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| Status |
Public on Feb 05, 2013 |
| Title |
HIF- and non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by array
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| Summary |
Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1 in the adaptive response, however, has not been determined. Here we investigate how HIF-1 influences hypoxic adaptation throughout Drosophila development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in dHIF mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIF and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIF in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs.
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| Overall design |
Fly eggs were collected onto egg caps with yeast paste . The caps were replaced after overnight incubation periods and kept at 25ºC, until mid-L2. At mid-L2, larvae were transferred to a fresh egg cap with blue yeast paste (0.3% bromophenol blue), and allowed to develop until achieving the partial clear gut L3 stage (-10 to -4 hrs prior to metamorphic onset). Appropriately staged animals were moved to fresh agar plates and allowed to age an additional 6 hours at 25ºC (normoxic treatment). Alternatively, animals were placed in an airtight Modular Incubator Chamber for 6 hours at 25ºC after a gas mixture containing 4% oxygen balanced with nitrogen was flashed into the chamber (hypoxic treatment). Mutant larvae were sorted for the absence of GFP expression using dissecting stereoscope with fluorescence at mid-L2. Microarray analyses were performed on at least three biological replicates of w1118, dHIF mutants, or dERR mutants at the partial clear gut third instar larval stage and were treated for 6 hours in normoxia or 4% O2. For each biological replicate, at least 10 larvae were collected and washed with 1×PBS. Larvae were placed in TRIzol (Invitrogen, Carlsbad, CA) and homogenized using VWR disposable pellet mixer. Total RNA was isolated using a TRIzol/RQ1 DNase (Promega, Madison, WI) hybrid extraction protocol. Template labelings were performed using the GeneChip 3' IVT Express Kit according to the manufacturer’s specifications (Affymetrix, Santa Clara, CA). Hybridizations to Affymetrix GeneChip Drosophila Genome 2.0 arrays were performed using the manufacturers recommendations. Every chip was scanned at a high resolution by the Affymetrix GeneChip® Scanner 3000 according to the GeneChip Expression Analysis Technical Manual procedures (Affymetrix, Santa Clara, CA).
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| Contributor(s) |
Baker KD, Li Y, Gentile LB, Dumur CI |
| Citation(s) |
23382692 |
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| Submission date |
Oct 19, 2011 |
| Last update date |
May 04, 2018 |
| Contact name |
Keith D Baker |
| E-mail(s) |
[email protected]
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| Phone |
8046284498
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| Organization name |
VCU School of Medicine
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| Department |
Biochemistry & Molecular Biology
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| Street address |
1220 E. Broad St.
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| City |
Richmond |
| State/province |
VA |
| ZIP/Postal code |
23298 |
| Country |
USA |
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| Platforms (1) |
| GPL1322 |
[Drosophila_2] Affymetrix Drosophila Genome 2.0 Array |
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| Samples (26)
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| GSM1019709 |
Control larvae at late-L3, normoxia biological rep1 |
| GSM1019710 |
Control larvae at late-L3, normoxia biological rep2 |
| GSM1019711 |
Control larvae at late-L3, normoxia biological rep3 |
| GSM1019712 |
Sima07607 mutant larvae at late-L3, normoxia biological rep1 |
| GSM1019713 |
Sima07607 mutant larvae at late-L3, normoxia biological rep2 |
| GSM1019714 |
Sima07607 mutant larvae at late-L3, normoxia biological rep3 |
| GSM1019715 |
ERR1/ERR2 mutant larvae at late-L3, normoxia biological rep1 |
| GSM1019716 |
ERR1/ERR2 mutant larvae at late-L3, normoxia biological rep2 |
| GSM1019717 |
ERR1/ERR2 mutant larvae at late-L3, normoxia biological rep3 |
| GSM1019718 |
Sima07607,ERR1/ERR2 double mutant larvae at late-L3, normoxia biological rep1 |
| GSM1019719 |
Sima07607,ERR1/ERR2 double mutant larvae at late-L3, normoxia biological rep2 |
| GSM1019720 |
Sima07607,ERR1/ERR2 double mutant larvae at late-L3, normoxia biological rep3 |
| GSM1019721 |
Control larvae at late-L3, hypoxia biological rep1 |
| GSM1019722 |
Control larvae at late-L3, hypoxia biological rep2 |
| GSM1019723 |
Control larvae at late-L3, hypoxia biological rep3 |
| GSM1019724 |
Control larvae at late-L3, hypoxia biological rep4 |
| GSM1019725 |
Control larvae at late-L3, hypoxia biological rep5 |
| GSM1019726 |
Sima07607 mutant larvae at late-L3, hypoxia biological rep1 |
| GSM1019727 |
Sima07607 mutant larvae at late-L3, hypoxia biological rep2 |
| GSM1019728 |
Sima07607 mutant larvae at late-L3, hypoxia biological rep3 |
| GSM1019729 |
ERR1/ERR2 mutant larvae at late-L3, hypoxia biological rep1 |
| GSM1019730 |
ERR1/ERR2 mutant larvae at late-L3, hypoxia biological rep2 |
| GSM1019731 |
ERR1/ERR2 mutant larvae at late-L3, hypoxia biological rep3 |
| GSM1019732 |
Sima07607,ERR1/ERR2 double mutant larvae at late-L3, hypoxia biological rep1 |
| GSM1019733 |
Sima07607,ERR1/ERR2 double mutant larvae at late-L3, hypoxia biological rep2 |
| GSM1019734 |
Sima07607,ERR1/ERR2 double mutant larvae at late-L3, hypoxia biological rep3 |
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| Relations |
| BioProject |
PRJNA149511 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE33100_RAW.tar |
82.0 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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