Haems are metalloporphyrins that serve as prosthetic groups for various biological processes including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control, metabolic reprogramming and microRNA processing [1][2][3][4] . With a few exceptions, haem is synthesized by a multistep biosynthetic pathway comprising defined intermediates that are highly conserved throughout evolution 5 . Despite our extensive knowledge of haem biosynthesis and degradation, the cellular pathways and molecules that mediate intracellular haem trafficking are unknown. The experimental setback in identifying haem trafficking pathways has been the inability to dissociate the highly regulated cellular synthesis and degradation of haem from intracellular trafficking events 6 . Caenorhabditis elegans and related helminths are natural haem auxotrophs that acquire environmental haem for incorporation into haemoproteins, which have vertebrate orthologues 7 . Here we show, by exploiting this auxotrophy to identify HRG-1 proteins in C. elegans, that these proteins are essential for haem homeostasis and normal development in worms and vertebrates. Depletion of hrg-1, or its paralogue hrg-4, in worms results in the disruption of organismal haem sensing and an abnormal response to haem analogues. HRG-1 andCorrespondence and requests for materials should be addressed to I.H. (hamza@umd.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Contributions Experimental design and execution were as follows: worm experiments and microarrays, A.R., A.U.R., M.K. and I.H.; mammalian experiments, A.R., A.U.R., M.T., C.H., S.U., M. HRG-4 are previously unknown transmembrane proteins, which reside in distinct intracellular compartments. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations and, most strikingly, profound defects in erythropoiesis-phenotypes that are fully rescued by worm HRG-1. Human and worm proteins localize together, and bind and transport haem, thus establishing an evolutionarily conserved function for HRG-1. These findings reveal conserved pathways for cellular haem trafficking in animals that define the model for eukaryotic haem transport. Thus, uncovering the mechanisms of haem transport in C. elegans may provide insights into human disorders of haem metabolism and reveal new drug targets for developing anthelminthics to combat worm infestations.In animals, the terminal enzyme in haem synthesis, ferrochelatase, is located on the matrix side of the inner mitochondrial membrane 8 . Most newly synthesized haem must be transported through mitochondrial membranes to haemoproteins found in distinct intracellular membrane compartments 6 . Haem synthesis is regulated at multiple steps by effectors including iron, haem and oxygen to prevent the uncoordinated accumulation of haem or its precursors 5 . C. elegans is a haem auxotroph and is therefore a unique genetic animal model in which to identify the molecule...

Background: Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), cause severe nonsyndromic myopia. Results: Collagens I and IV from P3h2-null mouse eye tissues were significantly reduced in 3-hydroxylation compared with wild-type littermates. Conclusion: Loss of P3h2 causes altered collagen prolyl 3-hydroxylation from multiple tissues. Significance: Improved understanding of molecular mechanisms of myopia could aid in early diagnosis and treatment of irreversible vision loss.

Osteogenesis imperfecta (OI) is an inherited brittle bone disorder characterized by bone fragility and low bone mass. Loss of function mutations in FK506-binding protein 10 (FKBP10), encoding the FKBP65 protein, result in recessive OI and Bruck syndrome, of which the latter is additionally characterized by joint contractures. FKBP65 is thought to act as a collagen chaperone, but it is unknown how loss of FKBP65 affects collagen synthesis and extracellular matrix formation. We evaluated the developmental and postnatal expression of Fkbp10 and analyzed the consequences of its generalized loss of function. Fkbp10 is expressed at low levels in E13.5 mouse embryos, particularly in skeletal tissues, and steadily increases through E17.5 with expression in not only skeletal tissues, but also in visceral tissues. Postnatally, expression is limited to developing bone and ligaments. In contrast to humans, with complete loss of function mutations, Fkbp10(-/-) mice do not survive birth, and embryos present with growth delay and tissue fragility. Type I calvarial collagen isolated from these mice showed reduced stable crosslink formation at telopeptide lysines. Furthermore, Fkbp10(-/-) mouse embryonic fibroblasts show retention of procollagen in the cell layer and associated dilated endoplasmic reticulum. These data suggest a requirement for FKBP65 function during embryonic connective tissue development in mice, but the restricted expression postnatally in bone, ligaments and tendons correlates with the bone fragility and contracture phenotype in humans.

Heme is a cofactor in proteins that function in almost all sub-cellular compartments and in many diverse biological processes. Heme is produced by a conserved biosynthetic pathway that is highly regulated to prevent the accumulation of heme—a cytotoxic, hydrophobic tetrapyrrole. Caenorhabditis elegans and related parasitic nematodes do not synthesize heme, but instead require environmental heme to grow and develop. Heme homeostasis in these auxotrophs is, therefore, regulated in accordance with available dietary heme. We have capitalized on this auxotrophy in C. elegans to study gene expression changes associated with precisely controlled dietary heme concentrations. RNA was isolated from cultures containing 4, 20, or 500 µM heme; derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. We identified 288 heme-responsive genes (hrgs) that were differentially expressed under these conditions. Of these genes, 42% had putative homologs in humans, while genomes of medically relevant heme auxotrophs revealed homologs for 12% in both Trypanosoma and Leishmania and 24% in parasitic nematodes. Depletion of each of the 288 hrgs by RNA–mediated interference (RNAi) in a transgenic heme-sensor worm strain identified six genes that regulated heme homeostasis. In addition, seven membrane-spanning transporters involved in heme uptake were identified by RNAi knockdown studies using a toxic heme analog. Comparison of genes that were positive in both of the RNAi screens resulted in the identification of three genes in common that were vital for organismal heme homeostasis in C. elegans. Collectively, our results provide a catalog of genes that are essential for metazoan heme homeostasis and demonstrate the power of C. elegans as a genetic animal model to dissect the regulatory circuits which mediate heme trafficking in both vertebrate hosts and their parasites, which depend on environmental heme for survival.

The homozygous G>A transition that results in the substitution of histidine for arginine at position 495 of the renal sodium-dependent phosphate transporter, SLC34A1, is involved in disease pathogenesis in these patients. Our report of the second family with two mutated SLC34A1 alleles expands the known phenotype of this rare condition.

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient Pi. Pi-limitation induces upregulation of inositol heptakisphosphate (IP7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate the PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Furthermore, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of Pi starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.

Angiotensin receptor blockers (ARBs) are a group of anti-hypertensive drugs that are widely used to treat pediatric hypertension. Recent application of ARBs to treat diseases such as Marfan syndrome or Alport syndrome has shown positive outcomes in animal and human studies, suggesting a broader therapeutic potential for this class of drugs. Multiple studies have reported a benefit of ARBs on adult bone homeostasis; however, its effect on the growing skeleton in children is unknown. We investigated the effect of Losartan, an ARB, in regulating bone mass and cartilage during development in mice. Wild type mice were treated with Losartan from birth until 6 weeks of age, after which bones were collected for microCT and histomorphometric analyses. Losartan increased trabecular bone volume vs. tissue volume (a 98% increase) and cortical thickness (a 9% increase) in 6-weeks old wild type mice. The bone changes were attributed to decreased osteoclastogenesis as demonstrated by reduced osteoclast number per bone surface in vivo and suppressed osteoclast differentiation in vitro. At the molecular level, Angiotensin II-induced ERK1/2 phosphorylation in RAW cells was attenuated by Losartan. Similarly, RANKL-induced ERK1/2 phosphorylation was suppressed by Losartan, suggesting a convergence of RANKL and angiotensin signaling at the level of ERK1/2 regulation. To assess the effect of Losartan on cartilage development, we examined the cartilage phenotype of wild type mice treated with Losartan in utero from conception to 1 day of age. Growth plates of these mice showed an elongated hypertrophic chondrocyte zone and increased Col10a1 expression level, with minimal changes in chondrocyte proliferation. Altogether, inhibition of the angiotensin pathway by Losartan increases bone mass and accelerates chondrocyte hypertrophy in growth plate during skeletal development.

. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/338350 doi: bioRxiv preprint first posted online Jun. 4, 2018; 3 Nitrogen-containing bisphosphonates (N-BPs) are the standard treatment for osteoporosis and several other bone diseases 11,12 . Certain N-BPs (pamidronate (Aredia®), zoledronate (Zometa®)) are also routinely prescribed to prevent skeletal complications in patients with multiple myeloma and with bone metastases from other malignancies, including breast and prostate cancer 13 . However, because N-BPs cause rare yet serious side-effects, such as atypical fractures and osteonecrosis of the jaw, many patients avoid taking them [5][6][7]11 , causing the number of prescriptions to plummet over 50% in the last decade 7,14 . Thus, there is a sizeable and growing need to better understand the genetic factors that might underlie the onand off-target clinical effects of the N-BPs.A goal of personalized medicine is to identify biomarkers that underlie drug responsiveness. In the case of the N-BPs, it can be said that there are limited personalization options owing to the limited number of genes implicated in the mechanism of action of N-BPs on bone. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/338350 doi: bioRxiv preprint first posted online Jun. 4, 2018; 4 understanding of the molecular mechanisms by which N-BPs regulate the major bone cell types would be improved by further studies.To provide insight into the mechanism(s) of N-BPs action, we performed a genetic screen to identify human genes required for the anti-proliferative effects of N-BPs (Fig. 1a) Other genes identified in our alendronate haploid screen (SNTG1, PLCL1, and EPHB1) have been previously connected to N-BP action on bone cells and/or human bone diseases [28][29][30] . To systematically evaluate all "hits" we identified (i.e., the 185 genes with FDR p-value < 0.05, see Fig. 1b), we developed a computational approach utilizing a molecular biology-optimized version of PubMed (currently at ~27M records) to assess to what extent our haploid screen identified genes were cited as relevant to human studies that focus on bone (see "PubMed citation analysis" in the Methods section for details). We asked whether our alendronate haploid screen hits were mentioned in publications co-occurring with the term "bone" vs. other tissues along with identifiers of genome-wide screening, namely the strings, "GWAS" or "genome-wide" (Fig. 1c). Indeed, our alendronate screen hits were enriched in publications co-mentioning "bone" and genome-wide studies compared to control gene sets (Fig. 1d, Supplementary Table 1b). This . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed...