Severe anemia in the Nan mutant mouse caused by sequence-selective disruption of erythroid Krüppel-like factor Miroslawa Siateckaa, Kenneth E. Sahrb, Sabra G. Andersenb, Mihaly Mezeic, James J. Biekera,d,e,1, and Luanne L. Petersb,1 aDepartment of Developmental and Regenerative Biology, cDepartment of Structural and Chemical Biology, dBlack Family Stem Cell Institute, and eTisch Cancer Institute, Mount Sinai School of Medicine, New York, NY 10029; and bThe Jackson Laboratory, Bar Harbor, ME 04609 Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved July 13, 2010 (received for review April 13, 2010) Studies of mouse models of anemia have long provided funda- directly influence this bipotential lineage decision by repressing mental insights into red blood cell formation and function. Here megakaryopoiesis while accentuating erythropoiesis (10–12). we show that the semidominant mouse mutation Nan (“neonatal The activation function of EKLF has been analyzed most ex- anemia”) carries a single amino acid change (E339D) within the tensively at the β-globin locus, where its plays critical roles in ge- second zinc finger of the erythroid Krüppel-like factor (EKLF), a crit- netic regulation of the adult β-globin promoter (13, 14). First, ical erythroid regulatory transcription factor. The mutation alters specific amino acids within its three C2H2 zinc fingers interact with the DNA-binding specificity of EKLF so that it no longer binds pro- guanosine residues at its cognate DNA binding site, leading to moters of a subset of its DNA targets. Remarkably, even when mu- precise and high-affinity binding (5, 15). Second, it integrates tant Nan and wild-type EKLF alleles are expressed at equivalent chromatin remodeling and transcriptional activities via critical levels, the mutant form selectively interferes with expression of protein–protein interactions (16–19). Consequently, EKLF plays EKLF target genes whose promoter elements it no longer binds. This a central role in establishing the precise 3D looping of the distant interference yields a distorted genetic output and selective protein locus control region with the proximal β-globin promoter in adult deficiencies that differ from those seen in EKLF-heterozygous and erythroid cells (20), resulting in formation of an active chromatin EKLF-null red blood cells and presents a unique and unexpected hub. Third, EKLF is a regulator of β-like globin switching (21). mechanism of inherited disease. Although EKLF heterozygotes are phenotypically normal, in the total absence of EKLF the active chromatin hub is not properly mouse model | neonatal anemia | red blood cells | zinc finger mutation formed (20), and adult β-globin is not expressed, leading to em- bryonic lethality by E14.5 caused by profound β-thalassemia (22, ereditary spherocytosis (HS) is the most common cause of 23). At the same time, murine embryonic ßh1-globin and (trans- Hinherited hemolytic anemia in Northern Europeans, with an genic) human fetal γ-globin genes are not switched off properly estimated frequency of ∼1/5,000 (1). Defects in the structural and remain expressed at higher levels and for a longer time during components of the red blood cell membrane skeleton comprising development (24–26). In humans, point mutations in the EKLF vertical interactions with the overlying lipid bilayer (spectrin, cognate binding site lead to β-thalassemia (15) with markedly el- ankyrin, protein 4.2, band 3) underlie HS, which follows from evated fetal hemoglobin levels (27). mechanically weakened red blood cells that lose membrane sur- We have identified a single amino acid substitution (E339D) in face area, become increasingly spheroidal, and are removed by the the second zinc finger of EKLF in Nan mutants. The earlier em- spleen (1). bryonic lethality of Nan homozygotes and severe anemia in Nan Spontaneous and targeted mutations in membrane skeleton heterozygotes distinguish Nan mutants from targeted EKLF genes in mice have been instrumental in defining the components, knockout strains and led us to investigate the molecular nature of GENETICS interactions, and functions of membrane skeleton proteins (2). The the EKLF mutation in detail. We find that the E339D change in mouse mutation Nan (for “neonatal anemia”) is an ethylnitrosourea EKLF results in failure to bind and transactivate a subset of fi (ENU)-induced semidominant hemolytic anemia rst described by downstream targets in Nan erythroid cells in a manner dependent Nan Mary Lyon more than 25 y ago (3). homozygotes die at em- on the cognate EKLF DNA binding sequence, in which a single – “ ” bryonic day (E)10 11 from a severe lack of haemopoiesis; with no nucleotide makes the difference between recognition by both WT Nan overt, nonerythroid defects evident. Heterozygotes ( /+) survive and Nan-EKLF versus recognition by WT EKLF alone. An EKLF- with a life-long, severe hemolytic anemia that displays many features DNA interaction model provides a structural basis for the failure of HS including increased osmotic fragility, splenomegaly, and iron of Nan-EKLF to bind selective recognition sites. Moreover, the Nan deposition in the kidney, liver, and spleen (12). is transferable Nan-EKLF variant distorts the expression pattern of EKLF target through hematopoietic stem cells and localizes to mouse chromo- genes even in the presence of WT EKLF. Quite unexpectedly, the some (Chr) 8 (4). Here, we describe positional cloning of Nan. fi Nan Nan genes de cient in expression in heterozygotes are those whose Unlike other models of HS, does not carry a primary defect in EKLF binding elements are not recognized by Nan-EKLF. Such a membrane skeleton gene. Rather, Nan is caused by a mutation in Klf1 target-selective effects lead to a distorted genetic readout and the Krüppel-like factor 1 ( ) gene encoding erythroid Krüppel- account for the severe anemia in Nan/+ mice. Our findings have like factor (EKLF) (14). The mutation causes HS by a mechanism that is unique not only to HS but to genetic disease in general. EKLF, the founding member of the KLF family of transcription fi Author contributions: M.S., J.J.B., and L.L.P. designed research; M.S., K.E.S., S.G.A., and factors (5, 6), is a hematopoietic C2H2 zinc nger transcriptional M.M. performed research; M.S., J.J.B., and L.L.P. analyzed data; and M.S., J.J.B., and L.L.P. factor that plays a global role in erythroid gene expression by wrote the paper. regulating expression of heme biosynthetic, red blood cell mem- The authors declare no conflict of interest. – brane, globin stabilizing, and cell-cycle proteins (7 9). During This article is a PNAS Direct Submission. fi hematopoiesis, EKLF rst is expressed at low levels in common 1To whom correspondence may be addressed. E-mail: [email protected] or luanne. myeloid progenitors but achieves a high level of expression solely [email protected]. within the megakaryocyte-erythroid progenitor, remaining ele- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vated selectively in its red blood cell progeny (10). EKLF levels 1073/pnas.1004996107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1004996107 PNAS | August 24, 2010 | vol. 107 | no. 34 | 15151–15156 Downloaded by guest on October 2, 2021 general implications for the structural parameters involved in We fine-mapped Nan to a 1.3-megabase interval between DNA recognition by KLF proteins and the unanticipated selective D8Mit78 and D8Mit79 on Chr 8 (Ensemble, http://www.ensembl. effects on target gene regulation that can arise after mutation. org) containing 46 known or predicted genes (Mouse Genome Informatics, http://www.informatics.jax.org). More than 600 kb of Results sequence were obtained, including all exons and intron–exon Phenotyping and Mapping of Nan. In agreement with original reports boundaries. Only Klf1 showed a sequence change in Nan: an A-to- (3, 4), we find that Nan homozygotes die in utero at ∼E10–11. T transversion at base pair 1064 (reference sequence NM_010635) Heterozygotes are severely anemic at birth and throughout life. Loss changes glutamic acid to aspartic acid in exon 3 (E339D) within the of heterozygotes in utero occurs also; reciprocal Nan/+ × WT second zinc finger required for DNA binding (14). The change matings produce only 34% affected offspring. The red blood cell creates an MboI site that segregates 100% with the Nan mutation. count, hemoglobin, and hematocrit are decreased significantly in Sequencing of Klf1 in E9.5 WT, Nan/+, and Nan/Nan DNA Nan/+ adults as compared with WT (Table S1). Circulating retic- showed the expected sequence change (Fig. 1G). ulocytes and spleen weights are increased dramatically (Table S2) Peripheral blood smears (Fig. 1A) reveal extensive anisocytosis with E339D Selectively Alters Target Gene Binding. Each EKLF zinc fin- many microcytes, spherocytes, and hypochromic cells, consistent ger interacts primarily with the G-rich strand of its DNA target via with the increased red blood cell distribution width and hemoglobin critical basic amino acid–guanosine interactions that yield a highly distribution width. Platelet levels are slightly but significantly in- specific consensus interaction sequence (5) (Fig. 2A). Mutations in creased (Table S1). this sequence lead to β-thalassemia in humans (HbVar: http:// Nan/+ bilirubin and iron levels do not differ significantly from globin.bx.psu.edu/cgi-bin/hbvar/query_vars3). The E339D muta- controls, but zinc protoporphyrin is dramatically increased (Table tion is located in the second zinc finger of EKLF in position +3 of S2), suggesting that a heme biosynthesis defect is unlikely (28). its α-helix, within the structural motif of the C2H2-type finger that Consistent with severe hemolysis, iron accumulates in Nan/+ fits into the DNA major groove (15). Based on structural data from kidney (Fig. 1B), liver (Fig. 1C), and spleen (Fig. 1D). Erythro- other C2H2 zinc finger proteins (31, 32), this position is involved in poietic foci are observed in the mutant liver (Fig.