SUMMARY Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis is the homologue of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of the murine homologue of EntA results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.
Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.DOI: http://dx.doi.org/10.7554/eLife.09406.001
SUMMARY Nuclear pore complexes (NPCs) are multiprotein channels connecting the nucleus with the cytoplasm. NPCs have been shown to have tissue-specific composition, suggesting that their function can be specialized. However, the physiological roles of NPC composition changes and their impacts on cellular processes remain unclear. Here we show that the addition of the Nup210 nucleoporin to NPCs during myoblast differentiation results in assembly of an Mef2C transcriptional complex required for efficient expression of muscle structural genes and microRNAs. We show that this NPC-localized complex is essential for muscle growth, myofiber maturation, and muscle cell survival and that alterations in its activity result in muscle degeneration. Our findings suggest that NPCs regulate the activity of functional gene groups by acting as scaffolds that promote the local assembly of tissue-specific transcription complexes and show how nuclear pore composition changes can be exploited to regulate gene expression at the nuclear periphery.
TATA-box-binding protein (TBP)-related factor 3, TRF3 (also called TBP2), is a vertebrate-specific member of the TBP family that has a conserved C-terminal region and DNA binding domain virtually identical to that of TBP 1 . TRF3 is highly expressed during embryonic development, and studies in zebrafish and Xenopus have shown that TRF3 is required for normal embryogenesis 2,3 . Here we show that Trf3-depleted zebrafish embryos exhibit multiple developmental defects and, in particular, fail to undergo hematopoiesis. Expression profiling for Trf3-dependent genes identified mespa, which encodes a transcription factor whose murine orthologue is required for mesoderm specification 4 , and chromatin immunoprecipitation verified that Trf3 binds to the mespa promoter. Depletion of Mespa resulted in developmental and hematopoietic defects strikingly similar to those induced by Trf3 depletion. Injection of mespa mRNA restored normal development to a Trf3-depleted embryo, indicating mespa is the single Trf3 target gene required for zebrafish embryogenesis. Zebrafish embryos depleted of Trf3 or Mespa also failed to express cdx4, a caudalrelated gene required for hematopoiesis. Mespa binds to the cdx4 promoter, and epistasis analysis revealed an ordered trf3-mespa-cdx4 pathway. Thus, in zebrafish commitment of mesoderm to the hematopoietic lineage occurs through a transcription factor pathway initiated by a TBP-related factor.To analyze the role of TRF3 during embryonic development, we used antisense morpholino oligonucleotides (MOs) to ablate Trf3, and as a control Tbp, function in zebrafish embryos. MOs were injected into wild-type one-cell stage fertilized embryos and depletion of Trf3 and Tbp was analyzed by immunoblotting at 6 hours post-fertilization (hpf), a time at which expression of both proteins was readily detectable (Supplementary Fig. 1a). Immunoblot analysis confirmed that injection of each MO efficiently and specifically depleted its target gene ( Supplementary Fig. 1b). Consistent with previous studies 5 , Tbp-depleted embryos appeared to initiate gastrulation but failed to progress past 50% epiboly (Supplementary Fig. 2; n=122/150). By contrast, Trf3-depleted embryos appeared to develop normally until the tailbud stage, but by 14 hpf exhibited delayed development and necrosis compared to siblings injected with a randomized control MO (n=166/177). Inspection of Trf3-depleted embryos at
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