Severe anemia caused by dominant mutations in Krüppel-like factor 1 (KLF1).

The etiology and severity of anemia, a common blood disorder, are diverse. Dominant mutations in Krüppel-like factor 1 (KLF1/EKLF) underlie the molecular basis for some of them. KLF1 is a zinc finger transcription factor that plays an essential role in red blood cell proliferation and differentiation. Mutations have been identified in the KLF1 gene that cause hematologic diseases. Two of these alter one allele but generate an extreme phenotype: the mouse Nan mutation (E339D) leads to hemolytic neonatal anemia with hereditary spherocytosis, and the human CDA mutation (E325K) causes congenital dyserythropoietic anemia (CDA) type IV. These modify functionally important amino acids in the zinc finger DNA-binding domain at positions involved in direct interactions with regulatory elements of KLF1's target genes. Although the two dominant mutations alter the same evolutionarily conserved glutamic acid residue, the substitutions are not equivalent and lead to divergent consequences for the molecular mechanisms underlying activity of these mutants, particularly in recognition and interaction with their unique binding sites. Consequently, the properties of the protein are transformed such that it acquires novel dominant characteristics whose effects may not be limited to the erythroid compartment. KLF1 mutants cause loss-of-function/haploinsufficiency effects on some KLF1 wild-type target genes, while at the same time gain-of-function effects activate ectopic sites and neomorphic gene expression. Such anomalies not only lead to intrinsic red cell problems, but also to expression of non-erythroid genes that systemically disturb organ development. This review highlights recent molecular, biochemical, and genetic studies of KLF1 mutants, particularly the dramatic consequences that come from just a single amino acid change. The study of these variants provides an important contribution to the overall understanding of the DNA-protein interface of the zinc finger subtype of transcription factors, and the potential clinical consequences of what might appear to be a minor change in sequence.

A Krüppel-like factor 1 (KLF1) Mutation Associated with Severe Congenital Dyserythropoietic Anemia Alters Its DNA-Binding Specificity.

Krüppel-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.

Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development.

Transcription factor control of cell-specific downstream targets can be significantly altered when the controlling factor is mutated. We show that the semi-dominant neonatal anemia (Nan) mutation in the EKLF/KLF1 transcription factor leads to ectopic expression of proteins that are not normally expressed in the red blood cell, leading to systemic effects that exacerbate the intrinsic anemia in the adult and alter correct development in the early embryo. Even when expressed as a heterozygote, the Nan-EKLF protein accomplishes this by direct binding and aberrant activation of genes encoding secreted factors that exert a negative effect on erythropoiesis and iron use. Our data form the basis for a novel mechanism of physiological deficiency that is relevant to human dyserythropoietic anemia and likely other disease states.

A regulatory function of long non-coding RNAs in red blood cell development.

In recent years it has been discovered that long non-coding RNAs are important regulators in many biological processes. In this review, we summarize the role of lncRNA in erythropoiesis. lncRNA are crucial for regulation of gene expression during both, proliferation and differentiation stages of red blood cell development. Many are regulated by erythroid-specific transcription factors and some are expressed in a developmental stage-specific manner. The majority of individually studied lncRNAs are involved in regulating the terminal maturation stages of red cell differentiation. Their regulatory function is accomplished by various mechanisms, including direct regulation in cis or trans by the lncRNA product or by the cis-localized presence of the lncRNA transcription itself. These add additional levels of regulation of gene expression during erythropoiesis.

Transcriptional activity of erythroid Kruppel-like factor (EKLF/KLF1) modulated by PIAS3 (protein inhibitor of activated STAT3).

Erythroid Kruppel-like factor (EKLF or KLF1) is a transcription factor crucial for red cell development that is directly involved in regulation of a large number of erythroid genes. EKLF serves mostly as an activator of expression of these genes; however, it can act also as a repressor. Here, we present evidence that EKLF interacts with proteins from the PIAS (protein inhibitor of activated STAT) family that convey repressive activity to EKLF in the absence of sumoylation. Our studies identify PIAS3 as a transcriptional corepressor of EKLF for at least a subset of its target genes during erythropoiesis (e.g. ß-globin, α-hemoglobin stabilizing protein). We demonstrate an interaction between EKLF and PIAS proteins confirmed by in vivo coimmunoprecipitation assays with both exogenous and endogenous proteins. We identified an LXXLL signature motif located near the N terminus of PIAS proteins that, although not involved in the EKLF-PIAS3 interaction, is required for the transrepression activity. Knockdown of endogenous PIAS3 accelerates differentiation of both murine erythroleukemia cells, as well as fetal liver cells, whereas an increase in PIAS3 levels inhibits this increase. Using chromatin immunoprecipitation assays, we show that PIAS3 preferentially occupies the ß-globin promoter in undifferentiated murine erythroleukemia cells. Together these results demonstrate that an interaction between EKLF and PIAS3 provides a novel mode of regulation of EKLF activity in the absence of sumolylation and furthermore shows an important involvement of PIAS proteins in erythropoiesis.

The multifunctional role of EKLF/KLF1 during erythropoiesis.

The cellular events that lead to terminal erythroid differentiation rely on the controlled interplay of extra- and intracellular regulatory factors. Their downstream effects are highly coordinated and result in the structural/morphologic and metabolic changes that uniquely characterize a maturing red blood cell. Erythroid Krüppel-like factor (EKLF/KLF1) is one of a very small number of intrinsic transcription factors that play a major role in regulating these events. This review covers 3 major aspects of erythropoiesis in which EKLF plays crucial functions: (1) at the megakaryocyte-erythroid progenitor stage, where it is involved in erythroid lineage commitment; (2) during the global expansion of erythroid gene expression in primitive and definitive lineages, where it plays a direct role in globin switching; and (3) during the terminal maturation of red cells, where it helps control exit from the cell cycle. We conclude by describing recent studies of mammalian EKLF/KLF1 mutations that lead to altered red cell phenotypes and disease.

Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Kruppel-like factor.

Studies of mouse models of anemia have long provided fundamental insights into red blood cell formation and function. Here we show that the semidominant mouse mutation Nan ("neonatal anemia") carries a single amino acid change (E339D) within the second zinc finger of the erythroid Krüppel-like factor (EKLF), a critical erythroid regulatory transcription factor. The mutation alters the DNA-binding specificity of EKLF so that it no longer binds promoters of a subset of its DNA targets. Remarkably, even when mutant Nan and wild-type EKLF alleles are expressed at equivalent levels, the mutant form selectively interferes with expression of EKLF target genes whose promoter elements it no longer binds. This interference yields a distorted genetic output and selective protein deficiencies that differ from those seen in EKLF-heterozygous and EKLF-null red blood cells and presents a unique and unexpected mechanism of inherited disease.

EKLF directly activates the p21WAF1/CIP1 gene by proximal promoter and novel intronic regulatory regions during erythroid differentiation.

The switch from proliferation to differentiation during the terminal stages of erythropoiesis is a tightly controlled process that relies in part on transcription factor-mediated activation of cell cycle components. EKLF is a key transcription factor that is necessary for the initial establishment of the red cell phenotype. Here, we find that EKLF also plays a role during the subsequent differentiation process, as it induces p21(WAF1/CIP1) expression independent of p53 to regulate the changes in the cell cycle underlying erythroid maturation. EKLF activates p21 not only by directly binding to an EKLF site within a previously characterized GC-rich region in the p21 proximal promoter but also by occupancy at a novel, phylogenetically conserved region that contains consensus CACCC core motifs located downstream from the p21 TATA box. Our findings demonstrate that EKLF, likely in coordination with other transcription factors, directly contributes to the complex set of events that occur at the final erythroid cell divisions and accentuates terminal differentiation directly by activation of CDK inhibitors such as p21.

Sumoylation of EKLF promotes transcriptional repression and is involved in inhibition of megakaryopoiesis.

Erythroid Krüppel-like factor (EKLF [KLF1]) is a transcriptional regulator that plays a critical role within a specific subset of hematopoietic cells, particularly in the erythroid lineage and its immediate precursor, the megakaryocyte-erythroid progenitor (MEP). We find that EKLF is posttranslationally modified by sumoylation at a single site near its amino terminus and that PIAS1 plays a critical role in this process. Mutation of this site has little effect on EKLF's ability to function as a transcriptional activator; however, it has a dramatic effect on its repressive abilities. The mechanism of repression likely involves a novel small ubiquitin-related modifier (SUMO)-dependent EKLF interaction with the Mi-2beta component of the NuRD repression complex. Mutated EKLF is attenuated in its ability to repress megakaryocyte differentiation, implicating EKLF sumoylation status in differentiative decisions emanating from the MEP. These studies demonstrate a novel mechanism by which transcription factor sumoylation can alter protein-protein interactions and bipotential lineage decisions.