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| Status |
Public on Jun 13, 2008 |
| Title |
Heme homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins |
| Organism |
Caenorhabditis elegans |
| Experiment type |
Expression profiling by array
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| Summary |
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.
Keywords: dose-response
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| 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 µM 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 ‰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.
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| Contributor(s) |
Rajagopal A, Rao AU, Amigo J, Hall C, Uhm S, Paw BH, Krause M, Hamza I |
| Citation(s) |
18418376, 20686661 |
| Submission date |
Aug 06, 2007 |
| Last update date |
Mar 19, 2020 |
| Contact name |
WeiPing Chen |
| E-mail(s) |
weipingChen@niddk.nih.gov
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| Phone |
301-496-0175
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| Organization name |
NIDDK/NIH
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| Department |
GCL
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| Lab |
Genomics Core Lab
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| Street address |
Bldg 8, Room 1A11, NIDDK/NIH
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL200 |
[Celegans] Affymetrix C. elegans Genome Array |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA101921 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE8696_RAW.tar |
29.2 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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