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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.

We describe a case of severe neonatal anemia with kernicterus caused by compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1-null human. The phenotype of severe nonspherocytic hemolytic anemia, jaundice, hepatosplenomegaly, and marked erythroblastosis is more severe than that present in congenital dyserythropoietic anemia type IV as a result of dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found that human KLF1 acts like mouse Klf1 to coordinate expression of many genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell-cycle regulators, and blood group antigens. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. We also identify new roles for KLF1 in autophagy, global transcriptional control, and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.

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

Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis.

KLF1 (formerly known as EKLF) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle. We have recently described the full repertoire of KLF1 binding sites in vivo by performing KLF1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the KLF1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1+/+ and Klf1−/− erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology, and provided novel insights into the function of KLF1 as a transcriptional activator. We define a cis-regulatory module bound by KLF1, GATA1, TAL1, and EP300 that coordinates a core set of erythroid genes. We also describe a novel set of erythroid-specific promoters that drive high-level expression of otherwise ubiquitously expressed genes in erythroid cells. Our study has identified two novel lncRNAs that are dynamically expressed during erythroid differentiation, and discovered a role for KLF1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.

KLF1 regulates a diverse suite of genes to direct erythroid cell differentiation from bipotent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as Hbb-b1, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of alpha- and beta-globin protein chains, heme biosynthesis, coordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 cooperation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment.

The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.

Alpha haemoglobin stabilising protein (AHSP) binds free alpha-globin chains and plays an important role in the protection of red cells, particularly during beta-thalassaemia. Murine ASHP was discovered as a GATA-1 target gene and human AHSP is directly regulated by GATA-1. More recently, AHSP was rediscovered as a highly erythroid Kruppel-like factor (EKLF) -dependent transcript. We have determined the organisation of the murine AHSP gene and compared it to orthologs. There are two CACC box elements in the proximal promoter. The proximal element is absolutely conserved, but does not bind EKLF as it is not a canonical binding site. In rodents, the distal element contains a 3 bp insertion that disrupts the typical EKLF binding consensus region. Nevertheless, EKLF binds this atypical site by gel mobility shift assay, specifically occupies the AHSP promoter in vivo in a chromatin immunoprecipitation assay, and transactivates AHSP through this CACC site in promoter-reporter assays. Our results suggest EKLF can occupy CACC elements in vivo that are not predictable from the consensus binding site inferred from structural studies. We also propose that absence of AHSP in EKLF-null red cells exacerbates the toxicity of free alpha-globin chains, which exist because of the defect in beta-globin gene activation.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells and an increased risk of late transformation to acute myeloid leukemia or primary myelofibrosis. Approximately 15% of MPN cases do not carry mutations in JAK2, CALR, or MPL and are thus often referred to as triple-negative cases. These are caused by a diverse set of rare mutations in cytokine receptors, JAK-STAT signaling pathway components, or epigenetic modifiers. In addition, some cases diagnosed as MPN are reactive rather than clonal disorders, so a negative result from a genetic screen can be informative. To obtain a comprehensive rapid molecular diagnosis for most MPNs, we developed an assay to detect genetic mutations (single nucleotide variants and/or small insertions/deletions) in 86 genes using targeted exon resequencing (AmpliSeq) and a bench-top semiconductor machine (Ion Torrent Personal Genome Machine). Our assay reliably detects well characterized mutations in JAK2, CALR, and MPL, but also rarer mutations in ASXL1, TET2, SH2B3, and other genes. Some of these mutations are novel. We find multiple mutations in advanced cases, suggesting co-operation between Janus kinase-STAT pathway mutations and epigenetic mutations in disease progression. This assay can be used to follow molecular progression, clonal heterogeneity, and drug resistance in MPNs.

The vascular adhesion molecule E-selectin is a key component of the Bone Marrow (BM) Haematopoietic Stem Cell (HSC) niche prompting HSC to proliferate at the expense of self-renewal (Winkler, Nat Med 2012).Only 3 - 5% of BM endothelial cells express E-selectin in steady state however E-selectin is greatly upregulated (5 - 10 fold) in BM of mice with acute myeloid leukemia (AML) raising the question; how do AML stem cells (LSC) respond to E-selectin at the vascular niche & does E-selectin signalling in AML & HSC differ? Using models of murine AML generated by retroviral transduction of MLL-AF9 or AML1-ETO oncogenes, we found leukemic blasts rapidly upregulate E-selectin-binding upon oncogenic transformation. Furthermore E-selectin adhesion promoted LSC survival to cytarabine in vitro as well as in vivo. LSC survival to chemotherapy in wildtype compared to E-selectin knockout (E-/-) mice quantified by rigorous limiting-dilution transplantation assay of 1%, 0.1%, 0.01% femur BM demonstrated that E-selectin deletion increased sensitivity of LSC to high-dose cytarabine therapy ~11-fold (900mg/kg cytarabine n=6 donors &15 recipients/gp p=0.0037). Thus E-selectin is a critical vascular niche component mediating LSC chemo resistance. Importantly these findings could be replicated by administration of a potent small molecule glycomimetic E-selectin antagonist (GMI-1271) to wt mice. Furthermore treatment with GMI-1271 (40mg/kg bidaily) for 10 days in combination with standard mouse version of 7+3 induction chemotherapy (5 days cytarabine 100mg/kg; 3 days doxorubicin 1mg/kg) was able to significantly double mouse survival over chemotherapy alone (p=0.0054; no chemotherapy median survival 25 d, AraC/Dox alone 32 d, AraC/Dox plus GMI-1271 survival 41 d; n=8 mice/gp). To understand mechanisms of this chemo-sensitisation, mice with MLL-AF9 monomyelocytic (11q23 translocation) or AML1-ETO granulocytic t(8;21) -induced AML were administered GMI-1271 or vehicle control for 5 days before sorting BM AML blasts for RNA sequencing. Analysis of differentially expressed transcripts by CuffDiff / DSeq2 revealed 170 RNAs differed following in vivo E-selectin blockade. KEGG pathway analysis indicated a pathway potentially dampened in AML blasts following GMI-1271 administration was PI3K - NF-kB signalling - raising the hypothesis that adhesion to E-selectin activates pro-survival NF-kB signalling in AML cells leading to enhanced chemoresistance. Using two in vitro assays, we confirmed E-selectin to be unique among vascular adhesion molecules tested in being able to directly activate NF-kB. Activation of NF-kB was only observed upon E-selectin mediated adhesion & was not observed following adhesion to P-selectin, PECAM-1 or VCAM-1 using either myeloid NF-kB GFP reporter cell lines or by induction of p65 NF-kB (Ser 536). Importantly E-selectin mediated NF-kB activation was completely inhibited when E-selectin antagonist GMI-1271 added. Assays repeated in presence of a specific NF-kB activation antagonist (BMS-345541 10uM 24hrs) demonstrated that NF-kB blockade alone reversed E-selectin-mediated chemoresistance in vitro. Upstream blockade of E-selectin by GMI-1271 not only inhibits NF-kB activation but also mobilizes LSC out of the protective BM niche & prevents re-entry thereby breaking the chemo resistance observed with these cells. A Phase I/II Clinical trial to study efficacy of GMI-1271 in combination with chemotherapy in AML patients (NCT02306291) is currently in progress Disclosures Winkler: GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Levesque:GlycoMimetics: Equity Ownership.

The molecular events and transcriptional mechanisms that underlie erythropoiesis are of great interest to biologists and hematologists since disorders of erythrocytes are common and remain relatively poorly understood. Kruppel-like factor 1 (KLF1) is a critical transcription factor for erythropoiesis in mice and man. Recently the use of chromatin immunoprecipitation (ChIP) coupled to next-generation DNA sequencing (ChIP-seq) has led to an updated understanding of how KLF1 functions in vivo. The full extent of KLF1 target genes have provided new insights into erythropoiesis, and have established that KLF1 controls almost all aspects of erythroid cell development and maturation.

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

The human beta globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5'-HBE1, HBG2, HBG1, HBD, HBB-3'. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the beta-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down-regulation of HBB, and up-regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long-distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage-specific assembly of the beta-globin ACH, and HPFH.

Erythroid Kruppel-like factor (EKLF, KLF1) is an essential erythroid cell specific C(2)H(2) zinc finger transcription factor that binds CACC box elements in promoters and distant regulatory elements to activate transcription. Forced expression of EKLF arrests cell division. The cyclin dependent kinase (Cdk) inhibitor p18(INK4c) was identified as a potential novel EKLF target gene from an expression profiling study. The p18(INK4c) protein functions as an inhibitor of Cdk4 and Cdk6 activity during early G1 phase of the cell cycle, thus acting as a physiological brake on cell division. We confirmed p18(INK4c) is downregulated in EKLF null mice by real-time PCR and Western blotting, and identified three closely associated and highly conserved EKLF binding sites (CCNCNCCCN) approximately 1 kb upstream of the p18(INK4c) transcriptional start site. We showed that EKLF binds to one of these elements by gel shift assay and demonstrated this site is capable of driving EKLF dependent transcription. We also determined by chromatin immunoprecipitation (ChIP) that this region of the p18(INK4c) promoter is bound by EKLF in erythroid cells. Thus, EKLF is a direct regulator of p18(INK4c) gene expression, and much of EKLF's role in driving erythroid cell differentiation may occur via p18(INK4c).

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated a-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for Modifiers of murine metastable epi-alleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation; i.e. an increased percentage of GFP+ circulating red blood cells. Both strains harbour point mutations in the erythroid specific transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain. D11 homozygous mice die in utero of anaemia at 14.5DPC. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have mild compensated microcytic anaemia which models the phenotype in a recently described human family. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/- mice have severe perinatal haemolytic anaemia and marked splenomegaly. Furthermore blood haemoglobin content, haematocrit and red blood cell size (MCV) were significantly reduced in Klf1H350R/- mice compared to wildtype and D45 homozygous offspring of the same age. Analysis of Klf1H350R/- by flow cytometry showed an increase in circulating immature red blood cells. In the bone marrow, a lack of mature red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells and a relative reduction in B and T Cells. Gel shifts assays of a recombinant Klf1 zinc finger protein with the H350R mutation showed normal binding to the b -globin promoter sequence but weak binding to the Alas2 intronic enhancer site. Furthermore b -globin gene expression was near normal whereas expression of other known Klf1 target genes was decreased. We will discuss how H350R disrupts function from ChIP-seq and RNA-seq in primary fetal liver tissue. Previous studies of the linkers in C2H2 zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C2H2 class of transcription factors. Our results thus far show that the second linker of Klf1 has a role in maintaining the integrity of Klf1 function (at a subset of Klf1-occupied sites,) and does not act just as a spacer for the zinc fingers. Disclosures Perkins: Novartis Oncology: Honoraria.

Krûppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor [1, 2]. A number of studies have shown up to ~700 genes are poorly expressed when KLF1 is absent [3-6]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF1 [7]. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF1 [8]. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (= E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation [9]. To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1nan into an erythroid cell line derived from Klf1-/- fetal liver cells [10]. We performed ChIP-seq to determine differences in genome occupancy in vivo, and identified novel sites occupied by EKLF-E339D but not by wild type KLF1. Using de novo motif discovery [11], we find KLF1nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to any known TFs, so we think it represents a sequence specificity not normally encoded in mammals. Ectopic binding to non-erythroid gene promoters is accompanied by aberrant gene expression as determined by 4sU labelling and deep sequencing of tamoxifen-induced primary nuclear RNAs. We find a 4-fold greater number of genes induced by KLF1-nan compared with wild type KLF1 which is consistent with degenerate genome occupancy. We compared the KLF1-nan dependent genes with RNA-seq performed in primary fetal liver for KLF1+/nan versus KLF1+/- mice. We confirmed aberrant binding using EMSA and surface plasmon resonance (SPR) using recombinant GST-Klf1 zinc finger domains expressed in E.coli. The degenerate motif is consistent with structural models of how the second zinc finger of KLF1 specifically interacts with its binding site [12, 13]. We are undertaking structural studies to confirm this modelling. Together RNA-seq, ChIP-seq and SPR studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. To our knowledge this mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel. References: 1.Perkins, A.C., A.H. Sharpe, and S.H. Orkin. Nature, 1995. 375(6529): p. 318-22. 2.Nuez, B., et al., Nature, 1995. 375(6529): p. 316-8. 3.Pilon, A.M., et al., Mol Cell Biol, 2006. 26(11): p. 4368-77. 4.Drissen, R., et al., Mol Cell Biol, 2005. 25(12): p. 5205-14. 5.Hodge, D., et al., Blood, 2006. 107(8): p. 3359-70. 6.Tallack, M.R., et al., Genome Res, 2012. 22(12):2385-98 7.Arnaud, L., et al., Am J Hum Genet. 87(5): p. 721-7. 8.Siatecka, M., et al., Proc Natl Acad Sci U S A. 2010. 107(34):15151-6 9.Heruth, D.P., et al., Genomics, 2010. 96(5): p. 303-7. 10.Coghill, E., et al., Blood, 2001. 97(6): p. 1861-1868. 11.Bailey, T.L., et al., Nucleic Acids Res, 2009. 37(Web Server issue): p. W202-8. 12.Schuetz, A., et al., Cell Mol Life Sci, 2011. 68(18): p. 3121-31. 13.Oka, S., et al., Biochemistry, 2004. 43(51): p. 16027-35. Disclosures No relevant conflicts of interest to declare.

We describe a case of severe neonatal anemia with kernicterus due to compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1 null human. The phenotype of severe DAT-negative non-spherocytic hemolytic anaemia (NSHA), jaundice, hepato-splenomegaly, and marked erythroblastosis is more severe than that present in CDA type IV due to dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found human KLF1 acts like mouse Klf1 to coordinate expression of >500 genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell cycle regulators, and blood group antigens. We interrogated recently published KLF1 ChIP-seq to determine directly regulated KLF1 genes in man. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. KIF23 mutations result in CDA type III so our finding provides a direct link btween CDA IV adn CDA III. We also identify new roles for KLF1 in autophagy, global transcriptional control and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis. Disclosures No relevant conflicts of interest to declare.

Iron is a critical element for erythroid development and the function of many proteins, but is toxic in excessive amounts, most often by inducing oxidative stress that may play a role in vasculopathy. We have shown that markers of high iron burden such as serum ferritin have been associated with excessive production of the angiogenic factor placenta growth factor (PlGF), high estimated pulmonary artery pressure and death in adults with sickle cell disease. Enforced PlGF expression in mice promotes high circulating levels of the potent vasoconstrictor endothelin-1 and pulmonary hypertension. We find that heme-bound iron stimulates PlGF gene transcription by Erythroid Krüppel‐like Factor (EKLF). Heme-bound iron (hemin) induces PlGF mRNA in erythroid cells more than 200-fold in a dose-dependent and time-dependent fashion. PlGF mRNA is induced by other complexes of iron, but not iron-free heme or zinc protoporphyrin, suggesting that PlGF induction specifically requires iron. In murine and human cell lines, expression of EKLF developmentally precedes PlGF, and enforced expression of EKLF in human erythroid progenitor cells induces PlGF mRNA. Now we further report that hemin-induced expression of PlGF is abolished in EKLF-deficient mouse erythroid cells but rescued by conditional expression of EKLF. By chromosome immunoprecipitation we find that EKLF binds directly to the PlGF promoter region that contains an EKLF consensus sequence. We find that SCD patients have higher level expression than healthy controls of both EKLF and PlGF mRNA in peripheral blood cells, and expression of the two are correlated with each other (r=0.822, p Demonstrating the generalizability of this link of iron overload to PlGF expression, we find that patients with hereditary hemochromatosis have a high plasma level of PlGF, which falls after correction of iron overload by periodic phlebotomy therapy (p=0.018). Reductant N-acetyl cysteine completely blocks hemin induction of PlGF, supporting the participation of oxidant stress response pathways already known to mediate signaling by iron on other promoters such as heme oxygenase-1. Our results for the first time demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to vasculopathy and pulmonary hypertension, suggesting that aggressive use of clinically approved iron chelation drugs should be investigated as adjunctive therapy to reduce the risk of pulmonary hypertension in SCD patients with associated iron overload. Linkage of iron overload in patients without hemolysis or anemia to abnormally high level expression of an angiogenic factor shown to induce pulmonary hypertension suggests a general pathobiological pathway connecting iron and vasculopathy. Disclosures: No relevant conflicts of interest to declare.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells, increased risk of thrombotic complications and slow progression to myelofibrosis or, less often, leukemia. Activation of the JAK-STAT signaling pathway is a common underlying feature of these diseases and JAK kinase inhibitors are efficacious in the more advanced forms of disease. Most cases of polycythemia vera (PV) and approximately 60% of essential thrombocythemia (ET) and primary myelofibrosis (MF) harbor a point mutation in JAK2 (V617F) which leads to constitutive JAK-STAT signaling and factor independent cell growth. The remaining 40% of cases of MF and ET harbor a broad range of mutations in many genes including those involved in cytokine receptor signaling, other components or the JAK-STAT pathway or epigenetic regulators. This poses a challenge for rapid molecular diagnosis. Also, since ET is essentially a diagnosis of exclusion of reactive causes of thrombocytosis, many cases of chronic ‘ET’ may not be clonal hematological neoplasms but reactive conditions. We have developed a rapid deep sequencing pipeline to detect mutations in 65 genes which have been implicated in MPN through previous reports of human mutations, mouse models of MPN, or other known components of hematopoietic cytokine receptor signaling. We used 10ng of DNA from blood to amplify and sequence all the exons of these 65 genes using Ampliseq and Ion Torrent PGM. Using 318 PGM chips and 8-fold multiplexing we achieved on average 200 fold coverage of the target exome. The bioinformatics of SNP validation and rapid generation of reports will be presented. From a pilot study of 30 cases referred for molecular diagnosis, we have detected the likely causative mutation in approximately 80% of ET and MF where JAK2 is wild type. Many of these mutations are known to be causative in MPN, including those in MPL, ASXL1, SET2, SH2B3 (LNK), EZH2, CBL, DNMT3A and other genes. We have identified a novel inherited mutation in a family with MPN and validated it in BAF3 factor-independency assays. We have identified further novel mutations in JAK3, EED, DNMT3A, APC and two phosphatases involved in silencing activated JAK-STAT pathway components. The biological significance of these is under investigation and progress will be reported. In many cases we find evidence for clonal evolution involving secondary mutations in epigenetic modifying proteins on top of driver mutations in the JAK-STAT pathway. In short, targeted exome re-sequencing using Ampliseq and Ion Torrent PGM provides a rapid and relatively cheap method for molecular diagnosis and characterization of most cases of MPN. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5′-ε-Gγ-Aγ- δ- β-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of γ-globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the δ-globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the δ-globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease.

Erythroid Kruppel-like Factornext term (EKLF) is a zinc finger transcription previous termfactornext term that is expressed specifically in erythrocytes throughout development. Its major function is the activation of β-globin gene expression, by binding to CACCC box motifs, however further roles in erythrocyte development appear likely. A transcriptional profiling experiment comparing the global gene expression in EKLF-null and wild-type erythrocytes has identified many differentially expressed genes. The Cyclin-dependent kinase (previous termCdk) inhibitor p18next term was identified as a potential EKLF target gene, and found to be down-regulated in EKLF-null mice as confirmed by real-time PCR. The previous termp18next term protein functions as an previous terminhibitornext term of Cdk4 and Cdk6 activity during early previous termG1next term phase of the cell cycle to control its progression. The search throughout the previous termp18next term gene locus for phylogenetically conserved extended CACC box elements (CCNCNCCC) found two closely associated CACC sites not, vert, similar 1 kb upstream of the transcriptional start site. We show EKLF binding to these sites by gel shift assay and have demonstrated that these sites are capable of driving EKLF-dependent transcription in luciferase reporter assays. We also show by chromatin immunoprecipitation (ChIP) assay that this region of the previous termp18next term promoter is specifically occupied by EKLF in vivo. These results suggest that EKLF acts to control the switch from proliferation to differentiation in erythrocytes by regulating previous termp18next term gene expression and thereby controlling passage through the previous termG1next term–S transition.

Erythroid Kruppel-Like Factor (EKLF) is a zinc finger transcription factor which is essential for β-globin gene expression. Knockout mice die from anemia at E15, but restoration of globin chain imbalance does not rescue anemia or increase survival. Cell lines derived from EKLF null mice undergo proliferation arrest upon reactivation of a conditional EKLF-ER fusion protein, suggesting a role in cell cycle control. A transcriptional profiling experiment comparing the global gene expression in EKLF null and wild type fetal liver identified many differentially expressed genes, a number of which function in G1 and at the G1/S checkpoint of the cell cycle. The Cyclin dependent kinase (Cdk) inhibitor, p18, and the S phase transcription factor E2F4 were both found to be significantly down regulated in EKLF null mice and this result was confirmed by real-time PCR. Interestingly, E2F4 knockout mice have a similar phenotype to EKLF knockout mice. Bioinformatic searches of the p18 and E2F4 genes shows that each contains phylogenetically conserved CACC box motifs capable of binding EKLF within longer regions of conservation in promoter and intron regions. The p18 gene contains two conserved CACCC sites upstream of the start of transcription, which are required for EKLF dependent promoter activity in luciferase reporter assays. The transcription factor E2F4 contains a conserved EKLF-binding CACC site within an intron that is closely associated with two conserved GATA1 binding sites. We show by a chromatin immunoprecipitation (ChIP) assays that the E2F4 intron and p18 promoter are occupied by EKLF in vivo. Together, these results suggest that EKLF is likely to directly regulate expression of key cell cycle genes in vivo to drive the switch from proliferation to differentiation of erythrocytes. The loss of EKLF is likely to result in aberrant proliferation and predisposition to leukemia.

Krûppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor1, 2. A number of studies have shown up to ∼700 genes are poorly expressed when KLF1 is absent3-6. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF17. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF18. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (i.e. E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation9. To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1nan into an erythroid cell line derived from Klf1-/- fetal liver cells10. We performed ChIP-seq to determine genome occupancy site preferences for KLF1 and KLF1nan. We identified about 4-fold the number of binding sites within the genome for KLF1nan versus KLF1; many of these are ectopic or promiscuous. Using de novo motif discovery11, we find KLF1nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to known TFs, so we think it represents specificity not normally present in mammals. The degenerate motif is consistent with models of how the second zinc finger of KLF1 specifically interacts with the 9bp consensus binding site12,13. We also isolated nascent RNA from wild type and mutant cells, to identify primary transcriptional targets of KLF1 and aberrant targets of the KLF1nanmutation. We performed primary transcript RNA-seq and validation using RT-PCR of pre-processed nuclear transcripts. Together the RNA-seq and ChIP-seq studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. This mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel. References: 1. Perkins, A.C., A.H. Sharpe, and S.H. Orkin. Nature, 1995. 375(6529): p. 318-22. 2. Nuez, B., et al., Nature, 1995. 375(6529): p. 316-8. 3. Pilon, A.M., et al., Mol Cell Biol, 2006. 26(11): p. 4368-77. 4. Drissen, R., et al., Mol Cell Biol, 2005. 25(12): p. 5205-14. 5. Hodge, D., et al., Blood, 2006. 107(8): p. 3359-70. 6. Tallack, M.R., et al., Genome Res, 2012. 22(12):2385-98 7. Arnaud, L., et al., Am J Hum Genet. 87(5): p. 721-7. 8. Siatecka, M., et al., Proc Natl Acad Sci U S A. 2010. 107(34):15151-6 9. Heruth, D.P., et al., Genomics, 2010. 96(5): p. 303-7. 10. Coghill, E., et al., Blood, 2001. 97(6): p. 1861-1868. 11. Bailey, T.L., et al., Nucleic Acids Res, 2009. 37(Web Server issue): p. W202-8. 12. Schuetz, A., et al., Cell Mol Life Sci, 2011. 68(18): p. 3121-31. 13. Oka, S., et al., Biochemistry, 2004. 43(51): p. 16027-35. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

Krûppel-like factor-1 (KLF1) is a C2H2 zinc finger transcription factor which is essential for broad erythroid gene expression and erythropoiesis in vivo. A number of studies have shown ∼700 genes are poorly expressed when KLF1 is absent [1-8]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice [9,10], and partly responsible for congenital anemia in humans and mice with dominant mutations in KLF1 [11,12]. To determine whether KLF1-dependent genes are direct or indirect targets of KLF1, we have previously performed global ChIP-seq experiments identifying 945-1350 regions of KLF1 occupancy in the mouse genome [7]. About 15% of these regions fall within the promoters of KLF1 target genes but surprisingly, most are thousands of kilobases distant from any known gene. Many of these distant sites exhibit co-occupancy with other transcriptional regulators involved in erythropoiesis, including GATA1. Approximately half of the KLF1 occupied sites are found within regions of mono-methylation of lysine 4 on histone 3 (H3K4me1). These regions are devoid of histones tri-methylated at the same residue (H3K4me3). This methylation signature is commonly associated with regions of the genome that act as transcriptional enhancers [13,14] and many are also bound by the co-activator, p300. The nature and function of these distant sites, particularly those without enhancer marks, is interesting as they may shed light on novel mechanisms of action of KLF1 and associated transcription factors. The transcriptional machinery of the cell, including many transcription factors is found in large sub-nuclear compartments called transcriptional factories [15]. KLF1 has been found localized to a subset of these in erythroid cells. KLF1 is also required for long-range looping of the β-globin gene into these transcription factories [16]. Other erythroid genes involved in the production of a functional haemoglobin molecule such as α-globin and haem synthesis enzymes are often found in the same transcription factory. This strongly suggests KLF1 can employ this sub-nuclear machine to co-ordinate the transcriptional output from many genes and thereby direct erythroid cell differentiation. To explore the function of KLF1-bound loci, we have performed multiplexed chromosome conformation capture (3C) coupled with sequencing (Capture-seq) using a tamoxifen responsive, KLF1 inducible cell line to investigate the role of KLF1 in chromosomal looping. In addition, we have analysed primary transcriptional output of KLF1 target genes by nascent RNA-seq. As expected β-globin and a-globin transcription is rapidly induced, becoming detectable within 5 minutes. However, the transcriptional response of dematin and a set direct KLF1 target genes is much slower. Thus, the mechanism of KLF1 transcriptional activation differs between target gene loci. We find a dynamic role of KLF1-dependent chromosomal looping and transcriptional co-factor recruitment required to effect gene transcription during erythropoiesis. We will discuss models of differentiation transcription regulation by KLF1. References: 1. Drissen R, et al. (2005). Molecular and Cellular Biology 25: 5205–5214. 2. Funnell APW, et al. (2007). Molecular and Cellular Biology 27: 2777–2790. 3. Hodge D, et al. (2006). Blood 107: 3359–3370. 4. Pilon AM, et al. (2008). Molecular and Cellular Biology 28: 7394–7401. 5. Siatecka M, et al. (2010). PNAS 107: 15151–15156. 6. Siatecka M, Bieker JJ (2011). Blood 118: 2044–2054. 7. Tallack MR, et al. (2010). Genome Res 20: 1052–1063. 8. Tallack MR, Perkins AC (2010). IUBMB Life 62: 886–890. 9. Perkins AC, Sharpe AH, Orkin SH (1995). Nature 375: 318–322. 10. Nuez B, et al. (1995). Nature 375: 316–318. 11. Arnaud L, S et al. (2010). Am J Hum Genet 87: 721–727. 12. Borg J, et al. (2011). Haematologica 96: 635–638. 13. Zentner GE, et al. (2011). Genome Res 21: 1273–1283. 14. Pekowska A, et al. (2011). EMBO J 30: 4198–4210. 15. Osborne CS, et al. (2004). Nat Genet 36: 1065–1071. 16. Schoenfelder S, et al. (2010). Nat Genet 42: 53–61. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

The vascular adhesion molecule E-selectin is a key component of the bone marrow (BM) vascular niche, awakening otherwise dormant hematopoietic stem cells (HSC) (Winkler et al., Nat Med 2012). Vascular niches also mediate malignant cell survival. We find E-selectin becomes upregulated on BM vasculature during leukemia and that adhesion of BM AML blasts to E-selectin promotes survival signaling. This is unique to E-selectin - not observed with adhesion to P-selectin, PECAM1, or integrin ligands ICAM1, VCAM1. In vivo we find absence (in SELE-/- mice) or therapeutic blockade of E-selectin by administration of E-selectin antagonist GMI-1271 to mice, induces 9-fold greater chemosensitivity in 11q23-rearranged AML Initiating Cells (LIC) to cytarabine therapy in vivo and doubles survival over that achieved by chemotherapy alone. Together these data support the Phase I/II clinical trial of GMI-1271 in combination with intensive chemotherapy to improve efficacy of therapy for AML (NCT02306291), and raise the further questions: 1) what are the AML LIC ligands interacting with E-selectin at the vascular niche and 2) what are the pathways initiated by E-selectin adhesion that mediate chemoresistance. Using murine models of AML generated by retroviral transduction of the 11q23 fusion oncogene MLL-AF9 into HSC, we find leukemic blasts rapidly upregulate E-selectin binding potential upon oncogenic transformation. To investigate if this is due to aberrant glycosylation facilitating the generation of de novo AML cell surface receptors, we generated AML from HSC knocked-out for the two canonical E-selectin receptors PSGL1 and CD44. Surprisingly we found although HSC from CD44-/-PSGL1-/- mice still bound to E-selectin (indicating HSC express additional E-selectin ligands), AML cells generated from these same HSCs no longer did. Thus the functional E-selectin ligands utilized by murine HSC and AML blasts differ. When transplanted into recipient mice CD44-/-PSGL1-/- AML engrafted with similar disease progression to matching wildtype AML but demonstrated ~100-fold greater sensitivity to high-dose cytarabine chemotherapy. When GMI-1271 was co-administered together with chemotherapy no further boost in chemosensitivity was observed in CD44-/-PSGL1-/- AMLs. This complements in vitro data showing E-selectin adhesion-mediated chemoresistance is lost when CD44 and/orPSGL1are absent in AML cells. Together suggesting that CD44 and/or PSGL1 are the key receptors involved in E-selectin-mediated pro-survival signaling. To understand the pro-survival signaling pathways involved, E-selectin antagonist GMI1271 or saline control were administered 5 days to mice with AML and BM KIT+ leukemic blasts sorted. RNA sequencing revealed only a small number (~200) of mRNA transcripts differentially regulated following therapeutic E-selectin blockade. Of note these included down-regulation in several components of the PI3K/AKT/NF-kB signaling pathway frequently involved in cell survival. Indeed AKT Ser473 and NF-kB p65 Ser536 were found to be rapidly phosphorylated within 15 minutes of E-selectin adhesion in AML blasts in vitro. This phosphorylation was specific to E-selectin adhesion and not observed with other vascular adhesion molecules tested suggesting that direct E-selectin-mediated AKT activation in AML cells at the vascular niche may be the potential mechanism driving vascular mediated chemoresistance. To confirm whether therapeutic blockade of E-selectin alone dampens intracellular AKT signaling in AML blasts, cohorts of mice with wildtype or matching CD44-/-PSGL1-/- AML were administered GMI-1271 for 5 days then BM AML blasts collected for signaling studies. Both the administration of E-selectin antagonist or absence of CD44 and/or PSGL-1 dampened AKTSer473 phosphorylation in BM AML blasts in vivo, correlating with their heightened sensitivity to chemotherapy . In summary, these findings suggest E-selectin ligands on specific CD44 and/or PSGL1 glycoforms are the predominant determinants through which the induction/amplification of chemoresistant pathways in AML blasts occur following vascular adhesion and further that these pathways are coupled to an early phosphorylation event of AKT - and finally explain potential mechanism how GMI-1271 administration is able to attenuate vascular-mediated signaling and restore susceptibility of AML to chemotherapy. Disclosures Magnani: GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.