Regulation of heat shock transcription factors and their roles in physiology and disease.

The heat shock transcription factors (HSFs) were discovered over 30 years ago as direct transcriptional activators of genes regulated by thermal stress, encoding heat shock proteins. The accepted paradigm posited that HSFs exclusively activate the expression of protein chaperones in response to conditions that cause protein misfolding by recognizing a simple promoter binding site referred to as a heat shock element. However, we now realize that the mammalian family of HSFs comprises proteins that independently or in concert drive combinatorial gene regulation events that activate or repress transcription in different contexts. Advances in our understanding of HSF structure, post-translational modifications and the breadth of HSF-regulated target genes have revealed exciting new mechanisms that modulate HSFs and shed new light on their roles in physiology and pathology. For example, the ability of HSF1 to protect cells from proteotoxicity and cell death is impaired in neurodegenerative diseases but can be exploited by cancer cells to support their growth, survival and metastasis. These new insights into HSF structure, function and regulation should facilitate the development tof new disease therapeutics to manipulate this transcription factor family.

Interactions between copper homeostasis and the fungal cell wall affect copper stress resistance.

Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1Δ mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin / chitosan deposition and exposure. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein could be involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake.

Ironing out a midlife crisis.

There is a strong correlation between age, genomic instability, and the development of cancer. Working in yeast, Veatch et al. (2009) now propose that defects in the biogenesis of iron-sulfur clusters arising as a consequence of mitochondrial dysfunction contribute to the increase in genomic instability as cells age.

Transcription factor-driven alternative localization of Cryptococcus neoformans superoxide dismutase.

Cryptococcus neoformans is an opportunistic fungal pathogen whose pathogenic lifestyle is linked to its ability to cope with fluctuating levels of copper (Cu), an essential metal involved in multiple virulence mechanisms, within distinct host niches. During lethal cryptococcal meningitis in the brain, C. neoformans senses a Cu-deficient environment and is highly dependent on its ability to scavenge trace levels of Cu from its host and adapt to Cu scarcity to successfully colonize this niche. In this study, we demonstrate for this critical adaptation, the Cu-sensing transcription factor Cuf1 differentially regulates the expression of the SOD1 and SOD2 superoxide dismutases in novel ways. Genetic and transcriptional analysis reveals Cuf1 specifies 5'-truncations of the SOD1 and SOD2 mRNAs through specific binding to Cu responsive elements within their respective promoter regions. This results in Cuf1-dependent repression of the highly abundant SOD1 and simultaneously induces expression of two isoforms of SOD2, the canonical mitochondrial targeted isoform and a novel alternative cytosolic isoform, from a single alternative transcript produced specifically under Cu limitation. The generation of cytosolic Sod2 during Cu limitation is required to maintain cellular antioxidant defense against superoxide stress both in vitro and in vivo. Further, decoupling Cuf1 regulation of Sod2 localization compromises the ability of C. neoformans to colonize organs in murine models of cryptococcosis. Our results provide a link between transcription factor-mediated alteration of protein localization and cell proliferation under stress, which could impact tissue colonization by a fungal pathogen.

Comparative interactomes of HSF1 in stress and disease reveal a role for CTCF in HSF1-mediated gene regulation.

Heat shock transcription factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation, and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-translational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth and chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington's disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeneration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.

Copper Acquisition and Utilization in Fungi.

Fungal cells colonize and proliferate in distinct niches, from soil and plants to diverse tissues in human hosts. Consequently, fungi are challenged with the goal of obtaining nutrients while simultaneously elaborating robust regulatory mechanisms to cope with a range of availability of nutrients, from scarcity to excess. Copper is essential for life but also potentially toxic. In this review we describe the sophisticated homeostatic mechanisms by which fungi acquire, utilize, and control this biochemically versatile trace element. Fungal pathogens, which can occupy distinct host tissues that have their own intrinsic requirements for copper homeostasis, have evolved mechanisms to acquire copper to successfully colonize the host, disseminate to other tissues, and combat host copper bombardment mechanisms that would otherwise mitigate virulence.

A lytic polysaccharide monooxygenase-like protein functions in fungal copper import and meningitis.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.

Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.

Copper is required for oncogenic BRAF signalling and tumorigenesis.

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

Reconstitution of a thermophilic Cu+ importer in vitro reveals intrinsic high-affinity slow transport driving accumulation of an essential metal ion.

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu+ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu+ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu+ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu+ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag+ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu+ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu+ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu+ pools.

Genome-wide analysis of the regulation of Cu metabolism in Cryptococcus neoformans.

The ability of the human fungal pathogen Cryptococcus neoformans to adapt to variable copper (Cu) environments within the host is key for successful dissemination and colonization. During pulmonary infection, host alveolar macrophages compartmentalize Cu into the phagosome and C. neoformans Cu-detoxifying metallothioneins, MT1 and MT2, are required for survival of the pathogen. In contrast, during brain colonization the C. neoformans Cu+ importers Ctr1 and Ctr4 are required for virulence. Central for the regulation and expression of both the Cu detoxifying MT1/2 and the Cu acquisition Ctr1/4 proteins is the Cu-metalloregulatory transcription factor Cuf1, an established C. neoformans virulence factor. Due to the importance of the distinct C. neoformans Cu homeostasis mechanisms during host colonization and virulence, and to the central role of Cuf1 in regulating Cu homeostasis, we performed a combination of RNA-Seq and ChIP-Seq experiments to identify differentially transcribed genes between conditions of high and low Cu. We demonstrate that the transcriptional regulation exerted by Cuf1 is intrinsically complex and that Cuf1 also functions as a transcriptional repressor. The Cu- and Cuf1-dependent regulon in C. neoformans reveals new adaptive mechanisms for Cu homeostasis in this pathogenic fungus and identifies potential new pathogen-specific targets for therapeutic intervention in fungal infections.

Gene duplication and neo-functionalization in the evolutionary and functional divergence of the metazoan copper transporters Ctr1 and Ctr2.

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu+ across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu+ transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu+ transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1-/- mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins.

Rational design and screening of peptide-based inhibitors of heat shock factor 1 (HSF1).

Heat shock factor 1 (HSF1) is a stress-responsive transcription factor that regulates expression of protein chaperones and cell survival factors. In cancer, HSF1 plays a unique role, hijacking the normal stress response to drive a cancer-specific transcriptional program. These observations suggest that HSF1 inhibitors could be promising therapeutics. However, HSF1 is activated through a complex mechanism, which involves release of a negative regulatory domain, leucine zipper 4 (LZ4), from a masked oligomerization domain (LZ1-3), and subsequent binding of the oligomer to heat shock elements (HSEs) in HSF1-responsive genes. Recent crystal structures have suggested that HSF1 oligomers are held together by extensive, buried contact surfaces, making it unclear whether there are any possible binding sites for inhibitors. Here, we have rationally designed a series of peptide-based molecules based on the LZ4 and LZ1-3 motifs. Using a plate-based, fluorescence polarization (FP) assay, we identified a minimal region of LZ4 that suppresses binding of HSF1 to the HSE. Using this information, we converted this peptide into a tracer and used it to understand how binding of LZ4 to LZ1-3 suppresses HSF1 activation. Together, these results suggest a previously unexplored avenue in the development of HSF1 inhibitors. Furthermore, the findings highlight how native interactions can inspire the design of inhibitors for even the most challenging protein-protein interactions (PPIs).

Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain.

Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu(+) transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu(+) transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu(+) and cisplatin acquisition.

Characterization of a novel murine model for spontaneous hemorrhagic stroke using in vivo PET and MR multiparametric imaging.

The clinical use of Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) has proven to be a strong diagnostic tool in the field of neurology. The reliability of these methods to confirm clinical diagnoses has guided preclinical research to utilize these techniques for the characterization of animal disease models. Previously, we demonstrated that an endothelial cell-specific ablation of the murine Serum Response Factor (SrfiECKO) results in blood brain barrier (BBB) breakdown and hemorrhagic stroke. Taking advantage of this mouse model we here perform a comprehensive longitudinal, multiparametric and in vivo imaging approach to reveal pathophysiological processes occurring before and during the appearance of cerebral microbleeds using combined PET and MRI. We complement our imaging results with data regarding animal behavior and immunohistochemistry. Our results demonstrate diffusion abnormalities in the cortical brain tissue prior to the onset of cerebral microbleeds. Diffusion reductions were accompanied by significant increments of [18F]FAZA uptake before the onset of the lesions in T2WI. The Open Field behavioral tests revealed reduced activity of SrfiECKO animals, whereas histology confirmed the presence of hemorrhages in cortical regions of the mouse brain and iron deposition at lesion sites with increased hypoxia inducible factor 1α, CD31 and glial fibrillary acidic protein expression. For the first time, we performed a thorough evaluation of the prodromal period before the occurrence of spontaneous cerebral microbleeds. Using in vivo PET and MRI, we show the pathological tissue changes that occur previous to gross blood brain barrier (BBB) disruption and breakage. In addition, our results show that apparent diffusion coefficient (ADC) reduction may be an early biomarker of BBB disruption proposing an alternate clinical interpretation. Furthermore, our findings remark the usefulness of this novel SrfiECKO mouse model to study underlying mechanisms of hemorrhagic stroke.

Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

Copper at the Fungal Pathogen-Host Axis.

Fungal infections are responsible for millions of human deaths annually. Copper, an essential but toxic trace element, plays an important role at the host-pathogen axis during infection. In this review, we describe how the host uses either Cu compartmentalization within innate immune cells or Cu sequestration in other infected host niches such as in the brain to combat fungal infections. We explore Cu toxicity mechanisms and the Cu homeostasis machinery that fungal pathogens bring into play to succeed in establishing an infection. Finally, we address recent approaches that manipulate Cu-dependent processes at the host-pathogen axis for antifungal drug development.

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain.

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2(-/-) mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.

Genomic heat shock element sequences drive cooperative human heat shock factor 1 DNA binding and selectivity.

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer.

Intestinal DMT1 is critical for iron absorption in the mouse but is not required for the absorption of copper or manganese.

Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc.