The mitochondrial ATP-binding cassette transporter Abcb7 is essential in mice and participates in...

The mitochondrial ATP-binding cassettetransporter Abcb7 is essential in mice andparticipates in cytosolic iron–sulfur clusterbiogenesis

Corinne Pondarre1, Brendan B. Antiochos1,{, Dean R. Campagna1,{, Stephen L. Clarke2,

Eric L. Greer1, Kathryn M. Deck2, Alice McDonald3, An-Ping Han1, Amy Medlock4,

Jeffery L. Kutok5, Sheila A. Anderson2, Richard S. Eisenstein2 and Mark D. Fleming1,*

1Department of Pathology, Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA, 2Department of

Nutritional Sciences, University of Wisconsin, Madison, WI 53706, USA, 3Millennium Pharmaceuticals, Cambridge,

MA 01239, USA, 4Department of Biochemistry and Molecular Biology, The Center for Metalloenzyme Studies,

University of Georgia, Athens, GA 30602, USA and 5Department of Pathology, Brigham and Women’s Hospital and

Harvard Medical School, Boston, MA 02115, USA

Received December 7, 2005; Revised and Accepted February 1, 2006

Proteins with iron–sulfur (Fe–S) clusters participate in multiple metabolic pathways throughout the cell. Themitochondrial ABC half-transporter Abcb7, which is mutated in X-linked sideroblastic anemia with ataxia inhumans, is a functional ortholog of yeast Atm1p and is predicted to export a mitochondrially derived metab-olite required for cytosolic Fe–S cluster assembly. Using an inducible Cre/loxP system to delete exons 9 and10 of the Abcb7 gene, we examined the phenotype of mice deficient in Abcb7. We found that Abcb7 wasessential in extra-embryonic tissues early in gestation and that the mutant allele exhibits an X-linkedparent-of-origin lethality effect. Furthermore, using X-chromosome inactivation assays and tissue-specificdeletions, Abcb7 was found to be essential for the development and function of numerous other cell typesand tissues. A notable exception to this was liver, where loss of Abcb7 impaired cytosolic Fe–S clusterassembly but was not lethal. In this situation, control of iron regulatory protein 1, a key cytosolic modulatorof iron metabolism, which is responsive to the availability of cytosolic Fe–S clusters, was impaired and con-tributed to the dysregulation of hepatocyte iron metabolism. Altogether, these studies demonstrate theessential nature of Abcb7 in mammals and further substantiate a central role for mitochondria in the bio-genesis of cytosolic Fe–S proteins.

INTRODUCTION

Mitochondria play a central role in cellular iron metabolism.Not only do the initial and final steps of heme biosynthesisoccur in mitochondria, so does the biosynthesis of iron–sulfur (Fe–S) clusters, which perform essential structuraland catalytic roles in many mitochondrial enzymes (1–3).Studies in yeast have shown that the mitochondrial Fe–Scluster synthesis machinery also has an essential function in

providing a component needed for extramitochondrial Fe–Sprotein assembly (4,5). In fact, the yeast requirement for mito-chondria depends not on their function in energy metabolism,but instead is due to their necessity in forming an essentialcytosolic Fe–S protein, Rli1p, involved in ribosome biogen-esis (6,7). Although yeast is a genetically flexible system formodeling mammalian mitochondrial Fe–S cluster metab-olism, sparingly little of this work has been directly validatedin mammalian cells. The primary exception being the study of

# The Author 2006. Published by Oxford University Press. All rights reserved.For Permissions, please email: journals.permissions@oxfordjournals.org

{These authors contributed equally.

*To whom correspondence should be addressed at: Department of Pathology, Children’s Hospital and Harvard Medical School, Enders 1116.1,320 Longwood Avenue, Boston, MA 02115, USA. Email: mark.fleming@childrens.harvard.edu

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Friedreich ataxia, which is a neurodegenerative disorder due tomutations in a mitochondrial protein, frataxin, that influencesmitochondrial Fe–S cluster synthesis (8,9). Nonetheless, theparticular role and the extent to which extramitochondrialFe–S clusters contribute to this or other human diseases has,for the large part, not been investigated.

Studies in yeast also indicate that there is a complexrelationship between Fe–S cluster assembly pathways andiron metabolism as a whole. For instance, disruption of Fe–Scluster biogenesis in mitochondria impairs heme formationin yeast by inhibiting the activity of ferrochelatase (10). Fur-thermore, Aft1p and Aft2p, iron-regulated transcriptionfactors controlling yeast iron homeostasis, respond not to cyto-solic iron, but to the rate of mitochondrial Fe–S cluster syn-thesis (11–13). Similarly, in mammals, the action of ironregulatory protein 1 (IRP1), a cytosolic modulator of ironhomeostasis, is controlled in part by an Fe–S cluster-dependent mechanism (14,15). However, how perturbationsin cytosolic Fe–S cluster assembly alone directly affectIRP1 function and influence cellular and systemic mammalianiron metabolism have not been explored.

In yeast, Atm1p, an ATP-binding cassette (ABC) transpor-ter of the inner mitochondrial membrane, is required for matu-ration of cytosolic Fe–S apo-proteins to their holo-forms (4).Consequently, Atm1p links the mitochondrial and cytosolicpathways for Fe–S cluster assembly, presumably by mediatingthe transport of a component required for cytosolic Fe–Scluster assembly from the mitochondria to the cytosol. Yeastwith chromosomal deletions in ATM1 (Datm1) develops mito-chondrial iron overload, which can be fully rescued by thehuman ortholog ABCB7 (16–19). Mitochondrial iron overloadresembling the Datm1 phenotype is a feature of several humandiseases, most notably sideroblastic anemias (20). An unusualform of X-linked sideroblastic anemia, syndromically associ-ated with ataxia and severe developmental hypoplasia of thecerebellum [X-linked sideroblastic anemia with ataxia(XLSA/A)] has been described in four families and is dueto mutations in ABCB7 (16,21–25). Each of thethree-recorded disease-causing alleles is a missense mutationin exon 9 or 10 of the gene. In order to further explore thefunction of mammalian Abcb7 and the link between mitochon-drial and cytosolic Fe–S cluster biogenesis and cellular ironmetabolism, we have created a conditionally targeted alleleof the gene in mice. Here, we report that Abcb7 is an essentialgene in mice and, like its yeast counterpart, participates in thematuration of cytosolic Fe–S cluster proteins in mammals.

RESULTS

Abcb7 is an essential gene in mice associated withX-linked parent of origin lethality

We targeted a loxP site and a loxP-flanked neomycin-resistance cassette (NeoR) into introns 8 and 10 of Abcb7 ofembryonic stem (ES) cells to create a conditionally targetedallele flanked by loxP sites (‘floxed’ allele, Fig. 1A and B).Transient transfection of correctly targeted ES clones harbor-ing the floxed allele (including the NeoR cassette, Fig. 1C) witha plasmid encoding Cre-recombinase readily yieldedneomycin-sensitive subclones, but none of these .50 colonies

contained a null allele in which exons 9 and 10 were alsodeleted (Fig. 1D). As the ES cells are karyotypically male(i.e. 40, XY and hemizygous for the X-linked Abcb7gene),and we were able to obtain deletion of exons 9 and 10 invivo (Fig. 1E and discussed subsequently), we conclude thatAbcb7 is an essential gene in ES cells cultured in vitro.

Several independently targeted ES cell clones were injectedinto blastocysts and gave rise to chimeric males that trans-mitted the modified Abcb7 allele through the germline. Micehemi- and homozygous for the NeoR-less floxed allele(Fig. 1D, Abcb7 f l/Y and Abcb7 f l/f l, respectively) on mixed129S substrain, and 129S6/SvEvTac (N4) and C57BL/6J(N8) congenic backgrounds, were viable and fertile and hadno grossly visible neurological phenotype or measurable hem-atological abnormality (data not shown). In order to obtain agermline null allele, we crossed chimeric 129S4/SvJaeAbcb7 f l/Y males to a Cre-recombinase transgenic (Tg) line,FVB-Tg(Gata1-Cre), which typically behaves as a generalizeddeleter strain (26), to obtain [FVB � 129]F1 females nomin-ally heterozygous for an Abcb7 null allele (Abcb7þ/2, Sup-plementary Material, Fig. S1A). Breeding these females towild-type males yielded neither live born male nor femalepups with a germline null allele (Table 1). Similarly, the reci-procal intercross mating, in which an Abcb7 f l/f l female wascrossed to a Gata1-Cre male produced no animals with agermline null allele (Supplementary Material, Fig. S1B;Table 1). In both crosses, several females and males bearingthe conditional allele with no or partial somatic deletionwere born alive; in these cases, rearrangement was essentiallylimited to the bone marrow of Cre-positive female animals,consistent with the preferential bone marrow deletion inanimals escaping early embryonic deletion previously reportedwith this Cre transgene (26). Embryonic dissections indicatedthat both male and female Cre-positive embryos died at orprior to E6.5–E7.5 (Table 2). Histological examinationrevealed slightly growth-retarded, inviable embryos, typicallywith fibrin and hemorrhage in the region of the ectoplacentalcone, but without other distinctive histological features or his-tochemical evidence of excess iron deposition (Fig. 2 and datanot shown). Notably relevant to XLSA/A, yolk sac hemato-poietic progenitors could be identified histologically (Fig. 2C).

These data indicate that Abcb7 is essential for mouse devel-opment and provided evidence for a parent of origin effect ofexpression of the gene; inheritance of either a germline nullallele from the female or a conditional allele and aGata1-Cre transgene from the female and male parents,respectively, resulted in death whether the embryo was ahemizygous Abcb7 f l/Y male or a heterozygous Abcb7 f l/þ

female. Furthermore, this specifically implicated an abnormal-ity in the extra-embryonic tissues, as the female-derivedX-chromosome is preferentially active in the extra-embryonictissues of eutherian mammals (27).

To further explore the likelihood of an extra-embryonicdefect, we crossed Abcb7 f l/ f l females to males hemizygousfor a Sox2-Cre (Supplementary Material, Fig. S1C) transgene,which drives Cre expression only in the embryonic epiblast,sparing many of the extra-embryonic tissues (28); we also per-formed a cross with males hemizygous for a Villin-Cre trans-gene (Supplementary Material, Fig. S1D), which is selectivelyexpressed in ciliated cells in the extra-embryonic visceral

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endoderm (29,30). In toto, these Cre transgenic lines producenearly complementary, non-overlapping patterns of deletionin the embryo and extra-embryonic tissues. Consistent withour hypothesis, using the Sox2-Cre line, we were able toobtain healthy live-born females that appeared to have fullyrearranged the single conditional allele inherited from theirmother (Table 3 and data not shown). As with the Gata1-Cretransgene, these nominally Abcb7þ/2 females were unable totransmit the null allele to live-born progeny (data not shown).Dissection of embryos from Abcb7 f l/f l

� Sox2-Cre pairingsrevealed that transgene-positive Abcb7 f l/Y males died at

E7.5–8.5, slightly later than in the Gata1-Cre crosses, indicat-ing that sparing the extra-embryonic defect is succeeded by alethal Abcb7-dependent embryonic abnormality in early-midgestation. Furthermore, and supportive of the notion that theextra-embryonic tissues are the cause of the early lethality, weobserved no live born Villin-Cre positive males or females incrosses between Abcb7 f l/f l females and Villin-Cremales (Sup-plementary Material, Fig. S1D; Table 3). Taken together, thesedata demonstrate thatAbcb7 is an essential gene inmice becauseof a requirement for Abcb7 in the extra-embryonic tissues earlyin gestation.

Abcb7 is required for the development or maintenanceof multiple adult cell lineages

In order to evaluate the role of Abcb7 at later stages in devel-opment, we bred the Abcb7 f l allele to several tissue-specific orinducible Cre transgenic lines. We were particularly interested

Figure 1. Abcb7 gene targeting. (A) The endogenous wild-type Abcb7 locus. (B) ES cells were transfected with a targeting construct that introduces a neomycin-resistance cassette flanked by loxP sites into intron 10 and a solitary loxP site into intron 8. (C) Neomycin resistant clones were analyzed for homologous recom-bination by Southern blot using an external 50 (exon 5) probe and BglII and a 30 probe (exon 14) and KpnI. (D) Transient transfection with a Cre recombinaseexpression plasmid yielded subclones lacking the neomycin resistant cassette. (E) Rearrangement to the null allele lacking exons 9 and 10 could be obtainedin vivo in female animals carrying a Cre transgene.

Table 1. Global deletion of Abcb7 leads to X-linked parent of origin lethality:weaned animal analysis

Genotype % Expected % Observed

[Gata1-CreTg/Tg � Abcb7fl/Y]F1 � Abcb7þ/þ (n ¼ 59)Abcb7þ/Y+ Cre 25.0 52.5Abcb7þ/þ + Cre 25.0 44.1Abcb7fl/Y+Cre 25.0 1.7a

Abcb7fl/þ+Cre 25.0 1.7b

x2 P-value ,0.001

Abcb7fl/þ � Gata1-CreTg/Tg (n ¼ 56)Abcb7þ/Y

þ Cre 25.0 57.1Abcb7þ/þ

þ Cre 25.0 37.5Abcb7fl/Yþ Cre 25.0 1.8a

Abcb7fl/þ þ Cre 25.0 3.6b

x2 P-value ,0.001

aNeither of these animals carried Gata1-Cre.bNone of these animals demonstrated rearrangement of the conditionalallele in tail DNA.

Table 2. Global deletion of Abcb7 leads to X-linked parent of origin lethality:embryonic dissection analysis

Gestation day % Alive % Dead

[Gata1-CreTg/Tg � Abcb7fl/Y]F1 � Abcb7þ/þ

E6.5 (n ¼ 5) 40.0 60.0E7.5 (n ¼ 32) 50.0 50.0E9.5 (n ¼ 37) 51.4 49.6

Abcb7fl/þ � Gata1-CreTg/Tg

E6.5 (n ¼ 57) 54.2 45.8E7.5 (n ¼ 47) 53.2 46.8E9.5 (n ¼ 16) 56.3 43.7

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in the development of the central nervous system (CNS) andhematopoietic system, as these tissues are clinically affectedin the human disorder XLSA/A. Inducible deletion in thebone marrow with MX1-Cre (31) led to bone marrow failure(C. Pondarre and M.D. Fleming, unpublished data).Nestin-Cre mediated CNS deletion (32) resulted in no grossabnormalities in brain development, but proved to be lethalin the immediate perinatal period (Table 4). As cerebellardevelopment occurs primarily after birth in mice, the natureof the cerebellar dysgenesis seen in XLSA/A patients couldnot be evaluated.

Given the failure of the targeted, tissue-specific approachto produce viable animals in which we could study a bio-chemical phenotype, we sought to simultaneously evaluatethe requirement for Abcb7 in multiple tissues. In order to doso, we examined X-inactivation patterns in female mice har-boring a paternally inherited Abcb7 f l allele in cis with a ‘ubi-quitously’ expressed X-linked green fluorescent protein (GFP)transgene (33) in the presence or absence of a maternallyderived Gata1-Cre transgene. In female animals withoutCre, X-inactivation should be stochastic, and GFP should beexpressed in a variegated pattern in any tissue in which thepromoter is active. In contrast, in the presence of Cre, GFPshould not be expressed tissues in which Abcb7 is essentialat any point in development or for maintenance of that celllineage. We found that in heterozygous females lacking a

Cre transgene, GFP protein was expressed in a variegatedpattern, consistent with random X-inactivation, in mosttissues (Fig. 3A). Notably, however, GFP was not expressedin most bone marrow cells, as well as lymphoid cells in thethymus and spleen, and present in only a minor subset ofhepatocytes; the inactivity of the promoter precluded X-inactivation analysis in these tissues. However, in the presenceof Cre, essentially all organs lacked GFP-positive parenchymalcells, providing evidence that Abcb7 is essential for the devel-opment and/or maintenance of numerous cell types (Fig. 3Band C). This result is congruous with the widespread expressionof Abcb7 in embryonic and adult murine tissues (Fig. 3F andG). Remarkably, capillary endothelial cells in many tissues,including glomerular tufts in the kidney, retained the variegatedpattern of expression regardless of Cre status (Fig. 3D and E),suggesting that Abcb7 is not essential in this lineage.

Hepatocyte-specific Abcb7 deletion disturbs cellular andsystemic iron metabolism

Although the GFP transgene was expressed only in a minorsubset of hepatocytes in animals lacking Cre, in the presenceof Cre, GFP staining persisted in a fraction of these cells(data not shown). This suggested that the active X-chromosome in these cells expressed the modified Abcb7allele. In order to investigate this more thoroughly, we bredthe conditional deletion allele to a hepatocyte-specific Cre-transgenic line: B6.Cg-Tg(Alb-Cre)21Mgn/J [Alb-Cre]. Thealbumin promoter driving Cre expression in this line is gener-ally not substantially active until after birth, with completehepatocyte deletion not occurring until 4–6 weeks afterbirth (34 and data not shown). Male animals carrying theAbcb7 f l allele and Alb-Cre will be subsequently referred toas Abcb7lv/Y to reflect the hepatocyte-specific deletion.

Abnormal iron metabolism in XLSA/A and in yeastdeficient in Atm1p (Datm1) lead us first to examine systemiciron parameters in Abcb7lv/Y mice. We found that the serumiron and total iron binding capacity (TIBC) were unchanged,however, the transferrin saturation was significantly increasedin knockout animals (Fig. 4A). In order to investigate thecause of this difference, we examined liver iron parametersand found that total liver iron was increased by 76% in themutants (Fig. 4B). As XLSA/A and Datm1yeast are specifi-cally associated with increased mitochondrial iron, it was

Figure 2. Abcb7 deficiency leads to early-mid-gestational death. H&E stainedtissue sections of [Abcb7fl/fl

� Gata1-CreTg]F1 embryos, without (A) and with(B) Gata1-Cre. (C) Higher magnification of (B). Black arrowhead indicatesembryo, gray arrowhead yolk sac hematopoietic progenitors and asteriskhemorrhage and fibrin in the area of the ectoplacental cone.

Table 3. Abcb7 is required in the extra-embryonic tissues

Genotype % Expected % Observed

Abcb7fl/fl � Sox2-CreTg/þ (n ¼ 47)Abcb7fl/Y2 Cre 25.0 44.7Abcb7fl/þ2 Cre 25.0 27.7Abcb7fl/Yþ Cre 25.0 0.0Abcb7fl/þ þ Cre 25.0 27.7

x2 P-value ,0.001

Abcb7fl/fl � Villin-CreTg/þ (n ¼ 27)Abcb7fl/Y2 Cre 25.0 55.6Abcb7fl/þ2 Cre 25.0 44.4Abcb7fl/Yþ Cre 25.0 0.0Abcb7fl/þ þ Cre 25.0 0.0

x2 P-value ,0.001

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somewhat surprising that the increase in liver iron was notassociated with mitochondrial iron overload (Fig. 4B). Liveriron loading was accompanied by a trend (P ¼ 0.07) toward aslight increase in ferritin protein and a co-existent, apparentlyparadoxical, rise in transferrin receptor-l (TfR1) protein(Fig. 4C and discussed subsequently). Perl’s Prussian blueiron stain showed a small subset of hepatocytes, typicallylocated adjacent to a portal triad or central vein, that containedabundant, coarsely granular cytosolic iron deposits, often with a

central clearing, and that were morphologically distinct fromferritin (Fig. 4D). Electron microscopy showed that these struc-tures were electron-dense rings with a homogenous center mor-phologically typical of neutral lipid (Fig. 5B and D). We alsoobserved numerous cytosolic lipid droplets and pale, swollenmitochondria suggestive of metabolic and/or mitochondrialinjury (Fig. 5B and D); mitochondria with electron-densedeposits typical of iron were not present. Consistent with theimpression of mild hepatocellular injury, Abcb7lv/Y livers also

Table 4. Abcb7 is essential in the central nervous system

Genotype % Expected E11–E12(n ¼ 20)

E13–E14(n ¼ 32)

E15–E16(n ¼ 43)

E17–E18(n ¼ 48)

P1(n ¼ 92)

Weanlings(n ¼ 100)

Abcb7þ/þ or Abcb7þ/Y2 Cre 25.0 25.0 28.1 25.6 18.8 25.0 39.0Abcb7þ/þ or Abcb7þ/Y

þ Cre 25.0 20.0 15.6 37.2 31.3 37.0 25.0Abcb7fl/þ2 Cre 12.5 15.0 28.1 16.3 10.4 15.2 13.0Abcb7fl/þ

þ Cre 12.5 30.0 9.4 7.0 8.3 9.8 13.0Abcb7fl/Y2 Cre 12.5 10.0 9.4 4.7 18.8 8.7 10.0Abcb7fl/Y

þ Cre 12.5 0.0 9.4 9.3 12.5 4.3 0.0x2 P-value whole set — 0.17 0.14 0.25 0.57 0.03 ,0.01

x2 P-value Abcb7fl/Yþ Cre

versus rest— 0.09 0.59 0.52 1.00 0.02 ,0.01

Genotypes of embryos, newborns (P1) and weanlings from an Abcb7 fl/þ� Nestin-CreTg/þ cross are given.

Figure 3. X-inactivation and RNA expression patterns indicate that Abcb7 is essential in multiple tissues. (A) GFP immunostaining in pancreas: [Gata1-CreTg/þ � Abcb7 f l-GFP/Y]F1 females without Cre show random inactivation of the GFP-tagged Abcb7 f l chromosome as demonstrated by a mixture of cells posi-tive and negative for GFP marking an active conditional allele. (B) [Gata1-CreTg/þ � Abcb7 f l-GFP/Y]F1 females with Cre showed no staining in pancreatic par-enchymal cells. (C) Table summarizing GFP immunohistochemistry X-inactivation assay results. Values reflect number of animals positive for GFP in thattissue/total number examined. (Asterisk) A small subset of hepatocytes was positive in four of six Cre-positive animals analyzed. Glomerular tufts from (D)Gata1-Cre negative and (E) Gata1-Cre positive Abcb7 f l-GFP/þ females showing persistence of GFP positivity and presumptive activity of the null allele inendothelial cells even in Cre-positive animals. (F) Northern blot of Abcb7 in adult tissues. (G) In situ hybridization of E15.5 mouse embryo demonstrating wide-spread expression of Abcb7 with high-level expression present in the neuroectoderm of the forebrain and the fetal liver.

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showed mild hepatic architectural disarray and hepatocellularmultinucleation on routine hematoxylin and eosin (H&E)tissue sections (data not shown). Limited, ongoing liverdamage was further substantiated by a 2-fold increase inserum levels of liver-derived transaminases (data not shown).

Hepatocellular deletion of Abcb7 impairs the activityof cytosolic but not mitochondrial Fe–S proteins

Because the yeast ortholog of Abcb7, Atm1p, is required forformation of Fe–S clusters in the cytosol, but not in the mito-chondria (4), we determined the activity of Fe–S enzymes inwild-type and Abcb7lv/Y mice. Ferrochelatase is a mitochon-drial Fe–S enzyme in the heme biosynthetic pathway. Wefound that its activity was not different between wild-typeand Abcb7lv/Y mice (Fig. 6A). The activity of mitochondrialaconitase, a Fe–S enzyme in the tricarboxylic acid cycle, wasslightly increased in the mutant animals (Fig. 6A). Activity of

succinate dehydrogenase (SDH), another mitochondrial Fe–Senzyme that participates in the electron transport chain(complex II), was reduced by 20% in mutants when normalizedto the activity of cytochrome C oxidase (CCO) (complex IV)(35). As in liver, the activity of complex I is one-half ofcomplex II, the minor decrease in complex II activity observedhere is not likely to limit electron transport chain flux, at leastwhen NADH is the source of electrons (36). Overall, deletionof Abcb7 had little impact on hepatocyte mitochondrial Fe–Senzyme activity. In contrast, the activity of the cytosolic Fe–Sprotein xanthine oxidase (XO), which also contains a molyb-denum cofactor prosthetic group, whose biosynthesis alsoappears to be dependent on Fe–S proteins (37), was reducedby 50% (Fig. 7A). As disruption of Atm1p in yeast resultedin a loss in the activity of the cytosolic Fe–S protein Leu1p,without affecting its protein level (4), we determined theabundance of XO protein. XO protein level was not affectedby the loss of Abcb7 (Fig. 7A). In sum, these data support a

Figure 4. Abnormal iron metabolism in Abcb7lv/Y animals. (A) Liver total and mitochondrial iron (n ¼ 5), (B) serum iron, TIBC, and transferrin saturation(n ¼ 5) and (C) tissue ferritin and transferrin levels (n ¼ 5) were determined in wild-type (open bars) and Abcb7lv/Y mice (black bars). A significant(P, 0.05) difference between groups is indicated with an asterisk. P ¼ 0.07 for ferritin. (D) Perl’s Prussian blue stain of Abcb7 lv/Y animal showing periportalhepatocellular iron deposition with characteristic round structures with central clearing (arrow).

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role for Abcb7 in the assembly of cytosolic, but not mitochon-drial, Fe–S clusters in the liver.

Activation and altered regulation of IRP1 and IRP2in Abcb7 lv/Y mice

As the function of IRP1 (aconitate hydratase 1), a cytosolicregulator of mammalian iron metabolism, is controlledthrough gain and loss of its Fe–S cluster, we sought to deter-mine whether it too was influenced by the loss of Abcb7. Iniron deficiency, IRP1 lacks its [4Fe–4S] cluster and controlsthe synthesis of ferritin and TfR1 by binding iron responsiveelements in their mRNAs (14,15). When iron levels increase,assembly of the Fe–S cluster in IRP1 inactivates RNAbinding, thereby maintaining iron homeostasis; in the holo-form, IRP1 is the cytosolic isoform of aconitase (c-acon).Similar to XO, c-acon activity declined significantly (�90%)in the liver of Abcb7lv/Y mice relative to wild-type animals(Fig. 7B). This was accompanied by the expected reciprocalrise in IRP1 RNA binding activity, which increased by nearly6-fold (Fig. 7B). This provides a likely explanation for the60% increase in TfR1 protein in Abcb7 lv/Y liver (Fig. 4C).Unexpectedly, and in marked contrast to XO, IRP1 proteinlevel declined by nearly 60% in Abcb7lv/Y liver, suggestingregulation of IRP1 at the protein level. We have subsequentlygone on to show that this effect is likely a consequence of theunique role that IRP1 plays in iron metabolism and is, inlarge part, due to iron-dependent, cluster-independent mechan-isms of regulating IRP1 protein (38).

We also determined the activity of IRP2, an RNA bindingprotein functionally homologous to IRP1 whose activity iscontrolled through iron-dependent changes in protein turn-over. In Abcb7lv/Y liver, we found that IRP2 RNA bindingactivity was increased 50% and its protein level was increasedby 24% (Fig. 7C). This effect is contrary to the apparentcellular iron overload and suggests that the iron sensing

mechanism is reacting as if the cell is iron deficient (4,13).As recent evidence suggests that IRP2 protein turnover canbe directly dependent on cytosolic heme levels (39), it is poss-ible that these cells are heme deficient, yet iron overloaded.

DISCUSSION

Animal models of rare human disorders can provide uniqueinsights into the pathogenesis of disease and the normal func-tion of the mutated proteins. Our studies of Abcb7 have affordedan opportunity to examine the function of the protein in a mam-malian system. We found that Abcb7 is an essential gene inmice because of an early requirement for the protein in theextra-embryonic tissues and have provided additional geneticevidence that Abcb7 is indeed functionally orthologous toAtm1p, playing a role in the formation of cytosolic Fe–S pro-teins. Furthermore, we demonstrate that in mammals, as inyeast, there is a complex interplay between the mitochondriaand cytosol in sensing and controlling the iron status of the cell.

That ES cells and male mice deficient in Abcb7 were invi-able was not surprising, given the requirement for a similarprotein in lower eukaryotes. Unexpected, however, was thefact that a maternally inherited modified Abcb7 allele waslethal to female embryos. By breeding to a series of Cre-transgenic lines, we were able to demonstrate that the lethalitywas specifically due to a defect in the extra-embryonic visceralendoderm, which like all of the extra-embryonic tissues pre-ferentially maintains the female X-chromosome as the activeallele. This so-called X-linked parent of origin effect hasbeen reported for several other null or severely hypomorphicalleles of X-linked genes, including several modeling hemato-logical diseases in man (e.g. glucose-6-phosphate dehydro-genase deficiency and X-linked dyskeratosis congenita)(40,41). In each case, embryonic death can be attributed tomorphologically distinctive developmental defects in theextra-embryonic tissues. However, in most other examples,lethality occurs somewhat later in embryogenesis and at apoint where male and female embryos can be distinguishedby genotyping. In the case of Abcb7, we were unable to deter-mine the sex of the embryos because of their early demiseand cannot infer whether males were more severely affectedthan females. If male embryos did indeed die earlier thanfemale embryos, this would imply that Abcb7 was essentialfor an embryonic function prior to the requirement in the extra-embryonic tissues. However, the observation that male embryosin which deletion occurs only in the epiblast (using Sox2-Cre)die at a slightly later time would suggest that the immediatecause of death is truly due to a defect in the extra-embryonicvisceral endoderm.

Abcb7 mRNA is widely distributed and tissue-specific del-etions in the CNS and bone marrow are lethal (Table 1 andFig. 5) (data not shown). This confirms the suggestion thatXLSA/A in man is likely due to ABCB7 partial loss of func-tion mutations, as a complete loss of function allele wouldalmost certainly be lethal because of its effects on the extra-embryonic tissues, CNS and/or bone marrow at a minimum.Furthermore, X-inactivation analysis indicated that Abcb7was essential in nearly all tissues. The primary exception tothis is the observation that hepatocytes and endothelial cellscan apparently survive in the absence of the protein. Why

Figure 5. Electron microscopy of Abcb7 liver-specific deletion. (A and C) Wild-type and (B and D) Abcb7lv/Y livers. Pale, swollen mitochondria, lipid vacuolesand cytoplasmic, electron-dense circular inclusions are present in the mutant.

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hepatocytes, in particular, are spared from the lethal effects ofAbcb7 is unclear. Three possibilities include (i) that theAlb-Cre achieves only partial deletion of the conditionalallele, (ii) that Abcb7 is redundant in certain cell types and(iii) that certain cell types are more or less sensitive to ordependent on the downstream effects of loss of Abcb7. Geno-typing of Abcb7lv/Y livers by Southern blot or PCR demon-strates that 80–90% of the cells have rearranged theAbcb7 f l conditional allele to the null allele (data notshown). The small fraction of cells with a residual, unrear-ranged conditional allele is not inconsistent with the numberof Kupffer, vascular and other accessory cells present in theliver in which Alb-Cre is inactive, suggesting that there is acomplete or near-complete deletion in the hepatocytelineage. These cells undoubtedly contribute to the residualenzymatic activities (such as XO) measured in these samplesand consequently mitigate the effects on bulk tissue enzymeassays. Furthermore, there are at least four other mitochondrialABC half transporters in mammals (42). One of these, Abcb6(mtAbc3 and UMAT1), has previously been shown to partiallycomplement the Datm1 yeast phenotype (43–45). Furthermore,

the expression of Abcb6 and Abcb7 are largely overlapping inadult tissues, with Abcb6 being expressed at particularly highlevels in the liver (Supplementary Material, Fig. S2). Conse-quently, functional redundancy could both play a role in thesurvival of Abcb7 null hepatocytes and contribute to theincomplete loss of Fe–S protein activity. Alternatively, thehepatocyte may be less dependent on cytoplasmic Fe–S pro-teins such as the mammalian homolog of Rli1p.Furthermore, given the hepatocyte’s pivotal role in systemiciron metabolism, it may be uniquely adapted to accommodatemetabolic dysregulation brought on by Abcb7 deficiency. Inthis regard, the surprising decrease in IRP1 protein level inliver of Abcb7 mice suggests the presence of a compensatorymechanism to prevent excessive accumulation of RNAbinding activity when Fe–S cluster-insertion is impaired andthereby limit hepatocyte damage (38).

The localization of Abcb7 to the mitochondrial inner mem-brane with its ABC domain facing the matrix places theprotein at the gateway between the mitochondria and thecytosol and predicts that Abcb7 exports a mitochondriallygenerated metabolite to the cytosol (2,4). Interestingly, and

Figure 6. Mitochondrial Fe–S enzyme activities in Abcb7ly/Y liver. (A) Activity of ferrochelatase and mitochondrial aconitase (n ¼ 3) and (B) SDH, CCO and CS(n ¼ 7) were determined in isolated mitochondria. Enzyme activities determined in wild-type (open bars) and Abcb7ly/Y mice (black bars) are shown. A significant(P, 0.05) difference between groups is indicated with an asterisk.

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in some contrast to yeast cells and erythroid precursors inXLSA/A, Abcb7 deficiency in the liver yields a modest mito-chondrial phenotype; to the extent we examined mitochondrialfunction, we observed only mild morphologic and enzymaticdefects. The apparent discrepancy in mitochondrial iron over-load and dysfunction between yeast and XLSA/A patient ery-throid cells and Abcb7-deficient hepatocytes may, amongother possibilities, be due to functionally redundant proteins,tissue-specific effects resulting from a relatively high require-ment for mitochondrial heme biosynthesis in erythroid cells,secondary, compensatory effects unique to mammalian cellsthat are dependent on the persistence of at least some Abcb7activity or differences in iron requirements in proliferatingyeast cells relative to mature hepatocytes.

More apparent than the mitochondrial effects, however, werethe cytosolic effects of Abcb7 deletion, particularly cytosoliciron inclusions. Although the inclusions have some resem-blance in size and shape to mitochondria, we were unable toidentify intermediate structures, indicating that it is unlikelythat the inclusions were derived from mitochondria. Further-more, in no case were the inclusions seen to be associatedwith a membrane, indicating that they are not derived fromany another organelle. Lastly, review of electron micrographsof murine neurons lacking frataxin also shows cytosolic lipiddroplets with a similar, but somewhat eccentric, electron-denserim (46). These structures, it would appear, may not be uniqueto Abcb7-deficient liver and may instead be a more generaliz-able effect of cytosolic Fe–S cluster deficiency.

Figure 7. Activity and abundance of XO, IRP1 and IRP2 in Abcb7ly/Y liver. (A) Liver cytosolic XO activity was quantified enzymatically (n ¼ 8) and abundanceof XO protein (n ¼ 5) was determined by western blotting. (B) IRP1 RNA binding activity was determined in liver cytosol by quantitative gel shift assay (n ¼ 5).The enzymatic activity of cytosolic aconitase was determined in liver cytosol (n ¼ 3). IRP1 protein level was determined in liver cytosol by western blotting(n ¼ 5). (C) IRP2 RNA binding activity was determined in liver cytosol by quantitative gel shift assay (n ¼ 5). IRP2 protein level was determined in liver cytosolby western blotting (n ¼ 3). Activities determined in wild-type (open bars) and Abcb7ly/Y mice (black bars) are shown. A significant (P, 0.05) differencebetween groups is indicated with an asterisk.

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Fe–S cluster deficiency was accompanied by dysregulatedactivation of IRP1 and IRP2, which likely contributed toincreased TfR1 expression and iron uptake in Abcb7lv/Y

animals; the slight, but not statistically significant, simul-taneous rise in ferritin expression may reflect iron-regulationof ferritin gene transcription or protein stability (47,48). Inthe end, however, the increased liver iron appears to not beappropriately sensed by IRP and suggests that like yeastAft1p and Aft2p, IRP RNA binding activity may respondmore to the flux of iron through specific metabolic pathways(i.e. Fe–S cluster assembly) than to the absolute level of cellulariron. Similarly, recent observations in zebrafish with mutationsin the mitochondrial Fe–S cluster biogenesis protein glutare-doxin 5 indicate that deficiency of Fe–S clusters may lead toinappropriate activation of IRP1 and death not as a consequenceof mitochondrial deficiency of Fe–S clusters per se, but ratherbecause of inappropriate regulation of downstream targets ofcytosolic IRP1 (49). Hence, although the targeted deletion ofIRP1 alone may not have a substantial phenotype (50,51), dys-regulation of IRP1 by alterations in Fe–S metabolism may wellindeed contribute to the pathogenesis of Fe–S cluster disorders,such as Friedreich ataxia and XLSA/A.

MATERIALS AND METHODS

Animals

J1 ES (129S4/SvJae lineage) cells were transfected with thegene targeting construct shown in Figure 1, selected forG418 resistance and ganciclovir sensitivity, and analyzed bySouthern blot for homologous recombination using standardtechniques (52). The NeoR cassette was excised in vitro bytransient transfection of a Cre recombinase plasmid (DrCharles Roberts, Dana Farber Cancer Institute, Boston, MA,USA) and G418 sensitive subclones analyzed for recombina-tion of the locus by Southern blot using external probes 50

and 30 of the targeting construct (Fig. 1). Chimeric malemice were generated from individual recombinants with andwithout the NeoR cassette, and the modified Abcb7 allele trans-mitted through the germline. In many instances, experimentswere performed with and without the NeoR cassette in place;the results did not differ between these groups. C57BL/6J-Albumin-, Nestin- and Sox2-Cre-recombinase transgeniclines were obtained from the Jackson Laboratory (BarHarbor, ME, USA). FVB/J-Tg(Gata1-Cre) and outbredVillin-Cre lines were obtained from Drs Stuart Orkin (Chil-dren’s Hospital, Boston, MA, USA) and Sylvie Robine (Paris,FR), respectively. Embryo dissections were performed usingroutine techniques with the morning that the post-coital plugwas observed defined as E0.5. All experiments with Alb-Crewere performed in Abcb7 f l/Y males at 6 or 8 weeks of age.All animal procedures were reviewed and approved by theAnimal Care and Use Committee, Children’s Hospital Boston.

Gene expression and phenotypic and X-inactivationanalyses

An adult mouse multitissue poly-A selected northern blot(OriGene, Rockville, MD, USA) was probed with murineAbcb7, Abcb6 and b-actin ORF probes. In situ hybridization

of murine embryos was performed as previously described(53), using a 35S-labeled riboprobe corresponding to nucleo-tides 308–691 of the murine Abcb7 cDNA (Ensembl tran-script ID ENSMUST00000033695). Serum iron parameterswere determined using the serum iron/UIBC kit from SigmaDiagnostics (Sigma-Aldrich, St Louis, MO, USA). Tissueand mitochondrial iron were measured as previously described(54). All histological and immunohistochemical analyseswere performed on routine formalin-fixed, paraffin-embeddedsections. GFP immunohistochemistry was performed aspreviously described (55). Electron microscopy was per-formed using routine osmium tetroxide treated, uranyl/leadacetate stained thin sections in the Electron Microscopy Facil-ity in the Department of Pathology, Children’s HospitalBoston.

IRE RNA binding activity

IRP1 and IRP2 RNA binding assays were determined using aquantitative RNA binding assay with rat L-ferritin RNA asdescribed previously (57). After binding of [32P]IRE toprotein, heparin was added and bound and free RNA separatedby electrophoretic mobility shift assay as described (57).Results were quantified by phosphorimaging including theuse of an RNA standard curve (58).

Liver subcellular fractionation and enzyme assays

Cytosol and mitochondria were obtained as described (56).Aconitase assays were performed at 258C for 10 min asdescribed (59). For XO activity, a kit from MolecularProbes, Inc. (A-22182, Eugene, OR, USA) was used. Ferro-chelatase (60), citrate synthase (CS) (61), SDH (62) andCCO (Sigma-Aldrich, St Louis, MO, USA) assays were per-formed as described. Protein levels were quantified using theImageQuant software supplied with the BioRad ChemiDocgel documentation system (Hercules, CA, USA).

Immunoblotting and antibodies

For determination of protein levels, either whole-tissue homo-genate or cytosol was used. The IRP1 blotting was performedas described (57). Other antibodies included rabbit anti-bovineXO (cat. no. ab6194, Abcam, Cambridge, MA, USA), Ferritin(63) and monoclonal rat anti-TfR1 (rat anti-CD71, cat. no.MCA1033G, Accurate Chemical & Scientific Corp., Westbury,NY, USA).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

This work was supported by NIH DK62474, the Pew Bio-medical Scholars Program, the Wilkes Fund of the Children’sHospital, Department of Pathology (M.D.F.), NIH RO1DK47219 (R.S.E.), L’Asssociation Francaise Contre leCancer (C.P.) and NIH training grant T32 DK07665, whichpartially supported S.L.C. Transgenic core facilities were

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provided by the Mental Retardation Research Center (MRRC)at Children’s Hospital, supported by NIH P30-HD 18655.Howard Mulhern and James Edwards of the Children’s Hospi-tal, Department of Pathology, Electron Microscopy Facility,and Histology Laboratory, respectively, provided experttechnical assistance. Ronald Parsons, Kavita Sharma andKatherine Shea are acknowledged for technical assistanceearly in the project. Members of the Fleming, Eisenstein andAndrews laboratories, particularly Lance Lee and JeremyGoforth, are acknowledged for ongoing criticism of theproject and review of the manuscript.

Conflicts of Interest statement. The authors have no conflictsof interest to declare.

REFERENCES

1. Beinert, H., Holm, R.H. and Munck, E. (1997) Iron–sulfur clusters—nature’s modular, multipurpose structures. Science, 277, 653–659.

2. Lill, R. and Muhlenhoff, U. (2005) Iron–sulfur-protein biogenesis ineukaryotes. Trends Biochem. Sci., 30, 133–141.

3. Rouault, T.A. and Tong, W.H. (2005) Iron–sulphur cluster biogenesis andmitochondrial iron homeostasis. Nat. Rev. Mol. Cell Biol., 6, 345–351.

4. Kispal, G., Csere, P., Prohl, C. and Lill, R. (1999) The mitochondrialproteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/Sproteins. EMBO J., 18, 3981–3989.

5. Leighton, J. and Schatz, G. (1995) An ABC transporter in themitochondrial inner membrane is required for normal growth of yeast.EMBO J., 14, 188–195.

6. Kispal, G., Sipos, K., Lange, H., Fekete, Z., Bedekovics, T., Janaky, T.,Bassler, J., Netz-Aguilar, D.J., Balk, J., Rotte, C. et al. (2005) Biogenesisof cytosolic ribosomes requires the essential iron–sulphur protein Rli1pand mitochondria. EMBO J., 24, 589–598.

7. Yarunin, A., Pause, V.G., Petfalski, E., Dez, C., Tollervey, D. andHurt, E.C. (2005) Functional link between ribosome formation andbiogenesis of iron–sulfur proteins. EMBO J., 24, 580–588.

8. Stehling, O., Elasser, H.P., Bruckel, B., Muhlenhoff, U. and Lill, R.(2004) Iron–sulfur protein maturation in human cells: evidence for afunction of frataxin. Hum. Mol. Genet., 13, 3007–3015.

9. Seznec, H., Simon, D., Bouton, C., Reutenauer, L., Hertzog, A., Golik, P.,Procaccio, V., Patel, M., Drapier, J.C., Koenig, M. et al. (2005) Friedreichataxia: the oxidative stress paradox. Hum. Mol. Genet., 14, 463–474.

10. Lange, H., Muhlenhoff, U., Denzel, M., Kispal, G. and Lill, R. (2004) Theheme synthesis defect of mutants impaired in mitochondrial iron–sulfurprotein biogenesis is caused by reversible inhibition of ferrochelatase.J. Biol. Chem., 279, 29101–29108.

11. Chen, O., Crisp, R.J., Valachovic,M., Bard,M.,Winge, D.R. and Kaplan, J.(2004) Transcription of the yeast iron regulon does not directly respond toiron but rather to iron–sulfur cluster biosynthesis. J. Biol. Chem., 279,29513–29518.

12. Li, J., Kogan, M., Knight, S.A.B., Pain, D. and Dancis, A. (1999) Yeastmitochondrial protein, Nfs1p, coordinately regulates iron–sulfur clusterproteins, cellular iron uptake, and iron distribution. J. Biol. Chem., 274,33025–33034.

13. Rutherford, J.C., Ojeda, L., Balk, J., Muhlenhoff, U., Lill, R. and Winge,D.R. (2005) Activation of the iron regulon by yeast Aft1/Aft2transcription factors depends on mitochondria but not cytosolic iron–sulfur protein biogenesis. J. Biol. Chem., 280, 10135–10140.

14. Eisenstein, R.S. (2000) Iron regulatory proteins and the molecular controlof mammalian iron metabolism. Annu. Rev. Nutr., 20, 627–662.

15. Hentze, M.W., Muckenthaler, M.U. and Andrews, N.C. (2004) Balancingacts: molecular control of mammalian iron metabolism. Cell, 117,285–297.

16. Allikmets, R., Raskind, W.H., Hutchinson, A., Schueck, N.D., Dean, M.and Koeller, D.M. (1999) Mutation of a putative mitochondrial irontransporter gene (ABC7) in X-linked sideroblastic anemia and ataxia(XLSA/A). Hum. Mol. Genet., 8, 743–749.

17. Csere, P., Lill, R. and Kispal, G. (1998) Identification of a humanmitochondrial ABC transporter, the functional orthologue of yeast Atm1p.FEBS Lett., 441, 266–270.

18. Kispal, G., Csere, P., Guiard, B. and Lill, R. (1997) The ABC transporterAtm1P is required for mitochondrial iron homeostasis. FEBS Lett., 418,426–436.

19. Senbongi, H., Ling, F. and Shibata, T. (1999) A mutation in amitochondrial ABC transporter results in mitochondrial dysfunctionthrough oxidative damage of mitochondrial DNA. Mol. Gen. Genet., 262,426–436.

20. Fleming, M.D. (2002) The genetics of inherited sideroblastic anemias.Semin. Hematol., 39, 270–281.

21. Bekri, S., Kispal, G., Lange, H., Fitzsimmons, E., Tolmie, J., Lill, R. andBishop, D.F. (2000) Human ABC7 transporter: gene structure andmutation causing X-linked sideroblastic anemia with ataxia withdisruption of cytosolic iron–sulfur protein maturation. Blood, 96,3256–3264.

22. Hellier, K.D., Hatchwell, E., Duncombe, A.S., Kew, J. andHammans, S.R. (2001) X-linked sideroblastic anaemia with ataxia:another mitochondrial disease? J. Neurol. Neurosurg. Psychiatry, 70,65–69.

23. Maguire, A., Hellier, K., Hammans, S. and May, A. (2001) X-linkedcerebellar ataxia and sideroblastic anaemia associated with a missensemutation in the Abcb7 gene predicting V411L. Brit. J. Haematol., 115,910–917.

24. Pagon, R.A., Bird, T.D., Detter, J.C. and Pierce, I. (1985) Hereditarysideroblastic anaemia and ataxia: an X-linked recessive disorder.J. Med. Genet., 22, 267–273.

25. Raskind, W.H., Wijsman, E., Pagon, R.A., Cox, T.C., Bawden, M.J., May,B.K. and Bird, T.D. (1991) X-linked sideroblastic anemia and ataxia:linkage to phosphoglycerate kinase at Xq 13. Am. J. Hum. Genet., 48,335–341.

26. Jasinski, M., Keller, P., Fujiwara, Y., Orkin, S.H. and Bessler, M. (2001)GATA1-Cre mediates Piga gene inactivation in the erythroid/megakaryocytic lineage and leads to circulating red cells and a partialdeficiency in glycosyl phosphatidylinositol-linked proteins (paroxysmalnocturnal hemoglobinuria type II cell. Blood, 98, 2248–2255.

27. Huynh, K.D. and Lee, J.T. (2003) Inheritance of a pre-inactivated paternalX chromosome in early mouse embryos. Nature, 426, 857–862.

28. Hayashi, S., Lewis, P., Pevny, L. and McMahon, A.P. (2002) Efficientgene modulation in mouse epiblast using a Sox2Cre transgenic mousestrain. Mech. Dev., 119, S97–S101.

29. Donovan, A., Lima, C.A., Pinkus, J.L., Pinkus, G.S., Zon, L.I., Robine, S.and Andrews, N.C. (2005) The iron exporter ferroportin/Slc40a1 isessential for iron homeostasis. Cell Metab., 1, 191–200.

30. El-Marjou, F., Janssen, K.P., Chang, B.H., Li, M., Hindie, V., Chan, L.,Louvard, D., Chambon, P., Metzger, D. and Robine, S. (2004)Tissue-specific and inducible Cre-mediate recombination in the gutepithelium. Genesis, 39, 186–193.

31. Kuhn, R., Schwenk, F., Aguet, M. and Rajewsky, K. (1995) Induciblegene targeting in mice. Science, 269, 1427–1429.

32. Tronche, F., Kellendonk, C., Kretz, O., Gass, P., Anlag, K., Orban, P.C.,Bock, R., Klein, R. and Schutz, G. (1999) Disruption of the glucocorticoidreceptor gene in the nervous system results in reduced anxiety. NatureGenet., 23, 99–103.

33. Hadjantonakis, A.K., Cox, L.L., Tam, P.P. and Nagy, A. (2001) AX-linked GFP transgene reveals unexpected paternal X-chromosomeactivity in trophoblast giant cells of mouse placenta. Genesis, 29,133–140.

34. Postic, C. and Magnuson, M.A. (2000) DNA excision in liver by analbumin-Cre transgene occurs progressively with age. Genesis, 26,149–150.

35. Rustin, P., Chretien, D., Bourgeron, T., Gerard, B., Rotig, A.,Saudubray, J.M. and Munnich, A. (1994) Biochemical and molecularinvestigations in respiratory chain deficiencies. Clin. Chim. Acta, 228,35–51.

36. Masini, A., Salvioli, G., Cremonesi, P., Botti, B., Gallesi, D. andCeccarelli, D. (1994) Dietary iron deficiency in the rat. I. Abnormalities inenergy metabolism of the hepatic tissue. Biochim. Biophys. Acta, 1188,46–52.

37. Hanzelmann, P., Hernandez, H.K., Menzel, C., Garcia-Serres, R., Huynh,B.H., Johnson, M.K., Mendel, R.R. and Schindelin, H. (2004)Characterization of MOCS1A, an oxygen-sensitive iron–sulfur proteininvolved in human molybdenum cofactor biosynthesis. J. Biol. Chem.,279, 34721–34732.

Human Molecular Genetics, 2006, Vol. 15, No. 6 963

by guest on June 6, 2013http://hm

g.oxfordjournals.org/D

ownloaded from

38. Clarke, S.L., Vasanthakumar, A., Anderson, S.A., Pondarre, C., Koh,C.M., Deck, K.M., Pitula, J.S., Epstein, C.J., Fleming, M.D. andEisenstein, R.S. (2005) Iron-responsive degradation of iron regulatoryprotein 1 does not require the Fe–S cluster. EMBO J., 25, 544–553.

39. Ishikawa, H., Kato, M., Hori, H., Ishimori, K., Kirisako, T., Tokunaga, F.and Iwai, K. (2005) Involvement of the heme regulatory motif inheme-mediated ubiquitination and degradation of IRP2. Mol. Cell, 19,171–181.

40. He, J., Navarrete, S., Jasinski, M., Vulliamy, T., Dokal, I., Bessler, M. andMason, P.J. (2002) Targeted disruption of Dkc1, the gene mutated inX-linked dyskeratosis congenita, causes embryonic lethality in mice.Oncogene, 21, 7740–7744.

41. Longo, L., Venegas, O.C., Patel, M., Rosti, V., Li, H., Waka, J.,Merghoub, T., Pandolfi, P.P., Notaro, R., Manova, K. et al. (2002)Maternally transmitted severe glucose 6-phosphate dehydrogenasedeficiency is an embryonic lethal. EMBO J., 21, 4229–4239.

42. Lill, R. and Kispal, G. (2001) Mitochondrial ABC transporters.Res. Microbiol., 152, 331–340.

43. Emadi-Konjin, H.P., Zhang, H., Anandan, V., Sun, D., Schuetz, J. andFuruya, K.N. (2002) Isolation of a genomic clone containing the promoterregion of the human ATP binding cassette (ABC) transporter, ABCB6.Biochim. Biophys. Acta, 1574, 117–130.

44. Hirsch-Ernst, K.L., Gaini-Rahimi, S., Ernst, B.P., Schmitz-Salue, C.,Blume, S. and Kahl, G.F. (1998) Molecular cDNA cloning and tissuedistribution of mRNA encoding a novel ATP-binding cassette (ABC)half-transporter. Biochem. Biophys. Res. Commun., 249, 151–155.

45. Mitsuhashi, N., Miki, T., Senbongi, H., Yokoi, N., Yano, H.,Miyazaki, M., Nakajima, N., Iwanaga, T., Yokoyama, Y., Shibata, T.et al. (2000) MTABC3, a novel mitochondrial ATP-binding cassetteprotein involved in iron homeostasis. J. Biol. Chem., 275, 17536–17540.

46. Puccio, H., Simon, D., Cossee, M., Filipe-Criqui, P., Tiziano, F., Melki, J.,Hindelang, C., Matyas, R., Rustin, P. and Koenig, M. (2001) Mousemodels for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defectand Fe–S enzyme deficiency followed by intramitochondrial irondeposits. Nature Genet., 27, 181–186.

47. Truty, J., Malpe, R. and Linder, M.C. (2001) Iron prevents ferritinturnover in hepatic cells. J. Biol. Chem., 276, 48775–48780.

48. White, K. and Munro, H.N. (1988) Induction of ferritin subunit synthesisby iron is regulated at both the transcriptional and translational levels.J. Biol. Chem., 263, 8938–8942.

49. Wingert, R.A., Galloway, J.L., Barut, B., Foott, H., Fraenkel, P., Axe,J.L., Weber, G.J., Dooley, K., Davidson, A.J., Schmidt, B. et al. (2005)Deficiency of glutaredoxin 5 reveals Fe–S clusters are required forvertebrate haem synthesis. Nature, 436, 1035–1039.

50. Galy, B., Ferring, D., Benesova, M., Benes, V. and Hentze, M.W. (2004)Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals

context-dependent RNA processing differences in vivo. RNA, 10,1019–1025.

51. Meyron-Holtz, E.G., Ghosh, M.C., Iwai, K., LaVaute, T., Brazzolotto, X.,Berger, U.V., Land, W., Olivarerre-Wilson, H., Grinberg, A., Love, P.et al. (2004) Genetic ablations of iron regulatory protein 1 and 2 revealwhy iron regulatory protein 2 dominates iron homeostasis. EMBO J.,23, 386–395.

52. Hogan, B., Constantini, F. and Lacy, E. (1986) Manipulating the MouseEmbryo. Cold Spring Harbor Laboratory, Cold Spring Harbor.

53. Ross, J.S., Sheehan, C.E., Fisher, H.A., Kaufman, R.P., Jr, Kaur, P.,Gray, K., Webb, I., Gray, G.S., Mosher, R. and Kallakury, B.V. (2003)Correlation of primary tumor prostate-specific membrane antigenexpression with disease recurrence in prostate cancer. Clin. Cancer Res.,9, 6357–6362.

54. Torrance, J.D. and Bothwell, T.H. (1980) Tissue iron stores. In Cook, J.D.(ed.), Methods in Hematology. Churchill Livingstone Press, New York,Vol. 1, pp. 104–109.

55. Langenau, D.M., Traver, D., Ferrando, A.A., Kutok, J.L., Aster, J.C.,Kanki, J.P., Lin, S., Prochownik, E., Trede, N.S., Zon, L.I. et al. (2003)Myc-induced T cell leukemia in transgenic zebrafish. Science, 299,887–890.

56. Blemings, K.P., Crenshaw, T.D., Swick, R.W. and Benevenga, N.J.(1994) Lysine alpha-ketoglutarate reductase and saccharopinedehydrogenase are located only in the mitochondrial matrix in rat liver.J. Nutr., 124, 1215–1224.

57. Eisenstein, R.S., Tuazon, P.T., Schalinske, K.L., Anderson, S.A. andTraugh., J.A. (1993) Iron-responsive element binding protein:phosphorylation by protein kinase C. J. Biol. Chem., 268, 27363–27370.

58. Pitula, J.S., Deck, K.M., Clarke, S.L., Anderson, S.A., Vasanthakumar, A.and Eisenstein, R.S. (2004) Selective inhibition of the citrate-to-isocitratereaction of cytosolic aconitase by phosphomimetic mutation ofserine-711. Proc. Natl Acad. Sci. USA, 101, 10907–10912.

59. Kennedy, M.C., Emptage, M.H., Dryer, J.-L. and Beinert, H. (1983) Therole of iron in the activation–inactivation of aconitase. J. Biol. Chem.,258, 11098–11105.

60. Dailey, H.A. and Fleming, J.E. (1983) Bovine ferrochelatase. Kineticanalysis of inhibition by N-methylprotoporphyrin, manganese, and heme.J. Biol. Chem., 258, 11453–11459.

61. Srere, P.A., Brazil, H. and Gonen, L. (1963) The citrate condensingenzyme of pigeon breast muscle and moth flight muscle. Acta Chem.Scand., 17, S129–S134.

62. Bregman, A. (1990) Laboratory Investigations in Cell and MolecularBiology. 3rd ed. John Wiley and Sons, Inc., New York.

63. Chen, O.S., Schalinske, K.L. and Eisenstein, R.S. (1997) Dietary ironintake modulates the activity of iron regulatory proteins (IRPs) and theabundance of ferritin and mitochondrial aconitase in rat liver. J. Nutr.,127, 238–248.

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