Project description:Hemes are essential but potentially cytotoxic cofactors that participate in critical and diverse biological processes. Although the pathway and intermediates for heme biosynthesis have been well defined, the intracellular networks which mediate heme trafficking remain unknown. Caenorhabditis elegans and related helminths are natural heme auxotrophs requiring environmental heme for growth and development. We exploited this auxotrophy to identify HRG-1 and HRG-4 in C. elegans and show that they are essential for heme homeostasis and normal vertebrate development. We demonstrate that heme deficiency upregulates expression of hrg-4 and its evolutionarily conserved paralog hrg-1. Depletion of either HRG-1 or HRG-4 in worms results in disruption of organismal heme sensing and abnormal response to heme analogs. HRG-1 and HRG-4 are novel transmembrane proteins that bind heme and have evolutionarily conserved functions. 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. These findings reveal unanticipated and conserved pathways for cellular heme trafficking in animals that defines the paradigm for eukaryotic heme transport. Uncovering the mechanisms of heme transport in C. elegans will provide novel insights into human disorders of heme metabolism and generate unique anthelmintics to combat worm infestations. Experiment Overall Design: As a first-step toward understanding heme homeostasis at the molecular level, we performed genome-wide microarrays to identify genes that are transcriptionally regulated by heme. For microarray analysis, synchronized F2 larvae were re-inoculated in mCeHR-2 medium supplemented with 4, 20 or 500 uM hemin and harvested at the late L4 stage for mRNA prep and probe hybridization to Affymetrix C. elegans Whole Genome Expression Arrays. Total RNA from three biological replicates were used at each hemin concentration. Data from worms grown in mCeHR-2 medium with 4 and 500 uM hemin were compared to data from worms grown in 20 uM hemin. Microarray data were verified with Microarray Suite 5.0 (Affymetrix) and Robust Multichip Average Method (RMA, R package). Results from MAS 5.0 and RMA analyses provided with 375 genes that showed 1.6 fold change in 4 and 500 uM hemin when compared to data from 20 uM hemin samples. Statistical analyses identified changes in 375 genes from worms grown in either 4 or 500 ÂµM heme (see Methods). The microarray results were validated by qRT-PCR (Supplementary Fig. 1) and the 375 heme-responsive genes were classified into eight categories based on their relative changes in gene expression (Table I). The data from the microarray study show that â?°1.9 % of genes in the worm genome are transcriptionally responsive to heme. Notably, of the 375 genes, 164 had some sequence identity in human genome databases at the amino acid level, and >90 % of the genes had no ascribed function in the C.

Project description:Purpose: The goal of this study is to understand how dbl-1, which is made primarily in neurons, and hrg-7, which is exclusively made in the intestine, contribute to systemic heme homeostasis. Methods: mRNA profiles of late L4 dbl-1(nk3) and hrg-7(tm6801) mutant C. elegans fed OP50 E. coli or OP50 + 50µM heme were compared to mRNA profiles from wildtype (WT) broodmates. Profiles were generated with single-end 50 base reads obtained using Illumina’s HiSeq 2500. Bioinformatics quality control was performed followed by alignment of reads to the ce10 reference genome using Tophat2, version 2.1.0. We found differentially expressed genes using Cufflinks 2, version 2.2.1 with a cutoff of 0.05 on False Discovery Rate (FDR). Results: We found a substantial overlap of genes regulated by both dbl-1 and hrg-7, including 49 heme-responsive genes (hrgs) in low heme (OP50) and 11 hrgs in high heme (OP50 + 50µM). Additionally, our data indicate crosstalk between dbl-1 and hrg-7 signaling. dbl-1 directly regulates hrg-7 expression, while hrg-7 regulates three components of the dbl-1 signaling pathway. Conclusions: Our study demonstrates that communication between the neuron and intestine is essential for heme homeostasis. Specifically, we report that HRG-7 functions as a secreted signaling factor which communicates intestinal heme status with extraintestinal tissues by integrating a DBL-1/BMP -dependent response from the neurons to transcriptionally regulate genes involved in heme homeostasis. Cellular requirements for heme are fulfilled by a cell’s internal capacity to synthesize its own heme in a cell-autonomous manner. However, growing evidence in vertebrates predicts that cellular heme levels in animals are not only maintained by heme synthesis, but also by distally located proteins that could signal systemic heme requirements to an inter-organ heme trafficking network through cell-nonautonomous regulation. Using C. elegans, a genetically and optically amenable animal model for visualizing heme-dependent signaling, we show that HRG-7, an aspartic protease homolog, mediates inter-organ signaling between the intestine and neuron. Loss of hrg-7 results in robust expression of intestinal heme importers and, remarkably, this occurs even under heme replete conditions when such transporters are not normally expressed. HRG-7 functions as a secreted signaling factor, independent of a functional enzymatic active site, and communicates intestinal heme status with extraintestinal tissues by integrating a DBL-1/BMP -dependent response from the neurons to transcriptionally regulate intestinal heme homeostasis. Given the evidence indicating that mechanisms of heme transport are conserved across metazoa, it is conceivable that the cell-nonautonomous signaling framework that we uncovered in C. elegans may have functional relevance for inter-organ regulation of iron and heme metabolism in humans.

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Caenorhabditis elegans

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