A novel human cellular model of CDA IV enables comprehensive analysis revealing the molecular basis of the disease phenotype.

Red blood cell disorders can result in severe anemia. One such disease congenital dyserythropoietic anemia IV (CDA IV) is caused by the heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by the paucity of suitable and adequate quantities of material from patients with anemia and the rarity of the disease. We, therefore, took a novel approach, creating a human cellular disease model system for CDA IV that accurately recapitulates the disease phenotype. Next, using comparative proteomics, we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include downregulated pathways the governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking, and global transcription, and upregulated networks governing mitochondrial biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying the effects of a rare mutation can reveal fundamental biology.

Association of DDX5/p68 protein with the upstream erythroid enhancer element (EHS1) of the gene encoding the KLF1 transcription factor.

EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. Regulation of KLF1 is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the DDX5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We further show that this protein associates with DEK and CTCF. We postulate that the range of interactions of DDX5/p68 with these and other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1 and enables the formation of a proper chromatin configuration at the Klf1 locus. These individual interactions provide quantitative contributions that, in sum, establish the high-level activity of the Klf1 promoter and suggest they can be selectively manipulated for clinical benefit.

Patients with hypercortisolemic Cushing disease possess a distinct class of hematopoietic progenitor cells leading to erythrocytosis.

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

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.

KLF1/EKLF expression in acute leukemia is correlated with chromosomal abnormalities.

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.

Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient.

Congenital dyserythropoietic anemia type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. Molecular characteristics of this disease have not been clarified, partly due to its rarity. We expanded erythroid cells from a patient's peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain patients' clinical and phenotypic features, and elucidate the dominant character of the mutation.

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.

EKLF/KLF1-regulated cell cycle exit is essential for erythroblast enucleation.

The mechanisms regulating the sequential steps of terminal erythroid differentiation remain largely undefined, yet are relevant to human anemias that are characterized by ineffective red cell production. Erythroid Krüppel-like Factor (EKLF/KLF1) is a master transcriptional regulator of erythropoiesis that is mutated in a subset of these anemias. Although EKLF's function during early erythropoiesis is well studied, its role during terminal differentiation has been difficult to functionally investigate due to the impaired expression of relevant cell surface markers in Eklf(-/-) erythroid cells. We have circumvented this problem by an innovative use of imaging flow cytometry to investigate the role of EKLF in vivo and have performed functional studies using an ex vivo culture system that enriches for terminally differentiating cells. We precisely define a previously undescribed block during late terminal differentiation at the orthochromatic erythroblast stage for Eklf(-/-) cells that proceed beyond the initial stall at the progenitor stage. These cells efficiently decrease cell size, condense their nucleus, and undergo nuclear polarization; however, they display a near absence of enucleation. These late-stage Eklf(-/-) cells continue to cycle due to low-level expression of p18 and p27, a new direct target of EKLF. Surprisingly, both cell cycle and enucleation deficits are rescued by epistatic reintroduction of either of these 2 EKLF target cell cycle inhibitors. We conclude that the cell cycle as regulated by EKLF during late stages of differentiation is inherently critical for enucleation of erythroid precursors, thereby demonstrating a direct functional relationship between cell cycle exit and nuclear expulsion.

Krüppeling erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants.

Until recently our approach to analyzing human genetic diseases has been to accurately phenotype patients and sequence the genes known to be associated with those phenotypes; for example, in thalassemia, the globin loci are analyzed. Sequencing has become increasingly accessible, and thus a larger panel of genes can be analyzed and whole exome and/or whole genome sequencing can be used when no variants are found in the candidate genes. By using such approaches in patients with unexplained anemias, we have discovered that a broad range of hitherto unrelated human red cell disorders are caused by variants in KLF1, a master regulator of erythropoiesis, which were previously considered to be extremely rare causes of human genetic disease.

A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis.

Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production in vitro. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated Foxo3-/- erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued Foxo3-/- erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.

Orchestration of late events in erythropoiesis by KLF1/EKLF.

PURPOSE OF REVIEW: Transcriptional regulators provide the molecular and biochemical basis for the cell specific properties and characteristics that follow from their central role in establishing tissue-restricted expression. Precise and sequential control of terminal cell divisions, nuclear condensation, and enucleation are defining characteristics within erythropoietic differentiation. This review is focused on KLF1, a central global regulator of this process. RECENT FINDINGS: Studies in the past year have brought a number of proteins that are targets of KLF1 regulation into focus with respect to their roles in terminal erythroid differentiation. Many of these are involved in fine control of the cell cycle at both early (E2F2, Cyclin A2) and later (p18, p27, p19) stages of differentiation, or are directly involved in enucleation (p18, p27). Dramatic biophysical changes controlled at the nuclear lamin by caspase 3 enable histone release and nuclear condensation, whereas dematin association with structural proteins alters the timing of enucleation. Conditional ablation of mDia2 has established its role in late stage cell cycle and enucleation. SUMMARY: Transcription factors such as KLF1, along with epigenetic modifiers, play crucial roles in establishing the proper onset and progression of terminal differentiation events. Studies from the past year show a remarkable multifaceted convergence on cell cycle control, and establish that the orthochromatic erythroblast stage is a critical nodal point for many of the effects on enucleation. These studies are relevant to understanding the underlying causes of anemia and hematologic disease where defective enucleation predicts a poor clinical outcome.

Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche.

The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, and that these correspond to erythro-myeloid progenitors (EMPs) that first appear in the yolk sac of the early developing embryo. Erythroid Krüppel-like factor (EKLF; KLF1), a crucial zinc finger transcription factor, is expressed in the EMPs, and plays an extrinsic role in erythroid maturation by being expressed in the supportive macrophage of the erythroblastic island and regulating relevant genes important for island integrity within these cells. Together with its well-established intrinsic contributions to erythropoiesis, EKLF thus plays a coordinating role between two different cell types whose interaction provides the optimal environment to generate a mature red blood cell.

Transcription factor EKLF (KLF1) recruitment of the histone chaperone HIRA is essential for ß-globin gene expression.

The binding of chromatin-associated proteins and incorporation of histone variants correlates with alterations in gene expression. These changes have been particularly well analyzed at the mammalian ß-globin locus, where transcription factors such as erythroid Krüppel-like factor (EKLF), which is also known as Krüppel-like factor 1 (KLF1), play a coordinating role in establishing the proper chromatin structure and inducing high-level expression of adult ß-globin. We had previously shown that EKLF preferentially interacts with histone H3 and that the H3.3 variant is differentially recruited to the ß-globin promoter. We now find that a novel interaction between EKLF and the histone cell cycle regulation defective homolog A (HIRA) histone chaperone accounts for these effects. HIRA is not only critical for ß-globin expression but is also required for activation of the erythropoietic regulators EKLF and GATA binding protein 1 (GATA1). Our results provide a mechanism by which transcription factor-directed recruitment of a generally expressed histone chaperone can lead to tissue-restricted changes in chromatin components, structure, and transcription at specific genomic sites during differentiation.

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.

KLF1: when less is more.

In this issue of Blood, Liu et al gain an understanding of phenotypic variability in hemoglobinopathies. They find that mutations in Krüppel-like factor-1 (KLF1) are significantly more prevalent in patients with ß-thalassemia than previously recognized and correlate with a milder phenotype. This supports the emerging concept that monoallelic KLF1 mutations can play a modulatory role in hemoglobinopathies.

Structural and functional characterization of an atypical activation domain in erythroid Kruppel-like factor (EKLF).

Erythroid Krüppel-like factor (EKLF) plays an important role in erythroid development by stimulating ß-globin gene expression. We have examined the details by which the minimal transactivation domain (TAD) of EKLF (EKLFTAD) interacts with several transcriptional regulatory factors. We report that EKLFTAD displays homology to the p53TAD and, like the p53TAD, can be divided into two functional subdomains (EKLFTAD1 and EKLFTAD2). Based on sequence analysis, we found that EKLFTAD2 is conserved in KLF2, KLF4, KLF5, and KLF15. In addition, we demonstrate that EKLFTAD2 binds the amino-terminal PH domain of the Tfb1/p62 subunit of TFIIH (Tfb1PH/p62PH) and four domains of CREB-binding protein/p300. The solution structure of the EKLFTAD2/Tfb1PH complex indicates that EKLFTAD2 binds Tfb1PH in an extended conformation, which is in contrast to the α-helical conformation seen for p53TAD2 in complex with Tfb1PH. These studies provide detailed mechanistic information into EKLFTAD functions as well as insights into potential interactions of the TADs of other KLF proteins. In addition, they suggest that not only have acidic TADs evolved so that they bind using different conformations on a common target, but that transitioning from a disordered to a more ordered state is not a requirement for their ability to bind multiple partners.

Functional interactions between erythroid Krüppel-like factor (EKLF/KLF1) and protein phosphatase PPM1B/PP2Cß.

Erythroid Krüppel-like factor (EKLF; KLF1) is an erythroid-specific transcription factor required for the transcription of genes that regulate erythropoiesis. In this paper, we describe the identification of a novel EKLF interactor, Ppm1b, a serine-threonine protein phosphatase that has been implicated in the attenuation of NFκB signaling and the regulation of Cdk9 phosphorylation status. We show that Ppm1b interacts with EKLF via its PEST1 sequence. However, its genetic regulatory role is complex. Using a promoter-reporter assay in an erythroid cell line, we show that Ppm1b superactivates EKLF at the ß-globin and BKLF promoters, dependent on intact Ppm1b phosphatase activity. Conversely, depletion of Ppm1b in CD34(+) cells leads to a higher level of endogenous ß-globin gene activation after differentiation. We also observe that Ppm1b likely has an indirect role in regulating EKLF turnover via its zinc finger domain. Together, these studies show that Ppm1b plays a multilayered role in regulating the availability and optimal activity of the EKLF protein in erythroid cells.

T-cell acute leukemia 1 (TAL1) regulation of erythropoietin receptor and association with excessive erythrocytosis.

During erythropoiesis, erythropoietin stimulates induction of erythroid transcription factors that activate expression of erythroid genes including the erythropoietin receptor (EPO-R) that results in increased sensitivity to erythropoietin. DNA binding of the basic helix-loop-helix transcription factor, TAL1/SCL, is required for normal erythropoiesis. A link between elevated TAL1 and excessive erythrocytosis is suggested by erythroid progenitor cells from a patient that exhibits unusually high sensitivity to erythropoietin with concomitantly elevated TAL1 and EPO-R expression. We found that TAL1 regulates EPO-R expression mediated via three conserved E-box binding motifs (CAGCTG) in the EPO-R 5' untranslated transcribed region. TAL1 increases association of the GATA-1·TAL1·LMO2·LDB1 transcription activation complex to the region that includes the transcription start site and the 5' GATA and 3' E-box motifs flanking the EPO-R transcription start site suggesting that TAL1 promotes accessibility of this region. Nucleosome shifting has been demonstrated to facilitate TAL1 but not GATA-1 binding to regulate target gene expression. Accordingly, we observed that with induced expression of EPO-R in hemotopoietic progenitor cells, nucleosome phasing shifts to increase the linker region containing the EPO-R transcription start site and TAL1 binds to the flanking 5' GATA and 3' E-box regions of the promoter. These data suggest that TAL1 binds to the EPO-R promoter to activate EPO-R expression and provides a potential link to elevated EPO-R expression leading to hypersensitivity to erythropoietin and the resultant excessive erythrocytosis.

Erythroid transcription factor EKLF/KLF1 mutation causing congenital dyserythropoietic anemia type IV in a patient of Taiwanese origin: review of all reported cases and development of a clinical diagnostic paradigm.

KLF1 is an erythroid specific transcription factor that is involved in erythroid lineage commitment, globin switching and terminal red blood cell maturation. Various mutations of KLF1 have been identified in humans, which have led to both benign and pathological phenotypes. The E325K mutation, within the second zinc finger of the KLF1 gene, has been shown to cause a new form of congenital dyserythropoietic anemia (CDA) now labeled as CDA type IV. We report the fourth documented case of this mutation, and propose a clinical diagnostic model to better identify this disease in other patients. Our patient is a Taiwanese child who presented to us at 8years of age with severe hemolytic anemia, splenomegaly, elevated fetal hemoglobin (HbF), iron overload, and dyserythropoiesis in the bone marrow. KLF1 sequence analysis revealed a G-to-A transition in one allele of exon 3, which resulted in the substitution of a glutamate 325 by a lysine. Flow cytometry analysis revealed decreased protein expression of CD44 on the red blood cells, and decreased red blood cell deformability as measured using an ektacytometer. Blood typing revealed his red blood cells to be Co(a-b-), In(b-), LW(ab-) and Lu(b+), even though DNA testing predicted that he would be Co(a+b-) and LW(a+b-). This newly discovered CDA combines features of a hemoglobinopathy, RBC membrane defect and hereditary persistence of HbF (HPFH) which are not seen in the previous types of CDA. Increased awareness of this phenotype may improve the more prompt and accurate diagnosis of these patients.