptor (IRR) is controversial. On one hand, studies have shown a functional link between EPOR and ßc receptor in tissue protection while others have shown no involvement of the ßc receptor in tissue repair. To date there is no biophysical evidence to confirm a direct association of the two receptors either in vitro or in vivo. We investigated the existence of an interaction between the extracellular regions of EPOR and the ßc receptor in silico and in vitro (either in the presence or absence of EPO or EPO-derived peptide ARA290). Although a possible interaction between EPOR and ßc was suggested by our computational and genomic studies, our in vitro biophysical analysis demonstrates that the extracellular regions of the two receptors do not specifically associate. We also explored the involvement of the ßc receptor gene (Csf2rb) under anaemic stress conditions and found no requirement for the ßc receptor in mice. In light of these studies, we conclude that the extracellular regions of the EPOR and the ßc receptor do not directly interact and that the IRR is not involved in anaemic stress.

Direct targets of pSTAT5 signalling in erythropoiesis.

Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.