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. 2009 Dec 1;23(23):2711-6.
doi: 10.1101/gad.1833609.

The Drosophila DHR96 Nuclear Receptor Binds Cholesterol and Regulates Cholesterol Homeostasis

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Free PMC article

The Drosophila DHR96 Nuclear Receptor Binds Cholesterol and Regulates Cholesterol Homeostasis

Michael A Horner et al. Genes Dev. .
Free PMC article

Abstract

Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. Here we show that the Drosophila DHR96 nuclear receptor binds cholesterol and is required for the coordinate transcriptional response of genes that are regulated by cholesterol and involved in cholesterol uptake, trafficking, and storage. DHR96 mutants die when grown on low levels of cholesterol and accumulate excess cholesterol when maintained on a high-cholesterol diet. The cholesterol accumulation phenotype can be attributed to misregulation of npc1b, an ortholog of the mammalian Niemann-Pick C1-like 1 gene NPC1L1, which is essential for dietary cholesterol uptake. These studies define DHR96 as a central regulator of cholesterol homeostasis.

Figures

Figure 1.
Figure 1.
Mass spectrometry identifies cholesterol bound to the DHR96 LBD. (A) CID mass spectrum of the DHR96/cholesterol complex under nondenaturing conditions with a collision voltage of 10 V. At this voltage, a portion of the ions representing the 12+ charge state of the DHR96/ligand complex (2685.87 m/z) fragment to generate ions of the 12+ charge state of DHR96 (2653.66 m/z), losing the ligand as a free molecule. The charge states of the ions at 2685.87 and 2653.66 m/z were determined from full-range MS scan spectra (data not shown). (B) CID mass spectrum of the DHR96/ligand complex with a collision voltage of 50 V caused complete dissociation of the receptor/ligand complex into unbound receptor. (C–F) The elution time of the major peak on a gas chromatogram of a derivatized chloroform–methanol extraction of the DHR96 LBD (C) matches that of a derivatived cholesterol standard (E). The corresponding electron ionization spectrum from the major peak of the DHR96 LBD at 19 min generates major fragmentation ions (D) that correspond closely to the major fragmentation ions generated from the major peak of the derivatived cholesterol standard (F).
Figure 2.
Figure 2.
Most cholesterol-regulated genes depend on DHR96 for their proper expression. (A,B) Heat maps are depicted representing the top 50 genes that are either up-regulated by cholesterol (A) or down-regulated by cholesterol (B) in CanS wild-type larvae, along with the responses of these same genes in DHR961 mutants, as determined by microarray analysis. The heat maps are arranged from top to bottom by their fold response to cholesterol in wild-type larvae. The expression levels in columns 2–4 of each heat map are normalized to the expression level in column 1 (CanS −, cholesterol). Red represents increased transcript levels relative to the transcript level in column 1, while green represents lower transcript levels. (C) RNA isolated from CanS control larvae and DHR961 mutant larvae, grown in either the absence (−) or presence (+) of cholesterol, was analyzed by Northern blot hybridization for expression of npc2c, CG14745, npc1b, CG5932, CG31148, and npc2e. Hybridization to detect rp49 was used as a control for loading and transfer.
Figure 3.
Figure 3.
DHR96 regulates cholesterol homeostasis. (A–C) DHR96 mutants arrest development on a low-cholesterol medium. CanS control and DHR961 mutant larvae were maintained on a low-cholesterol medium without supplementation (−yeast) (A), supplemented with yeast (+yeast) (B), or supplemented with 0.03% cholesterol (+chol) (C), and scored for the percent of adults that eclosed. (D) DHR961 mutants carrying a heat-inducible wild-type DHR96 transgene (+hs-DHR96) were grown on the low-cholesterol medium without supplementation in either the absence (−heat) or presence (+heat) of heat treatment, and scored for the percent of adults that eclosed. (E) The lethality of DHR96 mutants maintained on the low-cholesterol medium is rescued by expressing DHR96 in the midgut. CanS control and DHR961 mutant larvae, maintained either without any transgenes (−), with CG-GAL4;UAS-DHR96 (CG > DHR96; fat body-specific), or with Mex-GAL4;UAS-DHR96 (Mex > DHR96; midgut-specific) were grown on the low-cholesterol medium without supplementation and were scored for the percent of adults that eclosed. (F,G) DHR96 mutants accumulate cholesterol when grown on a high-cholesterol diet. CanS control and DHR961 mutant larvae were grown on the low-cholesterol medium either without supplementation (−chol) (F) or in the presence of 0.03% cholesterol (+chol) (G). Total cholesterol levels were measured in larvae collected 2 d after hatching and normalized for total protein. Data were pooled from two experiments and are presented as normalized to a wild-type (minus added cholesterol) level of 100%. (H) CanS control and DHR961 mutant larvae, maintained either without any transgenes (−), with a wild-type DHR96 genomic construct (P[96]+), or with Mex-GAL4;UAS-DHR96 (Mex > DHR96) were grown in the presence of 0.03% cholesterol. Total cholesterol levels were measured in larvae collected 2 d after hatching and were normalized for total protein. Data are presented as normalized to a wild-type level of 100%. Error bars are ±SE. (*) P < 0.05; (**) P < 5 × 10−4.
Figure 4.
Figure 4.
npc1b contributes to the cholesterol accumulation phenotype in DHR96 mutants. (A) DHR96 and npc1b are both expressed in the midgut. Midguts were dissected from NPC1b-GAL4;UAS-nGFP third instar larvae (NCP1b > nGFP) (Voght et al. 2007) and stained with affinity-purified antibodies to detect DHR96 protein. DHR96 protein is shown in red, and NPC1b expression is shown in green. All images were taken from the same field and focal plane. (B) DHR96 regulates npc1b transcription. RNA was extracted from CanS control and DHR961 mutant larvae, maintained in the presence of 0.03% cholesterol either without any transgenes (−) or with Mex-GAL4;UAS-DHR96 (Mex > DHR96), and analyzed by quantitative RT–PCR for levels of npc1b transcript. npc1b expression levels are presented as normalized to the level in CanS. (C) An npc1b mutation rescues the cholesterol accumulation defect in DHR96 mutants. CanS control, DHR961 mutant larvae, and npc1b1;DHR961 double mutants were grown in the presence of 0.03% cholesterol. Total cholesterol levels were measured in larvae collected 2 d after hatching and were normalized for total protein. Data are presented as normalized to a wild-type level of 100%. The cholesterol levels in CanS and npc1b1;DHR961 double mutants are not significantly different (P = 0.19). (D) Sterol absorption, but not fatty acid absorption, is blocked by an npc1b mutation in a DHR96 mutant background. CanS control, DHR961 mutants, npc1b1 mutants, and npc1b1;DHR961 double mutants were grown on a low-cholesterol medium supplemented with either 3H-cholesterol, 3H-sitosterol, or 3H-oleic acid along with 14C-glucose. Levels of radioactive lipid were normalized to the 14C-glucose and are presented as normalized to a wild-type level of 100%. Error bars are SE. (*) P < 0.05; (**) P < 0.01; (***) P < 1 × 10−4.

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