See the embryonic expression pattern of Fer1HCH at FlyExpress.
Insect ferritins have subunits homologous to the heavy and light chains of vertebrate ferritins. Cloning and sequence of the heavy chain homologue (HCH) of Drosophila ferritin subunit has been reported earlier. When Northern blots of D. melanogaster RNA were probed with a cDNA for this HCH, three bands were observed. It was shown that these represented at least four classes of mRNA of various lengths. The polymorphism results from alternative splicing of an intron in the 5' untranslated region (UTR) that contains the iron-responsive element (IRE) and from two alternative polyadenylation sites in the 3' UTR. By hybridizing Northern blots with specific probes, it has been shown that the relative proportions of the messages vary with the life stage and especially with iron supplementation of the diet. Iron significantly increases the amount of ferritin HCH messages and dramatically shifts the balance toward those messages that lack an IRE and/or have a short 3' UTR. In the larvae this change takes place in the gut , but not in the fat body. It is speculated that this dramatic shift in message distribution may result from an effect of iron on the rate of transcription or message degradation, or from an effect on the splicing process itself. Synthesis of ferritin HCH subunit mRNAs that lack an IRE may be important under conditions of iron overload (Georgieva, 1999).
Drosophila melanogaster secreted ferritin, like the cytosolic ferritins of other organisms, is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). The cloning of a cDNA encoding the ferritin LCH is repored. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues (Georgieva, 2002).
The genomic proximity between Fer1HCH and Fer2LCH could facilitate similar expression patterns to coordinate biosynthesis of the ferritin heteropolymer. In situ hybridizations of anti-sense RNA probes against the two genes in whole mount preparations of embryos at different stages of development were performed. It was reasoned that if the predominant sites of Fer1HCH and Fer2LCH expression were different, it would be unlikely that they shared common regulatory enhancers, as previously suggested (Dunkov, 1999; Dunkov, 2006; Missirlis, 2007).
Nevertheless, it was found that the different transcripts are expressed in highly specific, yet similar, patterns during embryogenesis. Fer1HCH and Fer2LCH were expressed during oogenesis and the maternal transcripts became evenly distributed in the egg and early embryo, where they were detected in the blastoderm. Tissue-specific transcripts were first detected during germ band elongation in cells of the mesoderm, which are specified to give rise to the fat bodies and amnioserosa. Staining was also seen in cells destined to become macrophages in the anterior head region of embryos. During germ-band retraction and dorsal closure, the amnioserosa retained ferritin mRNAs. At late stages of embryogenesis, cells in the developing midgut initiate ferritin transcription. The similar embryonic expression patterns of Fer1HCH and Fer2LCH suggest that both ferritin subunits are expressed in each cell type where ferritin is required (Missirlis, 2007).
Ferritin is abundant in the midguts from several different insect species and its expression is induced by dietary iron (Capurro Mde, 1996; Dunkov, 2002; Georgieva, 2002; Kim, 2002). It has been shown that GFP-tagged ferritin stores iron in Fer2LCHG188/+ flies. The expression pattern of GFPtagged ferritin was determined to see if is similar to that of the endogenous non-tagged protein. Indeed, GFPtagged ferritin is most prominently expressed in a cluster of cells of the middle midgut. These cells have been identified as the iron region of the insect midgut on the basis of their positive stain with Prussian blue and the accumulation of exogenously administered radioactive iron. Mid-third instar larvae were administered a diet containing 5mM ferric ammonium citrate and their intestines were dissected for imaging. GFP-tagged ferritin was inducible only in cells of the anterior midgut, but was constitutively expressed in the iron-region of the middle midgut and no expression was detected in the copper cells that are present in-between the two regions (Missirlis, 2007).
Midguts from larvae subjected to the same treatment were also stained with Prussian blue. A light blue staining indicative of the presence of ferric iron was observed in the iron region of both iron-fed and control larvae and was largely unchanged by feeding on iron-enriched diet. In contrast, the anterior region was not stained in control larvae, but stained dark blue in iron-fed individuals, indicating that the ferritin that accumulates in the anterior midgut upon iron feeding is rich in iron content (Missirlis, 2007).
To assess the time frame of ferritin induction during iron feeding and to further demonstrate that a specific subset of cells in the anterior midgut shows a strong response to iron levels, endogenous ferritin levels were determined in a time course following iron feeding. For this experiment wild type third instar larvae (100 hours old at 25ƒC) were used, and their anterior and middle midguts were dissected for Western Blot analysis. The results showed a clear induction of the ferritin heteropolymers three hours post-feeding in the anterior midgut. Consistent with imaging and iron-staining results, the induction of ferritin was largely restricted to the anterior midgut and was much less pronounced in the iron region (Missirlis, 2007).
Another essential transition metal and dietary nutrient for Drosophila is copper. Copper-containing (cuprophilic) cells function in the acidification of the midgut and are present at different sites than the iron region. Coppermetallothionein complexes in these cells fluoresce orange-red upon ultraviolet illumination. The coppermetallothionein fluorescence and GFP-Ferritin were imaged simultaneously in midguts from larvae fed a diet containing 1mM Cu2+. The results showed that ferritin-expressing cells indeed form a distinct cellular population from cells that contain copper (Missirlis, 2007).
To investigate which cellular compartment accumulates iron-loaded ferritin in midgut cells, GFP-Fer1HCH expressing cells were co-stained with an antibody raised against the Golgi-associated protein Lava lamp. Confocal images from a single cell in the iron region clearly showed that all GFP-ferritin localized within the Golgi compartment, consistent with the images obtained by electron microscopy (Locke, 1984; Nichol, 1990). A few Golgi bodies in cells from the iron region were devoid of ferritin. The same cells were also imaged stained with antibodies against the Fer2LCH subunits. As expected from their tight association revealed by structural studies, biochemical analysis and electron microscopy, there was complete co-localization with GFP-Fer1HCH in the Golgi complex of these specialized cells. Finally, focus was placed on the cells of the anterior midgut that do not normally express ferritin, but potently do so in the presence of high iron levels. The cells were stained for both ferritin chains during the time course of ferritin induction. Prior to iron feeding, GFP-Fer1HCH was not expressed and low levels of Fer2LCH were detected. At one hour post-induction GFP-Fer1HCH was detected in a compartment resembling the endoplasmic reticulum, but Fer2LCH-positive Golgi that were devoid of GFP-Fer1HCH subunits could not be detected. In contrast, four hours post-induction the two subunits were strongly induced and were seen only in complex with one another within the Golgi. Collectively, these results have identified different specialized intestinal sites for iron and copper metabolism and shown that ferritin synthesis is differentially regulated along the intestine. Significantly, the results also validate the use of the Fer2LCHG188 line as a faithful reporter of endogenous ferritin expression (Missirlis, 2007).
Two types of mutations in the IRE of the human L-ferritin gene cause hereditary hyperferritinemia cataract syndrome: mutations that disrupt base pairing of the stem loop and mutations that directly affect the specific CAGUG sequence of the loop and the cytosine of the IRE bulge (Cazzola 2002; Rouault 2006). The mutations that cause disease are detrimental to the defining features of IREs that are conserved from humans to flies. A survey of many sequenced Drosophila species confirmed that Fer2LCH mRNAs lack IREs, which are present only in the 5íUTR of Fer1HCH transcripts. Comparison of the new IREs identified several non-conserved nucleotides between species, but in all cases the overall structure of the stem loop remained intact and the nucleotides implicated in disease are conserved or altered in ways that are compatible with base pairing. Considering the finding that Fer2LCH mRNA lacks the IRE and is not predictably regulated by IRP-1A, it was asked whether other post-transcriptional control mechanisms govern Fer2LCH expression (Missirlis, 2007).
In order to study post-transcriptional regulation of Fer1HCH and Fer2LCH in Drosophila, flies were used with P-element insertions in these genes. Focus was placed on three P-elements: Fer1HCH451 and Fer2LCH35, which result in a loss-of-function mutant for each of the respective ferritin chains, and Fer2LCHG188, which adds GFP to the H-subunit. Fer2LCH35 is predicted to cause a genetic null mutation because the transposable element disrupts the open reading frame, whereas in Fer1HCH451 the P-element resides in intronic sequences and the mechanism by which it disrupts gene function is unknown. When homozygous, these insertions are embryonic lethal. Each insertion present over a deficiency chromosome lacking both genes also resulted in embryonic lethality, indicating that both ferritin subunit chains perform essential functions in Drosophila (Missirlis, 2007).
To investigate possible homeostatic interactions between the two proteins, their expression in heterozygous adult flies was examined by Western blot analysis. As expected, Fer1HCH levels were markedly reduced in Fer1HCH451/+ heterozygotes and the same was true for Fer2LCH levels in Fer2LCH35/+ flies. Unexpectedly, endogenous levels of Fer2LCH were also low in Fer1HCH451/+, and Fer1HCH levels were low in Fer2LCH35/+ flies. Because trans-heterozygous (Fer1HCH451/Fer2LCH35) flies are fully viable, it was hypothesized that neither of these insertions would directly affect the transcriptional activity of both genes simultaneously (Missirlis, 2007).
To address the potential role of the P-element insertions on transcription at the locus, Fer1HCH was ubiquitously overexpressed in Fer1HCH451/451 homozygous flies and adult viability was restored. When Fer1HCH was overexpressed in homozygous Fer2LCH35/35 flies, viability was not restored. The converse was true for overexpression of Fer2LCH. These results indicate that the respective P-elements interfere specifically with Fer1HCH and Fer2LCH expression and that reduction in levels of the alternate chain in each mutant is most likely due to post-transcriptional regulation (Missirlis, 2007).
Flies were generated that express Fer1HCH with a mutation that inactivates the ferroxidase activity, based on analogy to the ferroxidase-null human allele (Cozzi, 2000). Expression of the mutated transgene was not able to functionally substitute for Fer1HCH and rescue the lethality of Fer1HCH451/451 flies. Thus, the ferroxidase activity of Fer1HCH provides an essential function in vivo and is likely required for iron loading of the ferritin shell (Missirlis, 2007).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Ferritin
Barnes, C. M., Theil, E. C. and Raymond, K. N. (2002). Iron uptake in ferritin is blocked by binding of [Cr(TREN)(H(2)O)(OH)](2+), a slow dissociating model for [Fe(H(2)O)(6)](2+). Proc. Natl. Acad. Sci. 99(8): 5195-200. Medline abstract: 11959967
Beutler, E., et al. (2002). Relationship of body iron stores to levels of serum ferritin, serum iron, unsaturated iron binding capacity and transferrin saturation in patients with iron storage disease. Acta Haematol 107: 145-149. Medline abstract: 11978935
Capurro Mde, L., et al. (1996). Musca domestica hemolymph ferritin. Arch. Insect Biochem Physiol. 32: 197-207. Medline abstract: 8785419
Cazzola, M. (2002). Hereditary hyperferritinaemia-cataract syndrome. Best Pract. Res. Clin. Haematol. 15: 385-398. Medline abstract: 12401313
Charlesworth, A., et al. (1997). Isolation and properties of Drosophila melanogaster ferritin--molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome. Eur. J. Biochem. 247: 470-475. Medline abstract: 9266686
Cooperman, S. S., et al. (2005) Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2. Blood 106: 1084-1091. Medline abstract: 15831703
Cozzi, A., et al. (2000). Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity. J. Biol. Chem. 275: 25122-25129. Medline abstract: 10833524
Cozzi, A., et al. (2004). Analysis of the biologic functions of H- and L-ferritins in HeLa cells by transfection with siRNAs and cDNAs: evidence for a proliferative role of L-ferritin. Blood 103(6): 2377-83. Epub 2003 Nov 13. Medline abstract: 14615379
Dunkov, B. C. and Georgieva, T. (1999). Organization of the ferritin genes in Drosophila melanogaster. DNA Cell Biol. 18(12): 937-44. Medline abstract: 10619605
Dunkov, B. C., Georgieva, T., Yoshiga, T., Hall, M. and Law, J. H. (2002). Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance. J. Insect Sci. 2: 7. Medline abstract: 15455041
Dunkov, B., and Georgieva, T. (2006). Insect iron binding proteins: insights from the genomes. Insect Biochem. Mol. Biol. 36: 300-309. Medline abstract: 16551544
Ferreira, C., et al. (2001). H ferritin knockout mice: a model of hyperferritinemia in the absence of iron overload. Blood 98(3): 525-32. Medline abstract: 11468145
Galy, B., et al. (2005). Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2). Blood 106: 2580-2589. Medline abstract: 15956281
Galy, B., et al. (2006). Iron homeostasis in the brain: complete iron regulatory protein 2 deficiency without symptomatic neurodegeneration in the mouse. Nat. Genet. 38: 967-969. Medline abstract: 16940998
Georgieva, T., Dunkov, B. C., Harizanova, N., Ralchev, K. and Law, J. H. (1999). Iron availability dramatically alters the distribution of ferritin subunit messages in Drosophila melanogaster. Proc. Natl. Acad. Sci. 96(6): 2716-21. Medline abstract: 10077577 [PubMed - indexed for MEDLINE]
Georgieva, T., Dunkov, B. C., Dimov, S., Ralchev, K. and Law, J. H. (2002). Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types. Insect Biochem. Mol. Biol. 32(3): 295-302. Medline abstract: 11804801
Ghosh, S., Hevi, S. and Chuck, S. L. (2004). Regulated secretion of glycosylated human ferritin from hepatocytes. Blood 103: 2369-2376. Medline abstract: 14615366
Goralska, M., Holley, B. L. and McGahan, M. C. (2003). Identification of a mechanism by which lens epithelial cells limit accumulation of overexpressed ferritin H-chain. J. Biol. Chem. 278(44): 42920-6. Medline abstract: 12920121
Gray, N. K., Pantopoulos, K., Dandekar, T., Ackrell, B. A. and Hentze, M. W. (1996). Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements. Proc. Natl. Acad. Sci. 93(10): 4925-30. Medline abstract: 8643505
Hamburger, A. E., et al. (2005). Crystal structure of a secreted insect ferritin reveals a symmetrical arrangement of heavy and light chains. J. Mol. Biol. 349: 558-569. Medline abstract: 15896348
Infante, A. A., et al. (2007). Ferritin associates with marginal band microtubules. Exp. Cell Res. 313(8): 1602-14. Medline abstract: 17391669
Ingrassia, R., Gerardi, G., Biasiotto, G. and Arosio, P. (2006). Mutations of ferritin H chain C-terminus produced by nucleotide insertions have altered stability and functional properties. J. Biochem. (Tokyo). 139(5): 881-5. Medline abstract:
Iwasaki, K., et al. (2006). Hemin-mediated regulation of an antioxidant-responsive element of the human ferritin H gene and role of Ref-1 during erythroid differentiation of K562 cells. Mol. Cell. Biol. 26(7):2845-56. Medline abstract: 16537925
Iwasaki, K., Hailemariam, K. and Tsuji, Y. (2007).
Protein inhibitor of activated STAT3 (PIAS3) interacts with activating transcription factor 1 (ATF1) and regulates the human ferritin H gene through an antioxidant responsive element. J. Biol. Chem. 282(31): 22335-43. Medline abstract: 17565989
Kaur, D., et al. (2003). Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson's disease. Neuron 37: 899-909. Medline abstract: 12670420
Kaur, D., et al. (2006). Chronic ferritin expression within murine dopaminergic midbrain neurons results in a progressive age-related neurodegeneration. Brain Res. 1140: 188-94. Medline abstract: 16631136
Kim, B. S., et al. (2002). Cloning and expression of 32
kDa ferritin from Galleria mellonella. Arch Insect Biochem Physiol 51: 80-90. Medline abstract: 12232875
Lavaute, T., et al. (2001). Targeted deletion of the
gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and
neurodegenerative disease in mice. Nat Genet 27: 209-214. Medline abstract: 11175792
Levi, S., Cozzi, A. and Arosio, P. (2005). Neuroferritinopathy: a neurodegenerative disorder associated with L-ferritin mutation. Best Pract. Res. Clin. Haematol. 18: 265-276. Medline abstract: 15737889
Lind, M. I., Ekengren, S., Melefors, O. and Soderhall, K. (1998).
Drosophila ferritin mRNA: alternative RNA splicing regulates the presence of the iron-responsive element. FEBS Lett. 436(3): 476-82. Medline abstract: 9801172
Lind, M. I., Missirlis, F., Melefors, O., Uhrigshardt, H. Kirby, K., Phillips, J. P., Soderhall, K. and Rouault, T. A. (2006). Of two
cytosolic Aconitases expressed in Drosophila, only one functions as an iron-regulatory
protein. J. Biol. Chem. 281: 18707-18714. Medline abstract: 16679315
Missirlis, F., et al. (2003) Compartment-specificprotection of iron-sulfur proteins by superoxide dismutase. J. Biol. Chem. 278: 47365-47369. Medline abstract: 12972424
Missirlis, F., Holmberg, S., Georgieva, T., Dunkov, B. C., Rouault, T. A. and Law, J. H. (2006). Characterization of mitochondrial ferritin in Drosophila.
Proc. Natl. Acad. Sci. 103(15): 5893-8. Medline abstract: 16571656
Missirlis, F., Kosmidis, S., Brody, T., Mavrakis, M., Holmberg, S., Odenwald, W. F., Skoulakis, E. M. and Rouault, T. A. (2007). Homeostatic mechanisms for iron storage revealed by genetic manipulations and live imaging of Drosophila ferritin.
Genetics [Epub ahead of print]. Medline abstract: 17603097
Muckenthaler, M., et al. (1998). Ironregulatory protein-1 (IRP-1) is highly conserved in two invertebrate species--characterization of IRP-1 homologues in Drosophila melanogaster and Caenorhabditis elegans. Eur. J. Biochem. 254: 230-237. Medline abstract: 9660175
Nichol, H. and Law, J. H. (1990). The localization of ferritin in insects. Tissue Cell 22: 767-777.
Odenwald, W. F., Rasband, W., Kuzin, A. and Brody, T. (2005). EVOPRINTER, a
multigenomic comparative tool for rapid identification of functionally important DNA.
Proc. Natl. Acad. Sci. 102: 14700-14705. Medline abstract: 16203978
Pantopoulos, K. (2004). Iron metabolism and the IRE/IRP regulatory system: an update. Annals of the New York Academy of Sciences 1012: 1-13. Medline abstract: 15105251
Pham, C. G., et al. (2004). Ferritin heavy chain
upregulation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing reactive
oxygen species. Cell 119: 529-542. Medline abstract: 15537542
Pietsch, E. C., Chan, J. Y., Torti, F. M. and Torti, S. V. (2003). Nrf2 mediates the induction of ferritin H in response to xenobiotics and cancer chemopreventive dithiolethiones. J. Biol. Chem. 278(4): 2361-9. Medline abstract: 12435735
Renaud, D. L., Nichol, H. and Locke, M. (1991). The visualization of apoferritin in the secretory pathway of vertebrate liver cells. J. Submicrosc. Cytol. Pathol. 23: 501-507. Medline abstract: 1764677
Rothenberger, S., Mullner, E. W. and Kuhn, L. C. (1990). The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution. Nucleic Acids Res. 18: 1175-1179. Medline abstract: 2157191
Rouault, T. A. (2006). The role of iron regulatory proteins in mammalian iron homeostasis and disease. Nat Chem Biol 2: 406-414. Medline abstract: 16850017
Smith, S. R., et al. (2004). Severity of neurodegeneration correlates with compromise of iron metabolism in mice with iron regulatory protein deficiencies. Ann. N. Y. Acad. Sci. 1012: 65-83. Medline abstract: 1510525
Thompson, K., et al. (2003). Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress. J Neurosci Res 71: 46-63. Medline abstract: 12478613
Thomson, A. M., et al. (2005). The acute box cis-element in human heavy ferritin mRNA 5'-untranslated region is a unique translation enhancer that binds poly(C)-binding proteins. J. Biol. Chem. 280(34): 30032-45. Medline abstract: 15967798
Torti, F. M. and Torti, S. V. (2002). Regulation of ferritin genes and protein. Blood 99: 3505-3516. Medline abstract: 11986201
Tran, T. N., et al. (1997). Secretion of ferritin by rat hepatoma cells and its regulation by inflammatory cytokines and iron.
Blood 90: 4979-4986. Medline abstract: 9389717
Tsuji, Y., Moran, E., Torti, S. V. and Torti, F. M. (1999). Transcriptional regulation of the mouse ferritin H gene. Involvement of p300/CBP adaptor proteins in FER-1 enhancer activity. J. Biol. Chem. 274(11): 7501-7. Medline abstract: 10066817
Tsuji, Y. (2005). JunD activates transcription of the human ferritin H gene through an antioxidant response element during oxidative stress.
Oncogene 24(51): 7567-78. Medline abstract: 16007120
Walden, W. E., et al. (2006). Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA. Science 314(5807): 1903-8. Medline abstract: 17185597
Wilkinson, J. T., et al. (2006). Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo. Biochem J. 395: 501-507. Medline abstract: 16448386
Yepiskoposyan, H., Egli, D., Fergestad, T., Selvaraj, A., Treiber, C., Multhaup, G., Georgiev, O. and Schaffner, W. (2006). Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc.
Nucleic Acids Res. 34(17): 4866-77. 16973896
date revised: 24 July 2007
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.