ABSTRACT

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.

INTRODUCTION

Direct activation of tissue-restricted gene expression relies on transcription factors that faithfully recognize their cognate binding sequence in DNA and influence the transcriptional and epigenetic outcome (Novershtern et al., 2011). Of special importance for erythropoiesis is EKLF/KLF1, which exerts a global role in tissue-specific gene regulation by virtue of its three-finger recognition of the 9 bp 5′CCMCRCCCN sequence at target gene promoters and enhancers (reviewed by Siatecka and Bieker, 2011; Tallack and Perkins, 2010; Yien and Bieker, 2013). Its effects are circumscribed owing to its tissue-restricted expression in erythroid cells and its bipotent myeloid progenitor. Genetic ablation studies in the mouse show that EKLF is absolutely essential because death results from a profound β-thalassemia and the low to virtually non-existent expression of global erythroid genes of all categories.

Monoallelic mutation of KLF1 in humans can lead to a benign outcome that is nonetheless phenotypically important (Borg et al., 2011; Helias et al., 2013; Singleton et al., 2012; Tallack and Perkins, 2013; Waye and Eng, 2015), as haploinsufficient levels of KLF1 lead to altered β-globin switching, which can be advantageous in individuals with β-thalassemia (Liu et al., 2014). However, some mutations lead to anemias (Arnaud et al., 2010; Huang et al., 2015; Jaffray et al., 2013; Singleton et al., 2011; Viprakasit et al., 2014; reviewed by Perkins et al., 2016). The human KLF1 mutation (E325K) in congenital dyserythropoietic anemia (CDA) (Arnaud et al., 2010; Jaffray et al., 2013; Singleton et al., 2011) is at the same amino acid as that seen in the mouse Nan mutant (Heruth et al., 2010; Siatecka et al., 2010b), albeit a different substitution. Nan is inherited in a semi-dominant fashion: homozygotes die in utero at E10-11, while heterozygous Nan/+ mice exhibit lifelong severe anemia that is characterized by reticulocytosis, splenomegaly, altered globin expression and cell membrane defects (Lyon, 1983; Siatecka et al., 2010b; White et al., 2009). Transfer of Nan mouse anemic hematological properties after bone marrow transplantation of Nan/+ cells to a wild-type recipient shows the dominant, cell-autonomous nature of the effect (Lyon, 1983; White et al., 2009).

The mutation in zinc finger 2 at E339D, although conservative, is at a universally conserved amino acid and leads to altered recognition properties such that Nan-KLF1 recognizes only the 5′CCMCGCCCN subset of wild-type sequences and leads to a distorted genetic output (Siatecka et al., 2010b). Thus, there are two types of binding sites: those able to bind both wild-type KLF1 and Nan-KLF1 (category I) and those able to bind only wild-type KLF1 (category II) (Siatecka et al., 2010b). Paradoxically, the mutant Nan-KLF1 form interferes with the expression of target genes with category II sites, to which it can no longer bind. The molecular explanation behind this remains unclear. However, the change also confers recognition of an altered sequence that is not normally associated with KLF1 recognition (Gillinder et al., 2016), such that genes that are never seen in the erythroid cell are ectopically expressed in the Nan/+ mutant.

An incompletely explained aspect of the Nan/+ mouse is that its anemia is wide-ranging during development in both the embryo and the adult, and may not be completely accounted for by the distortion in the intrinsic pattern of expression in the Nan/+ erythroid cell. This is directly relevant to the humans with CDA, who also exhibit a range of effects that appear extrinsic to those caused by a tissue-restricted factor such as KLF1. We have addressed these issues by testing the hypothesis that ectopic expression of a specific subset of genes leads to effects that are superimposed on the red cell defects and thus plays a significant contributory role to the Nan anemia phenotype.

RESULTS

Altered recognition sequences of Nan-EKLF lead to circumscribed and ectopic/neomorphic gene expression in the Nan/+ mouse fetal liver erythroid cell

Inspection of globally expressed genes whose levels are altered in Nan/+ E13.5 fetal livers compared with their wild-type littermates (Gillinder et al., 2016) support the initial genotypic analysis of the Nan mouse (Siatecka et al., 2010b), showing a dysregulated erythroid transcript pattern in spite of the heterozygosity of the KLF1 mutation. For example (Fig. 1A), expression of dematin (Epb4.9, also known as Dmtn) and E2f2 are virtually absent in KLF1 knockout cells, and this is mimicked in Nan/+ cells; these contain category II elements that are affected by the presence of Nan-KLF1. As a control, Alas2, which contains a category I-binding element, is not altered in Nan/+ cells compared with wild type. Finally, hemoglobin switching is altered, as embryonic Hbb-bh1 globin expression is retained at high levels in both knockout and Nan/+ E13.5 fetal liver, whereas adult Hbb-b1 and Hbb-b2 globin expression are 50-70% less (Fig. 1B), as previously observed (Siatecka et al., 2010b). Although many of these changes arise from the more-limited target DNA sequence recognized by Nan-KLF1 protein (i.e. 5′CCMCGCCCN) compared with wild type (i.e. 5′CCMCRCCCN) (Siatecka et al., 2010b), our recent studies show that there is a second consequence of the E339D mutation in Nan-KLF1, namely that a different recognition sequence (5′CCMNGCCCN) is newly recognized (Gillinder et al., 2016). We considered whether this might play an additional role in the anemia and the phenotypic properties observed throughout development in Nan/+ embryos and adults, specifically whether the new targets would give rise to aberrant expression of unusual/unexpected proteins in Nan erythroid cells that yield deleterious effects.

Fig. 1.

Expression of EKLF (KLF1) targets in fetal liver samples. (A-C) RNA-seq expression data of E13.5 fetal liver cells from wild-type (+/+) (dark gray) and Nan/+ (red) littermates (‘Nan’), or from wild-type (+/+) (light gray) and EKLF-null (−/−) (blue) littermates were analyzed for expression of target genes. Each is an average of biological triplicate samples. (A) Genes containing category II (Ebp4.9, E2f2) or category I (Alas2) target sites (as defined by Siatecka et al., 2010b; examples are shown in Table S1) were analyzed for expression. (B) Expression of embryonic βh1 globin (Hbb-bh1) and adult β globins (Hbb-b1 and Hbb-b2) show target gene dysregulation. (C) Expression level of novel targets hepcidin (Hamp) and Irf7, which are most dramatically increased in the Nan/+ samples, are shown next to a representative browser expression profile from one sample each [wild type (+/+) (dark gray) and Nan/+ (red)]. In each case, expression of the adjacent genes is unaffected by Nan-EKLF expression status. (D) Expression of total EKLF (Klf1) in the Nan littermates is shown for comparison; this analysis does not differentiate between wild-type and Nan-EKLF RNA. (E) Total RNA from adherent cell-depleted and expanded erythroid cells isolated from wild-type (+/+) or Nan/+ littermates (‘Nan’) were monitored by q-RT-PCR for expression of EKLF (Klf1), Ebp4.9, hepcidin (Hamp) or Irf7. Results are the average of three biological replicates each performed in triplicate.

Fig. 1.

Expression of EKLF (KLF1) targets in fetal liver samples. (A-C) RNA-seq expression data of E13.5 fetal liver cells from wild-type (+/+) (dark gray) and Nan/+ (red) littermates (‘Nan’), or from wild-type (+/+) (light gray) and EKLF-null (−/−) (blue) littermates were analyzed for expression of target genes. Each is an average of biological triplicate samples. (A) Genes containing category II (Ebp4.9, E2f2) or category I (Alas2) target sites (as defined by Siatecka et al., 2010b; examples are shown in Table S1) were analyzed for expression. (B) Expression of embryonic βh1 globin (Hbb-bh1) and adult β globins (Hbb-b1 and Hbb-b2) show target gene dysregulation. (C) Expression level of novel targets hepcidin (Hamp) and Irf7, which are most dramatically increased in the Nan/+ samples, are shown next to a representative browser expression profile from one sample each [wild type (+/+) (dark gray) and Nan/+ (red)]. In each case, expression of the adjacent genes is unaffected by Nan-EKLF expression status. (D) Expression of total EKLF (Klf1) in the Nan littermates is shown for comparison; this analysis does not differentiate between wild-type and Nan-EKLF RNA. (E) Total RNA from adherent cell-depleted and expanded erythroid cells isolated from wild-type (+/+) or Nan/+ littermates (‘Nan’) were monitored by q-RT-PCR for expression of EKLF (Klf1), Ebp4.9, hepcidin (Hamp) or Irf7. Results are the average of three biological replicates each performed in triplicate.

We began by focusing on the relatively small number (∼80) of genes that are newly and most highly expressed in Nan/+ cells compared with wild type (Gillinder et al., 2016), and further parsed this to genes that are secreted and thus could systemically affect normal erythropoiesis via the circulation. Two genes in particular caught our attention. Hepcidin is a regulator of cellular iron use by virtue of its interaction with ferroportin (Ganz, 2011), and is primarily expressed in the adult liver. Interferon regulatory factor 7 (IRF7) is a transcription factor primarily expressed by macrophages that mediates inflammation by virtue of its activation of interferon β (IFNβ) (Ford and Thanos, 2010; Honda et al., 2005). Misexpression of either of these would be detrimental to effective erythropoiesis (Ganz and Nemeth, 2012; Kim and Nemeth, 2015; Nagata, 2007; Yoshida et al., 2005a). Our data show little expression in the wild-type or knockout cells or in the wild-type cells from the Nan strain; however, their levels are dramatically increased in the Nan/+ cells (Fig. 1C) [also observed in an independent experiment (Fig. S1A), thus providing us with data from a total of 12 biological replicates]. These are not the result of global or regional changes; in each case, expression of the adjacent genes remain unaffected irrespective of the genotype (Fig. 1C). Total KLF1 levels are only slightly lower in Nan/+ compared with wild type (Fig. 1D and Fig. S1B).

Although EKLF expression is restricted to erythroid progenitors and their progeny, we verified that the ectopic expression results from the erythroid compartment by depleting adherent cells and expanding E13.5 fetal liver cells in erythropoietin. qRT-PCR analysis of RNA shows that these erythroid-enriched cells express KLF1 at similar levels, that Nan/+ cells exhibit the dramatic decrease in dematin (Epb4.9) expression previously seen with total fetal liver material, and that there is dysregulation of hepcicin (Hamp) and Irf7 (Fig. 1E). We conclude that Hamp and Irf7 are mis-expressed in the erythroid cell by virtue of the presence of the Nan mutant variant of KLF1.

Erythroferrone is a new EKLF target that is part of the disrupted intrinsic expression pattern generated in the Nan-EKLF red cell

Hepcidin levels are suppressed by erythroferrone (Fam132b) (Kautz et al., 2014), a gene whose expression in the erythroid cell is stimulated by erythropoietin when iron mobilization is desired. We found that erythroferrone is regulated by KLF1 levels in the erythroid cell, as its expression is virtually absent in the knockout (Fig. 2A). Of relevance here, its levels are also dramatically deficient in Nan/+ cells (Fig. 2A,B; Fig. S1C); as with other similarly affected genes (Siatecka et al., 2010b), this is not due to haploinsufficient levels of wild-type EKLF (not shown). The two likely KLF1-binding sites in this gene are category II (Siatecka et al., 2010b), and thus are predicted to be negatively affected by the presence of the Nan variant.

Fig. 2.

Altered expression of erythroferrone (Fam132b) in Nan mice fetal liver samples or rescued EKLF-null erythroid cells. (A) RNA-seq expression data from biological triplicates of wild-type (+/+) (dark gray) and Nan/+ (red) littermates (‘Nan’), or from wild-type (+/+) (light gray) and EKLF-null (−/−) (blue) littermates were analyzed for expression of Fam132b (erythroferrone). A representative browser expression profile from one sample each is shown [wild type (+/+) (dark gray) and Nan/+ (red)]; the adjacent gene is unaffected by Nan-EKLF expression status. A flow chart showing the relationship of erythroferrone to upstream and downstream effectors is shown on the right. (B) Total RNA from adherent cell-depleted erythroid cells isolated from wild-type (+/+) or Nan/+ littermates (‘Nan’) were monitored by q-RT-PCR for expression of Fam132b (erythroferrone). Results are the average of three biological replicates each performed in triplicate. (C) EKLF-null erythroid cells were used to monitor EKLF association with Fam132b chromatin (ChIP) after a 1 h tamoxifen-induced rescue with wild-type EKLF (K1ER-A) or Nan-EKLF (K1NanER-B) (Coghill et al., 2001); Alas2 was monitored as a positive control. Analysis is limited to 1 h post-treatment to avoid loss of cell number following KLF1 induction of CDK inhibitors and the ensuing cell cycle arrest (Pilon et al., 2008; Siatecka et al., 2010a; Tallack et al., 2009, 2007; Gnanapragasam et al., 2016). Results are representative of two experiments. (D) RNA expression was monitored across the Fam132b locus during a 1 h tamoxifen-induced rescue with the wild-type EKLF (K1ER-A), Nan-EKLF (K1NanER-B) or parental K1 line; treatment with ethanol (EtOH) is the negative control in each case. 4-Thiouridine tagging was used to identify nascent RNA (Gillinder et al., 2016). Results are representative of two experiments.

Fig. 2.

Altered expression of erythroferrone (Fam132b) in Nan mice fetal liver samples or rescued EKLF-null erythroid cells. (A) RNA-seq expression data from biological triplicates of wild-type (+/+) (dark gray) and Nan/+ (red) littermates (‘Nan’), or from wild-type (+/+) (light gray) and EKLF-null (−/−) (blue) littermates were analyzed for expression of Fam132b (erythroferrone). A representative browser expression profile from one sample each is shown [wild type (+/+) (dark gray) and Nan/+ (red)]; the adjacent gene is unaffected by Nan-EKLF expression status. A flow chart showing the relationship of erythroferrone to upstream and downstream effectors is shown on the right. (B) Total RNA from adherent cell-depleted erythroid cells isolated from wild-type (+/+) or Nan/+ littermates (‘Nan’) were monitored by q-RT-PCR for expression of Fam132b (erythroferrone). Results are the average of three biological replicates each performed in triplicate. (C) EKLF-null erythroid cells were used to monitor EKLF association with Fam132b chromatin (ChIP) after a 1 h tamoxifen-induced rescue with wild-type EKLF (K1ER-A) or Nan-EKLF (K1NanER-B) (Coghill et al., 2001); Alas2 was monitored as a positive control. Analysis is limited to 1 h post-treatment to avoid loss of cell number following KLF1 induction of CDK inhibitors and the ensuing cell cycle arrest (Pilon et al., 2008; Siatecka et al., 2010a; Tallack et al., 2009, 2007; Gnanapragasam et al., 2016). Results are representative of two experiments. (D) RNA expression was monitored across the Fam132b locus during a 1 h tamoxifen-induced rescue with the wild-type EKLF (K1ER-A), Nan-EKLF (K1NanER-B) or parental K1 line; treatment with ethanol (EtOH) is the negative control in each case. 4-Thiouridine tagging was used to identify nascent RNA (Gillinder et al., 2016). Results are representative of two experiments.

The implication that erythroferrone is a KLF1 target was directly tested using the KLF1-null rescue system with which stably expressed cytoplasmic ER/KLF1 or ER/ Nan-KLF1 can be induced to translocate to the nucleus by treatment of the cells with tamoxifen (Coghill et al., 2001; Hodge et al., 2006). Although the treatment protocol is limited to 1 h, these rescued cells show robust chromatin association by ER/KLF1 to one of these sites (a category II site in intron 1) but not by ER/Nan-KLF1 (Fig. 2C). In addition, RNA begins to accumulate within this short time period only in the wild-type rescue (Fig. 2D).

We found additional evidence for disarray in iron utilization components within the Nan/+ erythroid cell (Gillinder et al., 2016). For example, when comparing Nan/+ cells with wild type we see a slight decrease in transferrin receptor (Tfrc) levels, and a slight increase in ferroportin (Slc40a1) in spite of the increase in hepcidin (Fig. 3A). This yields a lower level of iron in the red cell (Ganz and Nemeth, 2012; Tolosano, 2015). Coupled to this, heme oxygenase (Hmox1) levels increase while heme transporter HRG1 (Slc48a1) levels decrease in the Nan/+ cells compared with wild type (Fig. 3B), yielding less heme in the erythroid cell (Korolnek and Hamza, 2015; Narla and Mohandas, 2015).

Fig. 3.

Expression of iron- and heme-utilization targets in fetal liver samples. (A,B) RNA-seq expression data from biological triplicates of wild-type (+/+) and Nan/+ littermates (‘Nan’) were analyzed for expression of genes coding for iron (A) and heme (B) transport proteins as indicated.

Fig. 3.

Expression of iron- and heme-utilization targets in fetal liver samples. (A,B) RNA-seq expression data from biological triplicates of wild-type (+/+) and Nan/+ littermates (‘Nan’) were analyzed for expression of genes coding for iron (A) and heme (B) transport proteins as indicated.

In light of this, we examined expression patterns of other genes involved in iron utilization or in the inflammation pathway (reviewed by Ganz, 2011; Ginzburg and Rivella, 2011; Guida et al., 2015; Kim and Nemeth, 2015; Li et al., 2014; Nagata, 2007; Rivella, 2015; Yoshida et al., 2005b) to address indirect or Nan-KLF1-independent causes for the inefficient erythropoiesis (Gillinder et al., 2016). Expression is either unchanged [hemochromatosis (Hfe), IRP1 (Aco1), aconitase (Aco2), Flvcr1 (Mfsd7b); Fig. S2A] or remains insignificantly expressed [Bmp6, hemojuvelin (Hfe2), Gdf11, Pigf; not shown]. Genes involved in regulation of hepcidin, such as Tmprss6, are not altered (Fig. S2B) or not expressed (Gdf15, Atoh8 and Sox2; not shown), although the level of Stat3, an upstream regulator of hepcidin, is increased in Nan/+ cells (Fig. S2B) in spite of undetectable Il6 (not shown). Levels of erythroid regulators such as Twsg1, HIF2α (Epas1), or IRP2 (Ireb2) are not altered (Fig. S2C). With respect to genes involved in a predicted inflammatory response due to Irf7 expression, we see no change in expression of Irf3, Irf2 or Tlr2 (Fig. S2D), and no expression of Tlr6 (not shown). We conclude that the misexpression of hepcidin and IRF7, and the depletion of erythroferrone expression in the red blood cell are not the result of a generalized response to external stimuli but rather are caused by the intrinsic presence and inappropriate binding of Nan-KLF1.

Effects of Nan-EKLF expression early in development

Embryos that express only the Nan-EKLF variant (i.e. Nan/Nan or Nan/−) are lethal by E10-11 and display a striking dysmorphology that extends beyond the fetal erythroid cell (Heruth et al., 2010). To model these early stages, we established embryonic stem cells(ESCs) from Nan/Nan and +/+ littermates and differentiated them to embryoid bodies (EBs). EBs synchronously recapitulate erythropoiesis (Choi et al., 2005; Kennedy and Keller, 2003); in our hands, the onset of Klf1 and primitive erythropoiesis begins at day 4 and peaks by day 6, when definitive erythropoiesis is well underway to eventual dominance (Adelman et al., 2002; Bruce et al., 2007; Lohmann and Bieker, 2008). Nan/Nan EBs demonstrate morphological defects by day 6 of differentiation, as they are smaller in comparison with +/+ EBs (Fig. 4A). Although the onset of the CD44 early definitive erythroid cell surface marker (Chen et al., 2009; Liu et al., 2013) is normal in both sets by day 6 (Fig. 4B, left), Nan/Nan EBs are defective in generating late (primitive or definitive) erythroid cells, as judged by the absence of Ter119 expression in these cells at day 6 or day 7 (Fig. 4B, left), which attains normal levels (Lohmann and Bieker, 2008; Soni et al., 2014) in the wild-type EBs.

Fig. 4.

Effects of Nan on embryoid body differentiation. Embryonic stem (ES) cells derived from wild-type (+/+) or Nan/Nan littermates were differentiated to embryoid bodies (EBs) and analyzed at day (d) 4, d6 or d7. (A) Photomicrographs of d6 EBs concurrently generated from wild-type or Nan/Nan ES cells (same magnification). (B) Cell-surface marker expression of CD44 (early erythroid) and Ter119 (late erythroid) was performed on d4, d6 or d7 EBs concurrently generated from wild-type or Nan/Nan ES cells. (Left) Results from individual cultures. (Right) Results after having mixed equivalent numbers of ES cells (wild type with Nan/Nan as indicated) at d0.

Fig. 4.

Effects of Nan on embryoid body differentiation. Embryonic stem (ES) cells derived from wild-type (+/+) or Nan/Nan littermates were differentiated to embryoid bodies (EBs) and analyzed at day (d) 4, d6 or d7. (A) Photomicrographs of d6 EBs concurrently generated from wild-type or Nan/Nan ES cells (same magnification). (B) Cell-surface marker expression of CD44 (early erythroid) and Ter119 (late erythroid) was performed on d4, d6 or d7 EBs concurrently generated from wild-type or Nan/Nan ES cells. (Left) Results from individual cultures. (Right) Results after having mixed equivalent numbers of ES cells (wild type with Nan/Nan as indicated) at d0.

Although this may result from an intrinsic erythroid deficit, we also tested whether the Nan cells could exert an extrinsic effect on wild-type EB differentiation by mixing equivalent numbers of +/+ and Nan/Nan cells at d0 and then monitoring erythroid status during differentiation via cell surface marker analysis as before. Again, although CD44 levels are not affected, no evidence of Ter119 expression is evident even though the number of +/+ cells included in the culture was no different from before (Fig. 4B, right). We conclude that the dysmorphology of the non-erythroid tissues seen in the Nan embryo may well result from the ectopic expression pattern and resultant secretory proteins seen within the Nan-expressing cell.

Effects of Nan-EKLF expression in the adult

We next addressed whether the altered expression patterns of Hamp and Irf7 seen in the fetal liver are also manifested in the Nan/+ adult, and could explain part of the anemia exhibited by the Nan mouse. The adult Nan/+ mouse exhibits low mean corpuscular volume and mean corpuscular hemoglobin, reticulocytosis and splenomegaly (Siatecka et al., 2010b), suggesting defective erythropoiesis possibly due to iron deficiency (Andrews, 2009). Consistent with this, circulating erythropoietin levels are higher in Nan/+ adults than in matched littermates (Fig. 5A). Hamp and Irf7 expression levels are significantly higher in spleen and bone marrow RNA from the Nan/+ mouse than in the wild type (Fig. 5B) (although hepcidin expression is lower in bone marrow than in spleen). To probe the possibility of systemic changes, we monitored serum levels of hepcidin and found that its levels are significantly increased in the circulation in Nan/+ mice compared with wild type (Fig. 5A). Analysis of serum IFNβ levels show an even more dramatic change (Fig. 5A). Serum analyses in the adult reflect alterations in the iron use components we had observed in the Nan/+ fetal erythroid cell (Fig. 3), as there is a slight but significant increase in serum bilirubin and a decrease in serum iron (Fig. 5C). Together, these add up to an iron- and heme-deficient red cell environment that contributes to the overall anemia of the mouse.

Fig. 5.

Analysis of expression changes in adult Nan/+ mice. (A) Sera from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) were monitored for expression of erythropoietin (EPO) (n=6, each analyzed at three dilutions, P<0.0001), hepcidin (n=11, each analyzed in duplicate, P<0.0001) or IFNβ (n=4, each analyzed in duplicate, P=0.049). Data are average±s.e.m. (B) Spleen and bone marrow RNA from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) was monitored by qRT-PCR for expression of hepcidin (Hamp) or Irf7. Results are from two experiments each performed in triplicate and analyzed at the same time. Although Irf7 is regulated differently in the adult bone marrow compared with the fetal liver (Kingsley et al., 2013; Liang et al., 2015), its levels are nonetheless significantly increased in the adult Nan/+ bone marrow. (C) Sera from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) were monitored for expression of bilirubin (n=10-12, P<0.0001) or iron (n=5-7, P=0.0025). Data are average±s.e.m. (D) Sera from wild-type (+/+) or Nan/+ adult littermates (‘Nan’), were monitored for expression of IL6 (n=8, each analyzed at two dilutions as indicated) and compared with IL6 standards (duplicates are shown).

Fig. 5.

Analysis of expression changes in adult Nan/+ mice. (A) Sera from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) were monitored for expression of erythropoietin (EPO) (n=6, each analyzed at three dilutions, P<0.0001), hepcidin (n=11, each analyzed in duplicate, P<0.0001) or IFNβ (n=4, each analyzed in duplicate, P=0.049). Data are average±s.e.m. (B) Spleen and bone marrow RNA from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) was monitored by qRT-PCR for expression of hepcidin (Hamp) or Irf7. Results are from two experiments each performed in triplicate and analyzed at the same time. Although Irf7 is regulated differently in the adult bone marrow compared with the fetal liver (Kingsley et al., 2013; Liang et al., 2015), its levels are nonetheless significantly increased in the adult Nan/+ bone marrow. (C) Sera from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) were monitored for expression of bilirubin (n=10-12, P<0.0001) or iron (n=5-7, P=0.0025). Data are average±s.e.m. (D) Sera from wild-type (+/+) or Nan/+ adult littermates (‘Nan’), were monitored for expression of IL6 (n=8, each analyzed at two dilutions as indicated) and compared with IL6 standards (duplicates are shown).

Increased hepcidin production can be achieved by IL6-mediated stimulation of the liver, particularly as a result of an inflammatory response (Nemeth et al., 2004a). As a result, we checked for IL6 in the serum, but did not find any detectable levels in either the wild-type or Nan/+ adult (Fig. 5D). This removes inflammation as a causal effector and negates a possible liver contribution to the circulatory hepcidin levels we see in the Nan/+ adult.

Collectively, the developmental data suggest that the anemia in adult Nan/+ mice is exacerbated not only by the intrinsic defects in erythroid expression based on the category I versus category II distinction, but also by the neomorphic genetic output via the new recognition site. This leads to expression of secreted molecules that work directly against establishing a steady-state replenishment of erythroid cells to combat the anemia.

In vitro and in vivo testing of altered DNA binding by Nan-EKLF protein in the Nan/+ adult

The DNA-recognition properties of the Nan-KLF1 protein variant (Gillinder et al., 2016) suggest a possible mechanism for the effect; i.e. by binding to novel target site(s) within the Hamp and Irf7 genes not normally recognized by the wild-type KLF1 protein. Inspection of the gene loci of Hamp and Irf7 reveal a number of potential Nan-KLF1 target sites (5′CCMNGCCCN) that meet this criterion (Fig. 6A). A subset were tested in vitro by quantitative gel shift assays for binding to wild-type or Nan-KLF1 proteins. As controls, we obtained dissociation constants for known category I (p21 site 3) and category II (dematin and E2F2 site 2) DNA sites binding to wild-type or Nan-KLF1 protein (Fig. 6B). Using this approach, we monitored binding to the predicted new sites in the hepcidin (Hamp) and Irf7 genes and found that binding affinities are substantially higher for Nan-KLF1 compared with wild type (Fig. 6C), supporting the predictions made from the global variant analysis.

Fig. 6.

Binding of Nan-EKLF to novel genomic regions. (A) Localization of wild-type EKLF binding consensus (5′CCMCRCCCN) or Nan-EKLF-binding consensus (5′CCMNGCCCN) across the hepcidin (Hamp) and Irf7 genomic loci are shown. Circled regions (red) show the location and sequences used for further analysis: Hamp-1, Hamp-2 and Irf7-5 (right). (B) In vitro quantitative gel-shift analyses were performed to assess the binding affinities of wild-type and Nan-EKLF proteins to representative category I (at the p21 gene) or category II (ECR1 at the dematin gene and E2f2 site 2) elements (Siatecka et al., 2010b). Multiple DNA concentrations were tested and selected, as described in the Materials and Methods: 12.5 to 50 nM for p21 (P=0.32, not significant), 25 nM for ECR1 (P=0.0045), and 0.5 to 12.5 nM for E2f2 site 2 (P=0.0052), all by unpaired t-test. Excel Solver software was used to determine Kd values along with the standard deviation (s.d.); SLS is the sum of the squares of the errors and indicates the range of error over the curves. Also shown (as inserts on the p21 data) are typical gel data generated and analyzed for all the experiments. GST alone showed no binding to any of these probes (not shown). These provide us with a range of well-discriminated affinities, with nanomolar binding constants that are equivalent at p21 but are 2- (E2F2) to 3.5- (dematin) fold higher with Nan-KLF1 compared with wild type. (C) In vitro quantitative gel shift analyses were performed to assess the binding affinities of wild-type and Nan-EKLF proteins to the novel Hamp-1, Hamp-2 and Irf7-5 elements. All were analyzed at 5 nM DNA, except Hamp1 ‘EKLF 2’ and IRF7-5 ‘EKLF 2’, which were at 25 nM. (D) In vivo chromatin immunoprecipitation was performed at selected regions (as shown in A) with IgG (I) or 7B2 anti-EKLF (E) antibodies. Necdin was included as a negative control. Spleens from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) were used for the analyses. Results are from two separate experiments, each performed in triplicate.

Fig. 6.

Binding of Nan-EKLF to novel genomic regions. (A) Localization of wild-type EKLF binding consensus (5′CCMCRCCCN) or Nan-EKLF-binding consensus (5′CCMNGCCCN) across the hepcidin (Hamp) and Irf7 genomic loci are shown. Circled regions (red) show the location and sequences used for further analysis: Hamp-1, Hamp-2 and Irf7-5 (right). (B) In vitro quantitative gel-shift analyses were performed to assess the binding affinities of wild-type and Nan-EKLF proteins to representative category I (at the p21 gene) or category II (ECR1 at the dematin gene and E2f2 site 2) elements (Siatecka et al., 2010b). Multiple DNA concentrations were tested and selected, as described in the Materials and Methods: 12.5 to 50 nM for p21 (P=0.32, not significant), 25 nM for ECR1 (P=0.0045), and 0.5 to 12.5 nM for E2f2 site 2 (P=0.0052), all by unpaired t-test. Excel Solver software was used to determine Kd values along with the standard deviation (s.d.); SLS is the sum of the squares of the errors and indicates the range of error over the curves. Also shown (as inserts on the p21 data) are typical gel data generated and analyzed for all the experiments. GST alone showed no binding to any of these probes (not shown). These provide us with a range of well-discriminated affinities, with nanomolar binding constants that are equivalent at p21 but are 2- (E2F2) to 3.5- (dematin) fold higher with Nan-KLF1 compared with wild type. (C) In vitro quantitative gel shift analyses were performed to assess the binding affinities of wild-type and Nan-EKLF proteins to the novel Hamp-1, Hamp-2 and Irf7-5 elements. All were analyzed at 5 nM DNA, except Hamp1 ‘EKLF 2’ and IRF7-5 ‘EKLF 2’, which were at 25 nM. (D) In vivo chromatin immunoprecipitation was performed at selected regions (as shown in A) with IgG (I) or 7B2 anti-EKLF (E) antibodies. Necdin was included as a negative control. Spleens from wild-type (+/+) or Nan/+ adult littermates (‘Nan’) were used for the analyses. Results are from two separate experiments, each performed in triplicate.

As a final test, we checked for in vivo interaction of KLF1 or Nan-KLF1 to these new sites. As we do not have an antibody that can distinguish wild-type KLF1 from Nan-KLF1, we simply monitored whether the adult Nan/+ spleen shows a novel KLF1 ChIP compared with wild type at any of the potential sites of Hamp and Irf7. KLF1 is well expressed within the red pulp of the normal adult spleen (Miller and Bieker, 1993; Southwood et al., 1996). The analyses (Fig. 6D) show that the putative sites are indeed occupied only in the chromatin derived from the Nan/+ mouse. Collectively, the in vitro and in vivo data demonstrate that the variant DNA recognition property of Nan-KLF1 is responsible for altered binding and subsequent ectopic protein expression in the Nan/+ erythroid cell.

DISCUSSION

Our studies demonstrate that an apparently minor conservative change in a single amino acid (E to D) of a crucial erythroid transcription factor can result in significant phenotypic changes. In Nan-KLF1, the residue in question is absolutely conserved across the whole KLF family in all species examined. This leads to a limitation on its ability to recognize the consensus wild-type DNA element (Siatecka et al., 2010b). However, a remarkable finding from the global analyses was that a novel, atypical site is newly recognized (Gillinder et al., 2016), leading to a significant number of expressed genes that are not normally present in the mammalian red cell. We have shown how this can lead to defects of gene expression within and outside the cell.

Within the wild-type erythroid compartment, neither hepcidin nor IRF7 is expressed. However, unanticipated phenotypic changes arise from two physiologically nonproductive cycles generated by the Nan/+ red cell (Fig. 7). In one case, misexpression of hepcidin and deficient expression of erythroferrone lead to a decrease in iron availability and lower red cell numbers. In the second case, misexpression of IRF7 induces IFNβ expression, which has a repressive effect on erythroid development. The organism responds to the defects by inducing erythropoietin to replenish erythropoiesis, leading to splenomegaly. However, this is futile, as the very cells being expanded are those that are secreting hepcidin and IFNβ, leading to an ill-designed feedback inhibition. This cycle is superimposed upon the intrinsic erythroid deficits, leading to a worsening of the red cell anemia. The increase in hepcidin is both direct (via binding of Nan-EKLF to its atypical site) and indirect (via low erythroferrone expression in the Nan/+ environment), as ablation of EKLF (and thus low erythroferrone) is not sufficient to substantially increase hepcidin levels. Remarkably, these deficits occur even in the presence of a normal wild-type copy of KLF1.

Fig. 7.

Summary and model of systemic and intrinsic effects of the Nan-EKLF mutation. (A) (Left) During normal wild-type steady-state, adult erythropoiesis is kept in balance by the levels of hepcidin, which negatively regulates iron availability when erythroblast levels are replete, but is negatively regulated by erythroid erythroferrone when iron is needed for erythroid expansion, such as after stimulation by erythropoietin. (Right) In the case of Nan/+ adults, the direct increase of hepcidin by Nan-EKLF (a) and the low level of expression of erythroferrone (b) decrease iron availability, negatively affecting erythroblast expansion. Coupled to this, misexpression of Irf7 (c) leads to secretion of IFNβ, superimposing another negative cycle on erythroid expansion. The net result is a stimulation of erythroblast proliferation and the accompanying splenomegaly that is non-productive and does not address the anemia, which remains chronic. These effects in the adult form part of an array of developmental deficits that occur in spite of the erythroid-restricted expression of Nan-EKLF, including dymorphology in the embryo and embryoid body, and defective erythropoiesis in the fetal red cell. (B) Coupled to the systemic effects summarized in A, intrinsic expression changes (based on fetal liver expression, although also likely in the adult) in the Nan/+ red cell contribute to defective erythropoiesis by altering genes that play roles in heme and iron uptake.

Fig. 7.

Summary and model of systemic and intrinsic effects of the Nan-EKLF mutation. (A) (Left) During normal wild-type steady-state, adult erythropoiesis is kept in balance by the levels of hepcidin, which negatively regulates iron availability when erythroblast levels are replete, but is negatively regulated by erythroid erythroferrone when iron is needed for erythroid expansion, such as after stimulation by erythropoietin. (Right) In the case of Nan/+ adults, the direct increase of hepcidin by Nan-EKLF (a) and the low level of expression of erythroferrone (b) decrease iron availability, negatively affecting erythroblast expansion. Coupled to this, misexpression of Irf7 (c) leads to secretion of IFNβ, superimposing another negative cycle on erythroid expansion. The net result is a stimulation of erythroblast proliferation and the accompanying splenomegaly that is non-productive and does not address the anemia, which remains chronic. These effects in the adult form part of an array of developmental deficits that occur in spite of the erythroid-restricted expression of Nan-EKLF, including dymorphology in the embryo and embryoid body, and defective erythropoiesis in the fetal red cell. (B) Coupled to the systemic effects summarized in A, intrinsic expression changes (based on fetal liver expression, although also likely in the adult) in the Nan/+ red cell contribute to defective erythropoiesis by altering genes that play roles in heme and iron uptake.

More globally, iron is indispensable for all tissues, being a crucial component of essential enzymes and multiprotein complexes that contain heme and iron-sulfur clusters (Zhang et al., 2014). As a result, heme-containing proteins play crucial roles in all cells (Sawicki et al., 2015); however, heme can also play lineage-specific roles in differentiation (Philip et al., 2015). Proper heme levels are also crucial for normal fetal development, as defects can lead to limb deformities in addition to abnormal erythropoiesis (Keel et al., 2008). Not surprisingly then, increased circulatory hepcidin can have systemic effects; for example, decreasing heme- and nonheme-iron absorption to the duodenum (Cao et al., 2014). In the present case, the levels of hepcidin in the Nan/+ mouse are high enough to exert a physiological effect (Nemeth et al., 2004b).

As a result, we suggest that Nan-EKLF expression contributes to the dysmorphology of Nan embryos seen during early stages of development and during ES cell differentiation, to the fetal liver erythroid misregulation, and to the chronic anemia of the adult (Fig. 7). In this context, it is of interest that a DAVID analysis of the most significantly upregulated gene categories in Nan/+ RNA shows that those in the ‘secreted’ and ‘signal’ groupings are the most numerous (Fig. S3).

Intriguingly, low iron availability via hepcidin expression coupled with expression of IFNβ resembles the anemia of chronic inflammation (Goodnough et al., 2010), where iron restriction sensitizes cells to the inhibitory effects of inflammatory cytokines (Richardson et al., 2013). However, we do not detect any IL6, the key inducer of hepcidin and mediator of inflammation (Hom et al., 2015; Wang and Babitt, 2016).

To our knowledge, finding genetic effects of a tissue-restricted factor that occur outside of its normal expression realm is novel, and thus may be an unrecognized contribution to human disease. In the present case, the most direct connection is with CDA type IV (Iolascon et al., 2012), which is caused by a mutation in human KLF1 at the same amino acid (Arnaud et al., 2010; Jaffray et al., 2013; Singleton et al., 2011). The change is non-conservative (E to K), so although not all characteristics of the Nan mouse may apply, individuals with CDA exhibit altered globin switching and membrane deficits that are highly reminiscent of the present model (discussed by Siatecka et al., 2010b). Of greater interest are the non-erythroid aspects of a subset of these individuals, such as short stature, high serum iron and bilirubin levels, and altered gonads. These suggest that there may very well be secreted factors that are aberrantly/ectopically expressed in these human cells as a direct result of the single amino acid change seen in the human KLF1 CDA mutant.

It may appear surprising that Nan-KLF1 is able to recognize its atypical site within genes that are presumably not easily accessible outside their normal cell of expression. However, recent studies with KLF4, which is highly related to KLF1 (Bieker, 2001), suggest that zinc fingers 2 and 3 play a necessary and dominant role in targeting its recognition site within nucleosomes (Soufi et al., 2015). In the present case, the amino acid change is located within zinc finger 2 and thus could enable Nan-KLF1 to play a role as a pioneer factor at ectopic sites in spite of their lower accessibility in the red cell.

Our present studies, as with the earlier analyses of the Nan-KLF1 protein gene regulation (Siatecka et al., 2010b), re-emphasize that the effects of KLF1 mutation are not strictly due to a dominant-negative outcome. This is clearly different from the cell-intrinsic defects seen with the haploinsufficient or most of the other mutant KLF1 proteins that have been identified in humans (Borg et al., 2011; Helias et al., 2013; Perkins et al., 2016; Singleton et al., 2012; Tallack and Perkins, 2013; Waye and Eng, 2015). What is seen in the present case is a new variant with novel properties that dramatically changes Nan/+ red cell identity.

MATERIALS AND METHODS

General protocols

Establishment of the Nan mouse strain has been previously described (Heruth et al., 2010; Lyon, 1983; Siatecka et al., 2010b). All protocols have been reviewed and approved by the IACUC at Mount Sinai. RNA was isolated from E13.5 fetal livers or adult spleens using Trizol. Total RNA was purified with a Qiagen RNeasy kit. qRT-PCR was performed using published procedures (Siatecka et al., 2010b). Global RNA-seq was performed by the Mount Sinai Facility or by the JAX facility and analyzed as described (Gillinder et al., 2016). The DAVID analysis tool v6.7 (https://david-d.ncifcrf.gov/) was used as described (Huang et al., 2009).

Cell isolation and manipulation

K1-ER or K1-Nan-ER cells (originally described by Coghill et al., 2001, recently authenticated and reanalyzed by Gillinder et al., 2016) were grown and treated with doxycycline as described (Coghill et al., 2001; Hodge et al., 2006). Analysis is limited to 1 h post-treatment to avoid loss of cell number following KLF1 induction of CDK inhibitors and the ensuing cell cycle arrest (Gnanapragasam et al., 2016; Pilon et al., 2008; Siatecka et al., 2010a; Tallack et al., 2009, 2007). As a result, only immediate-early effects can be detected. Nascent RNA expression was monitored after a concurrent 1 h treatment of cells with 4-thiouridine, as described previously (Gillinder et al., 2016).

Mouse ESCs derived from wild-type and Nan/Nan blastocysts (littermates) were established as described previously with minor modifications (Meissner et al., 2009). These were maintained and differentiated to embryoid bodies as per established protocols (Choi et al., 2005; Kennedy and Keller, 2003) described previously (Manwani et al., 2007). Ter119-PE and CD44-FITC antibodies were from eBiosciences.

Erythroblasts were derived from wild-type and Nan/+ E12.5 fetal livers and depleted of adherent cells as previously described (England et al., 2011; Gnanapragasam et al., 2016). Briefly, cells were harvested, and plated on gelatin dishes for 2 days. This was followed by a 1-day culture in erythroid growth media prior to harvest for RNA analysis.

ChIP analysis of EKLF binding was performed using a modification of previous procedures (Lohmann and Bieker, 2008; Siatecka et al., 2015). Briefly, cells from fetal livers or spleens (7-10×106 per immunoprecipitation) were crosslinked with 1.0% formaldehyde. Chromatin was sonicated, immunoprecipitated with antibodies against mouse IgG (557273; BD Pharmingen) or EKLF (7B2), and purified on magnetic protein G Dynabeads (10004D; Life Technologies). Eluted and precipitated genomic DNA was amplified and quantified by PCR.

Serum testing

Serum from adult mice was analyzed using ELISA kits for erythropoietin (R&D Systems), IL6 (Thermo Fisher), hepcidin (Intrinsic Lifesciences) or IFNβ (PBL Assay Science), following the manufacturers' instructions. Bilirubin and iron were measured on a Beckman Coulter AU680 Chemistry Analyzer; only samples with low hemolysis [i.e. hemoglobin value lower than 100 (Beckman-Coulter)] were used for these analyses.

Quantitative protein-DNA binding analysis

Quantitative gel shifts were performed using GST-tagged EKLF zinc fingers (Bieker and Southwood, 1995), purified as previously described (Soni et al., 2014) and incubated with 32P-labeled DNA oligonucleotide probes in 25 mM HEPES (pH 7.5), 16 mM KCl, 50 mM NaCl, 2 µM ZnCl2, 0.6 mM β-mercaptoethanol and 8% glycerol for 30 min on ice at various concentrations (see figures and legends). A 10-fold molar excess of polydeoxyinosinic-deoxycytidylic acid (dIdC, Sigma-Aldrich) relative to the DNA probe was used to reduce non-specific binding. Protein concentrations were determined by saturation binding to the p21 gene EKLF site, to which both EKLF ZF and Nan ZF bind similarly. A fixed amount of DNA was incubated with increasing amounts of protein and DNA, and protein concentrations were adjusted to maximize the curve fits. Samples were electrophoresed on 8% acrylamide gels in 0.5× TBE at 150 volts for 1.5 h at room temperature. Quantitation was performed on a Molecular Dynamics Phosphorimager. Kd values were determined by curve fitting with Excel Solver using the following equation and minimizing the squared error by varying Kdis:
formula
where Dtot is the DNA concentration, Ptot is the protein concentration, and Kdis is the dissociation constant. Results for E2f2 site 2 and wild-type EKLF in Fig. 6B appear more variable than others because different amounts of poly(dIdC) were used (20- to 400-fold molar excess) to counteract non-specific binding that occurred at high protein to DNA ratios. The four best fits (SLS≤0.045) using 0.5 to 2.5 nM DNA and poly(dIdC) were averaged. For high-affinity binding, DNA amounts were chosen such that 70 to 100% of the probe was bound at highest protein concentration and there was detectable binding in the low protein samples: once established, these same conditions were used for lower affinity interactions.

To generate probes, one strand of DNA oligonucleotide was end-labeled with polynucleotide kinase and γ-P32 ATP, and then annealed to the unlabeled complimentary strand. For some experiments, only labeled DNA was used and concentrations were determined with Quant-it PicoGreen from Invitrogen. For other experiments, the labeled DNA was used a tracer with known quantities of unlabeled DNA. Similar results were obtained with these methods.

Acknowledgements

We thank S. Soni for providing purified recombinant protein, and N. Gnanapragasam and C. Deligianni for primary cell protocol advice. We thank T. Ganz, S. Rivella, N. Andrews, D. Higgs, H. Drakesmith, C. Roy and A. Goldfarb for discussion. We thank S. Ghaffari, J. Little and C. Deligianni for comments on the manuscript.

Footnotes

Author contributions

A.P., L.X., C.D.T., M.D., K.G., M.S. and D.N. performed experiments. L.L.P. and A.C.P. provided data and analysis. J.J.B. directed experiments, provided data and analysis, and wrote the manuscript with input from all authors.

Funding

This work was supported by two National Institutes of Health Public Health Service grants (R01 DK46865 to J.J.B. and R01 DK100692 to L.L.P.), by a National Science Centre grant (2013/09/B/NZ1/01879 to M.S.), by a National Health and Medical Research Council grant (APP1082429 to A.C.P.) and by a grant from the National Cancer Institute to the Jackson Laboratory (P30 CA034196). C.D.T. was supported by the National Institutes of Health Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases. D.N. is an American Society for Hematology Scholar. Deposited in PMC for release after 12 months.

Data availability

The RNA-seq data referred to in this study (Gillinder et al., 2016) are available from Gene Expression Omnibus (GEO) under accession GSE71396.

References

Adelman
,
C. A.
,
Chattopadhyay
,
S.
and
Bieker
,
J. J.
(
2002
).
The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media
.
Development
129
,
539
-
549
.
Andrews
,
N. C.
(
2009
).
Genes determining blood cell traits
.
Nat. Genet.
41
,
1161
-
1162
.
Arnaud
,
L.
,
Saison
,
C.
,
Helias
,
V.
,
Lucien
,
N.
,
Steschenko
,
D.
,
Giarratana
,
M.-C.
,
Prehu
,
C.
,
Foliguet
,
B.
,
Montout
,
L.
,
de Brevern
,
A. G.
, et al. 
(
2010
).
A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia
.
Am. J. Hum. Genet.
87
,
721
-
727
.
Bieker
,
J. J.
(
2001
).
Kruppel-like factors: Three fingers in many pies
.
J. Biol. Chem.
276
,
34355
-
34358
.
Bieker
,
J. J.
and
Southwood
,
C. M.
(
1995
).
The Erythroid Krüppel-like Factor (EKLF) transactivation domain is a critical component for cell-specific inducibility of a β-globin promoter
.
Mol. Cell. Biol.
15
,
852
-
860
.
Borg
,
J.
,
Patrinos
,
G. P.
,
Felice
,
A. E.
and
Philipsen
,
S.
(
2011
).
Erythroid phenotypes associated with KLF1 mutations
.
Haematologica
96
,
635
-
638
.
Bruce
,
S. J.
,
Gardiner
,
B. B.
,
Burke
,
L. J.
,
Gongora
,
M. M.
,
Grimmond
,
S. M.
and
Perkins
,
A. C.
(
2007
).
Dynamic transcription programs during ES cell differentiation towards mesoderm in serum versus serum-freeBMP4 culture
.
BMC Genomics
8
,
365
.
Cao
,
C.
,
Thomas
,
C. E.
,
Insogna
,
K. L.
and
O'Brien
,
K. O.
(
2014
).
Duodenal absorption and tissue utilization of dietary heme and nonheme iron differ in rats
.
J. Nutr.
144
,
1710
-
1717
.
Chen
,
K.
,
Liu
,
J.
,
Heck
,
S.
,
Chasis
,
J. A.
,
An
,
X.
and
Mohandas
,
N.
(
2009
).
Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis
.
Proc. Natl. Acad. Sci. USA
106
,
17413
-
17418
.
Choi
,
K.
,
Chung
,
Y. S.
and
Zhang
,
W. J.
(
2005
).
Hematopoietic and endothelial development of mouse embryonic stem cells in culture
.
Methods Mol. Med.
105
,
359
-
368
.
Coghill
,
E.
,
Eccleston
,
S.
,
Fox
,
V.
,
Cerruti
,
L.
,
Brown
,
C.
,
Cunningham
,
J.
,
Jane
,
S.
and
Perkins
,
A.
(
2001
).
Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice
.
Blood
97
,
1861
-
1868
.
England
,
S. J.
,
McGrath
,
K. E.
,
Frame
,
J. M.
and
Palis
,
J.
(
2011
).
Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus
.
Blood
117
,
2708
-
2717
.
Ford
,
E.
and
Thanos
,
D.
(
2010
).
The transcriptional code of human IFN-beta gene expression
.
Biochim. Biophys. Acta
1799
,
328
-
336
.
Ganz
,
T.
(
2011
).
Hepcidin and iron regulation, 10 years later
.
Blood
117
,
4425
-
4433
.
Ganz
,
T.
and
Nemeth
,
E.
(
2012
).
Hepcidin and iron homeostasis
.
Biochim. Biophys. Acta
1823
,
1434
-
1443
.
Gillinder
,
K. R.
,
Ilsley
,
M. D.
,
Nébor
,
D.
,
Sachidanandam
,
R.
,
Lajoie
,
M.
,
Magor
,
G. W.
,
Tallack
,
M. R.
,
Bailey
,
T.
,
Landsberg
,
M. J.
,
Mackay
,
J. P.
, et al. 
(
2016
).
Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viabilty
.
Nucleic Acids Res
.
(in press), pii: gkw1014.
Ginzburg
,
Y.
and
Rivella
,
S.
(
2011
).
beta-thalassemia: a model for elucidating the dynamic regulation of ineffective erythropoiesis and iron metabolism
.
Blood
118
,
4321
-
4330
.
Gnanapragasam
,
M. N.
,
McGrath
,
K. E.
,
Catherman
,
S.
,
Xue
,
L.
,
Palis
,
J.
and
Bieker
,
J. J.
(
2016
).
EKLF/KLF1-regulated cell cycle exit is essential for erythroblast enucleation
.
Blood
128
,
1631
-
1641
.
Goodnough
,
L. T.
,
Nemeth
,
E.
and
Ganz
,
T.
(
2010
).
Detection, evaluation, and management of iron-restricted erythropoiesis
.
Blood
116
,
4754
-
4761
.
Guida
,
C.
,
Altamura
,
S.
,
Klein
,
F. A.
,
Galy
,
B.
,
Boutros
,
M.
,
Ulmer
,
A. J.
,
Hentze
,
M. W.
and
Muckenthaler
,
M. U.
(
2015
).
A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia
.
Blood
125
,
2265
-
2275
.
Helias
,
V.
,
Saison
,
C.
,
Peyrard
,
T.
,
Vera
,
E.
,
Prehu
,
C.
,
Cartron
,
J.-P.
and
Arnaud
,
L.
(
2013
).
Molecular analysis of the rare in(Lu) blood type: toward decoding the phenotypic outcome of haploinsufficiency for the transcription factor KLF1
.
Hum. Mutat.
34
,
221
-
228
.
Heruth
,
D. P.
,
Hawkins
,
T.
,
Logsdon
,
D. P.
,
Gibson
,
M. I.
,
Sokolovsky
,
I. V.
,
Nsumu
,
N. N.
,
Major
,
S. L.
,
Fegley
,
B.
,
Woods
,
G. M.
,
Lewing
,
K. B.
, et al. 
(
2010
).
Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model
.
Genomics
96
,
303
-
307
.
Hodge
,
D.
,
Coghill
,
E.
,
Keys
,
J.
,
Maguire
,
T.
,
Hartmann
,
B.
,
McDowall
,
A.
,
Weiss
,
M.
,
Grimmond
,
S.
and
Perkins
,
A.
(
2006
).
A global role for EKLF in definitive and primitive erythropoiesis
.
Blood
107
,
3359
-
3370
.
Hom
,
J.
,
Dulmovits
,
B. M.
,
Mohandas
,
N.
and
Blanc
,
L.
(
2015
).
The erythroblastic island as an emerging paradigm in the anemia of inflammation
.
Immunol. Res.
63
,
75
-
89
.
Honda
,
K.
,
Yanai
,
H.
,
Negishi
,
H.
,
Asagiri
,
M.
,
Sato
,
M.
,
Mizutani
,
T.
,
Shimada
,
N.
,
Ohba
,
Y.
,
Takaoka
,
A.
,
Yoshida
,
N.
, et al. 
(
2005
).
IRF-7 is the master regulator of type-I interferon-dependent immune responses
.
Nature (Lond.)
434
,
772
-
777
.
Huang
,
D. W.
,
Sherman
,
B. T.
and
Lempicki
,
R. A.
(
2009
).
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources
.
Nat. Protoc.
4
,
44
-
57
.
Huang
,
J.
,
Zhang
,
X.
,
Liu
,
D.
,
Wei
,
X.
,
Shang
,
X.
,
Xiong
,
F.
,
Yu
,
L.
,
Yin
,
X.
and
Xu
,
X.
(
2015
).
Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin
.
Eur. J. Hum. Genet.
23
,
1341
-
1348
.
Iolascon
,
A.
,
Esposito
,
M. R.
and
Russo
,
R.
(
2012
).
Clinical aspects and pathogenesis of congenital dyserythropoietic anemias: from morphology to molecular approach
.
Haematologica
97
,
1786
-
1794
.
Jaffray
,
J. A.
,
Mitchell
,
W. B.
,
Gnanapragasam
,
M. N.
,
Seshan
,
S. V.
,
Guo
,
X.
,
Westhoff
,
C. M.
,
Bieker
,
J. J.
and
Manwani
,
D.
(
2013
).
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
.
Blood Cells Mol. Dis.
51
,
71
-
75
.
Kautz
,
L.
,
Jung
,
G.
,
Valore
,
E. V.
,
Rivella
,
S.
,
Nemeth
,
E.
and
Ganz
,
T.
(
2014
).
Identification of erythroferrone as an erythroid regulator of iron metabolism
.
Nat. Genet.
46
,
678
-
684
.
Keel
,
S. B.
,
Doty
,
R. T.
,
Yang
,
Z.
,
Quigley
,
J. G.
,
Chen
,
J.
,
Knoblaugh
,
S.
,
Kingsley
,
P. D.
,
De Domenico
,
I.
,
Vaughn
,
M. B.
,
Kaplan
,
J.
, et al. 
(
2008
).
A heme export protein is required for red blood cell differentiation and iron homeostasis
.
Science
319
,
825
-
828
.
Kennedy
,
M.
and
Keller
,
G. M.
(
2003
).
Hematopoietic commitment of ES cells in culture
.
Methods Enzymol.
365
,
39
-
59
.
Kim
,
A.
and
Nemeth
,
E.
(
2015
).
New insights into iron regulation and erythropoiesis
.
Curr. Opin. Hematol.
22
,
199
-
205
.
Kingsley
,
P. D.
,
Greenfest-Allen
,
E.
,
Frame
,
J. M.
,
Bushnell
,
T. P.
,
Malik
,
J.
,
McGrath
,
K. E.
,
Stoeckert
,
C. J.
and
Palis
,
J.
(
2013
).
Ontogeny of erythroid gene expression
.
Blood
121
,
e5
-
e13
.
Korolnek
,
T.
and
Hamza
,
I.
(
2015
).
Macrophages and iron trafficking at the birth and death of red cells
.
Blood
125
,
2893
-
2897
.
Li
,
Y.
,
Esain
,
V.
,
Teng
,
L.
,
Xu
,
J.
,
Kwan
,
W.
,
Frost
,
I. M.
,
Yzaguirre
,
A. D.
,
Cai
,
X.
,
Cortes
,
M.
,
Maijenburg
,
M. W.
, et al. 
(
2014
).
Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production
.
Genes Dev.
28
,
2597
-
2612
.
Liang
,
R.
,
Campreciós
,
G.
,
Kou
,
Y.
,
McGrath
,
K.
,
Nowak
,
R.
,
Catherman
,
S.
,
Bigarella
,
C. L.
,
Rimmelé
,
P.
,
Zhang
,
X.
,
Gnanapragasam
,
M. N.
, et al. 
(
2015
).
A systems approach identifies essential FOXO3 functions at key steps of terminal erythropoiesis
.
PLoS Genet.
11
,
e1005526
.
Liu
,
J.
,
Zhang
,
J.
,
Ginzburg
,
Y.
,
Li
,
H.
,
Xue
,
F.
,
De Franceschi
,
L.
,
Chasis
,
J. A.
,
Mohandas
,
N.
and
An
,
X.
(
2013
).
Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis
.
Blood
121
,
e43
-
e49
.
Liu
,
D.
,
Zhang
,
X.
,
Yu
,
L.
,
Cai
,
R.
,
Ma
,
X.
,
Zheng
,
C.
,
Zhou
,
Y.
,
Liu
,
Q.
,
Wei
,
X.
,
Lin
,
L.
, et al. 
(
2014
).
Erythroid Kruppel-like factor mutations are relatively more common in a thalassemia endemic region and ameliorate the clinical and hematological severity of beta-thalassemia
.
Blood
124
,
803
-
811
.
Lohmann
,
F.
and
Bieker
,
J. J.
(
2008
).
Activation of Eklf expression during hematopoiesis by Gata2 and Smad5 prior to erythroid commitment
.
Development
135
,
2071
-
2082
.
Lyon
,
M. F.
(
1983
).
Dominant haemolytic anaemia
.
Mouse News Letter
68
,
68
.
Manwani
,
D.
,
Galdass
,
M.
and
Bieker
,
J. J.
(
2007
).
Altered regulation of beta-like globin genes by a redesigned erythroid transcription factor
.
Exp. Hematol.
35
,
39
-
47
.
Meissner
,
A.
,
Eminli
,
S.
and
Jaenisch
,
R.
(
2009
).
Derivation and manipulation of murine embryonic stem cells
.
Methods Mol. Biol.
482
,
3
-
19
.
Miller
,
I. J.
and
Bieker
,
J. J.
(
1993
).
A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins
.
Mol. Cell. Biol.
13
,
2776
-
2786
.
Nagata
,
S.
(
2007
).
Autoimmune diseases caused by defects in clearing dead cells and nuclei expelled from erythroid precursors
.
Immunol. Rev.
220
,
237
-
250
.
Narla
,
A.
and
Mohandas
,
N.
(
2015
).
Jekyll and Hyde: the role of heme oxygenase-1 in erythroid biology
.
Haematologica
100
,
567
-
568
.
Nemeth
,
E.
,
Rivera
,
S.
,
Gabayan
,
V.
,
Keller
,
C.
,
Taudorf
,
S.
,
Pedersen
,
B. K.
and
Ganz
,
T.
(
2004a
).
IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin
.
J. Clin. Invest.
113
,
1271
-
1276
.
Nemeth
,
E.
,
Tuttle
,
M. S.
,
Powelson
,
J.
,
Vaughn
,
M. B.
,
Donovan
,
A.
,
Ward
,
D. M.
,
Ganz
,
T.
and
Kaplan
,
J.
(
2004b
).
Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization
.
Science
306
,
2090
-
2093
.
Novershtern
,
N.
,
Subramanian
,
A.
,
Lawton
,
L. N.
,
Mak
,
R. H.
,
Haining
,
W. N.
,
McConkey
,
M. E.
,
Habib
,
N.
,
Yosef
,
N.
,
Chang
,
C. Y.
,
Shay
,
T.
, et al. 
(
2011
).
Densely interconnected transcriptional circuits control cell states in human hematopoiesis
.
Cell
144
,
296
-
309
.
Perkins
,
A.
,
Xu
,
X.
,
Higgs
,
D. R.
,
Patrinos
,
G. P.
,
Arnaud
,
L.
,
Bieker
,
J. J.
and
Philipsen
,
S.
(
2016
).
Kruppeling erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants
.
Blood
127
,
1856
-
1862
.
Philip
,
M.
,
Funkhouser
,
S. A.
,
Chiu
,
E. Y.
,
Phelps
,
S. R.
,
Delrow
,
J. J.
,
Cox
,
J.
,
Fink
,
P. J.
and
Abkowitz
,
J. L.
(
2015
).
Heme exporter FLVCR is required for T cell development and peripheral survival
.
J. Immunol.
194
,
1677
-
1685
.
Pilon
,
A. M.
,
Arcasoy
,
M. O.
,
Dressman
,
H. K.
,
Vayda
,
S. E.
,
Maksimova
,
Y. D.
,
Sangerman
,
J. I.
,
Gallagher
,
P. G.
and
Bodine
,
D. M.
(
2008
).
Failure of terminal erythroid differentiation in EKLF-deficient mice is associated with cell cycle perturbation and reduced expression of E2F2
.
Mol. Cell. Biol.
28
,
7394
-
7401
.
Richardson
,
C. L.
,
Delehanty
,
L. L.
,
Bullock
,
G. C.
,
Rival
,
C. M.
,
Tung
,
K. S.
,
Kimpel
,
D. L.
,
Gardenghi
,
S.
,
Rivella
,
S.
and
Goldfarb
,
A. N.
(
2013
).
Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response
.
J. Clin. Invest.
123
,
3614
-
3623
.
Rivella
,
S.
(
2015
).
beta-thalassemias: paradigmatic diseases for scientific discoveries and development of innovative therapies
.
Haematologica
100
,
418
-
430
.
Sawicki
,
K. T.
,
Chang
,
H.-C.
and
Ardehali
,
H.
(
2015
).
Role of heme in cardiovascular physiology and disease
.
J. Am. Heart Assoc.
4
,
e001138
.
Siatecka
,
M.
and
Bieker
,
J. J.
(
2011
).
The multifunctional role of EKLF/KLF1 during erythropoiesis
.
Blood
118
,
2044
-
2054
.
Siatecka
,
M.
,
Lohmann
,
F.
,
Bao
,
S.
and
Bieker
,
J. J.
(
2010a
).
EKLF directly activates the p21WAF1/CIP1 gene by proximal promoter and novel intronic regulatory regions during erythroid differentiation
.
Mol. Cell. Biol.
30
,
2811
-
2822
.
Siatecka
,
M.
,
Sahr
,
K. E.
,
Andersen
,
S. G.
,
Mezei
,
M.
,
Bieker
,
J. J.
and
Peters
,
L. L.
(
2010b
).
Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Kruppel-like factor
.
Proc. Natl. Acad. Sci. USA
107
,
15151
-
15156
.
Siatecka
,
M.
,
Soni
,
S.
,
Planutis
,
A.
and
Bieker
,
J. J.
(
2015
).
Transcriptional activity of erythroid Kruppel-like factor (EKLF/KLF1) modulated by PIAS3 (protein inhibitor of activated STAT3)
.
J. Biol. Chem.
290
,
9929
-
9940
.
Singleton
,
B. K.
,
Lau
,
W.
,
Fairweather
,
V. S. S.
,
Burton
,
N. M.
,
Wilson
,
M. C.
,
Parsons
,
S. F.
,
Richardson
,
B. M.
,
Trakarnsanga
,
K.
,
Brady
,
R. L.
,
Anstee
,
D. J.
, et al. 
(
2011
).
Mutations in the second zinc finger of human EKLF reduce promoter affinity but give rise to benign and disease phenotypes
.
Blood
118
,
3137
-
3145
.
Singleton
,
B. K.
,
Frayne
,
J.
and
Anstee
,
D. J.
(
2012
).
Blood group phenotypes resulting from mutations in erythroid transcription factors
.
Curr. Opin. Hematol.
19
,
486
-
493
.
Soni
,
S.
,
Pchelintsev
,
N.
,
Adams
,
P. D.
and
Bieker
,
J. J.
(
2014
).
Transcription factor EKLF (KLF1) recruitment of the histone chaperone HIRA is essential for beta-globin gene expression
.
Proc. Natl. Acad. Sci. USA
111
,
13337
-
13342
.
Soufi
,
A.
,
Garcia
,
M. F.
,
Jaroszewicz
,
A.
,
Osman
,
N.
,
Pellegrini
,
M.
and
Zaret
,
K. S.
(
2015
).
Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming
.
Cell
161
,
555
-
568
.
Southwood
,
C. M.
,
Downs
,
K. M.
and
Bieker
,
J. J.
(
1996
).
Erythroid Krüppel-like Factor (EKLF) exhibits an early and sequentially localized pattern of expression during mammalian erythroid ontogeny
.
Dev. Dyn.
206
,
248
-
259
.
Tallack
,
M. R.
and
Perkins
,
A. C.
(
2010
).
KLF1 directly coordinates almost all aspects of terminal erythroid differentiation
.
IUBMB Life
62
,
886
-
890
.
Tallack
,
M. R.
and
Perkins
,
A. C.
(
2013
).
Three fingers on the switch: Krüppel-like factor 1 regulation of gamma-globin to beta-globin gene switching
.
Curr. Opin. Hematol.
20
,
193
-
200
.
Tallack
,
M. R.
,
Keys
,
J. R.
and
Perkins
,
A. C.
(
2007
).
Erythroid Kruppel-like factor regulates the G1 cyclin dependent kinase inhibitor p18INK4c
.
J. Mol. Biol.
369
,
313
-
321
.
Tallack
,
M. R.
,
Keys
,
J. R.
,
Humbert
,
P. O.
and
Perkins
,
A. C.
(
2009
).
EKLF/KLF1 controls cell cycle entry via direct regulation of E2f2
.
J. Biol. Chem.
284
,
20966
-
20974
.
Tolosano
,
E.
(
2015
).
Increasing serum transferrin to reduce tissue iron overload due to ineffective erythropoiesis
.
Haematologica
100
,
565
-
566
.
Viprakasit
,
V.
,
Ekwattanakit
,
S.
,
Riolueang
,
S.
,
Chalaow
,
N.
,
Fisher
,
C.
,
Lower
,
K.
,
Kanno
,
H.
,
Tachavanich
,
K.
,
Bejrachandra
,
S.
,
Saipin
,
J.
, et al. 
(
2014
).
Mutations in Kruppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression
.
Blood
123
,
1586
-
1595
.
Wang
,
C.-Y.
and
Babitt
,
J. L.
(
2016
).
Hepcidin regulation in the anemia of inflammation
.
Curr. Opin. Hematol.
23
,
189
-
197
.
Waye
,
J. S.
and
Eng
,
B.
(
2015
).
Krüppel-like factor 1: hematologic phenotypes associated with KLF1 gene mutations
.
Int. J. Lab. Hematol.
37
Suppl 1
,
78
-
84
.
White
,
R. A.
,
Sokolovsky
,
I. V.
,
Britt
,
M. I.
,
Nsumu
,
N. N.
,
Logsdon
,
D. P.
,
McNulty
,
S. G.
,
Wilmes
,
L. A.
,
Brewer
,
B. P.
,
Wirtz
,
E.
,
Joyce
,
H. R.
, et al. 
(
2009
).
Hematologic characterization and chromosomal localization of the novel dominantly inherited mouse hemolytic anemia, neonatal anemia (Nan)
.
Blood Cells Mol. Dis.
43
,
141
-
148
.
Yien
,
Y. Y.
and
Bieker
,
J. J.
(
2013
).
EKLF/KLF1, a tissue-restricted integrator of transcriptional control, chromatin remodeling, and lineage determination
.
Mol. Cell. Biol.
33
,
4
-
13
.
Yoshida
,
H.
,
Kawane
,
K.
,
Koike
,
M.
,
Mori
,
Y.
,
Uchiyama
,
Y.
and
Nagata
,
S.
(
2005a
).
Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells
.
Nature (Lond.)
437
,
754
-
758
.
Yoshida
,
H.
,
Okabe
,
Y.
,
Kawane
,
K.
,
Fukuyama
,
H.
and
Nagata
,
S.
(
2005b
).
Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA
.
Nat. Immunol.
6
,
49
-
56
.
Zhang
,
D.-L.
,
Ghosh
,
M. C.
and
Rouault
,
T. A.
(
2014
).
The physiological functions of iron regulatory proteins in iron homeostasis - an update
.
Front. Pharmacol.
5
,
124
.

Competing interests

The authors declare no competing or financial interests.

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