The Drosophila DHR96 nuclear
receptor binds cholesterol
and regulates cholesterol
Michael A. Horner,1Keith Pardee,2Suya Liu,3
Kirst King-Jones,4Gilles Lajoie,3Aled Edwards,2
Henry M. Krause,2and Carl S. Thummel1,5
1Department of Human Genetics, University of Utah School of
Medicine, Salt Lake City, Utah 84112, USA;2Banting and Best
Department of Medical Research, University of Toronto, Toronto
Ontario M5G 1L6, Canada;3UWO Biological Mass Spectrometry
Laboratory, University of Western Ontario, London, Ontario
N6G 2V4, Canada;4Department of Biological Sciences,
University of Alberta, Edmonton, Alberta T6G 2E9, Canada
Cholesterol homeostasis is required to maintain normal
cellular function and avoid the deleterious effects of hy-
percholesterolemia. Here we show that the Drosophila
DHR96 nuclear receptor binds cholesterol and is required
regulated by cholesterol and involved in cholesterol up-
take, trafficking, and storage. DHR96 mutants die when
grown on low levels of cholesterol and accumulate excess
cholesterol when maintained on a high-cholesterol diet.
to misregulation of npc1b, an ortholog of the mammalian
Niemann-Pick C1-like 1 gene NPC1L1, which is essen-
tial for dietary cholesterol uptake. These studies define
DHR96 as a central regulator of cholesterol homeostasis.
Supplemental material is available at http://www.genesdev.org.
Received June 17, 2009; revised version accepted October 2, 2009.
Cholesterol is an essential component of cell membranes
that influences the permeability and fluidity of the lipid
bilayer. Cholesterol also acts as a precursor for steroid
hormone biosynthesis and contributes to cell–cell signal-
ing pathways. These critical cellular functions are sup-
ported by regulatory mechanisms that maintain normal
cholesterol levels and prevent hypercholesterolemia,
which is a major risk factor for cardiovascular disease
in humans. Cholesterol homeostasis in vertebrates is
achieved primarily through de novo synthesis and dietary
uptake (Ikonen 2008). Although extensive studies have
defined a central role for the sterol regulatory element-
binding protein (SREBP) family of transcription factors in
controlling cholesterol synthesis (Brown and Goldstein
1997), the mechanisms that regulate dietary cholesterol
absorption remain more poorly understood. One central
component of this pathway is the Niemann-Pick C1-like
1 gene NPC1L1, which encodes a plasma membrane pro-
teinthat mediatesthe uptakeof dietary cholesterol by the
intestine (Wang 2007; Ge et al. 2008). Mouse mutants for
NPC1L1 display significantly reduced levels of choles-
terol absorption and are insensitive to treatment with the
anti-hypercholesterolemia drug ezetimibe, which acts as
a specific NPC1L1 inhibitor (Davis et al. 2008). Another
major regulator of cholesterol homeostasis is the liver
X receptor a (LXRa) nuclear receptor, which binds cho-
lesterol metabolites and regulates the transcription of
genes that control cholesterol transport and metabolism,
including NPC1L1 (Duval et al. 2006; Kalaany and
Mangelsdorf 2006; Valasek et al. 2007).
We used the fruit fly, Drosophila, as a model system to
study the regulation of cholesterol homeostasis. Unlike
vertebrates, insects are cholesterol auxotrophs that are
unable to synthesize this essential compound (Va’nt
Hoog 1936). Little is known, however, about the mech-
anisms that regulate the uptake of dietary cholesterol in
Drosophila. A recent study showed that the fly ortholog
of NPC1L1, npc1b, is expressed specifically in the midgut
and is essential for dietary cholesterol absorption (Voght
et al. 2007). Other NPC disease gene homologs in Dro-
sophila also contribute to cholesterol homeostasis. The
Drosophila ortholog of vertebrate NPC1, npc1a, and two
of the eight fly NPC2 homologs, npc2a and npc2b, play
important roles in intracellular cholesterol trafficking
and synthesis of the steroid hormone 20-hydroxyecdy-
sone (20E) (Huang et al. 2005, 2007; Fluegel et al. 2006).
Other predicted regulators of cholesterol metabolism in
Drosophila, however, remain unstudied, and upstream
factors that might sense cholesterol levels and control
cholesterol homeostasis are undefined.
In this study, we show that the Drosophila DHR96
nuclear receptor binds cholesterol, is essential for sur-
vival on a low-cholesterol diet, and is required to main-
tain cholesterol homeostasis when animals are grown on
a high-cholesterol diet. We further show that dietary cho-
lesterol regulates the transcription of many genes that are
expressed in the midgut and that act in lipid metabolism,
and that this transcriptional response fails to occur in
DHR96 mutants. Misregulation of one of these genes,
npc1b, is sufficient to explain the cholesterol accumula-
tion defect seen in DHR96 mutants, defining npc1b as
a critical functional target of the receptor. This study
provides a new framework for understanding the molec-
ular mechanisms that regulate cholesterol homeostasis.
Results and Discussion
DHR96 binds cholesterol
Mass spectrometry (MS) was used to identify potential
ligands for DHR96. The DHR96 ligand-binding domain
(LBD) was overexpressed, purified from insect cells, and
subjected to electrospray ionization (ESI) MS, under both
denaturing and nondenaturing conditions. The full mass
range spectrum of the sample from the denaturing con-
dition had a series of peaks corresponding to the DHR96
LBD, with a measured molecular weight (MW) of
31,831.92 Da, close to the predicted theoretical mass
(31,830.84 Da for His6-DHR96S471-H723) (data not shown).
Under nondenaturing conditions, the full mass range scan
detected an additional series of peaks corresponding to
a MW of 32,218.44 Da. This mass shift indicates that the
[Keywords: Gene regulation; nuclear receptor signaling; cholesterol
metabolism; Niemann-Pick disease gene]
E-MAIL firstname.lastname@example.org; FAX (801) 581-5374.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1833609.
GENES & DEVELOPMENT 23:2711–2716 ? 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org 2711
DHR96 LBD copurifies with a molecule of 386.52 Da in
One of the presumptive DHR96/ligand complex peaks
(2685.87 m/z) was then selected for collision-induced
dissociation (CID) with a collision voltage of 10 V, result-
ing in the generation of two ions in the 12+charge state
(Fig. 1A).One ion represents the mass of the intact DHR96
LBD/ligand complex (2685.87 m/z; 32218.44 Da), while
the other represents the unbound DHR96 LBD (2653.66
m/z; 31831.92 Da). Using a higher collision voltage of 50 V
completely disrupted the DHR96 LBD/ligand complex to
generate a CID spectrum with only an unbound receptor
(2653.60 m/z; 31831.2 Da) (Fig. 1B). The charge state of the
receptor ion did not change upon loss of the ligand and no
peakwasobserved inthelower massrange,indicatingthat
the ligandis a neutral molecule. These observations, along
with the mass shift of 386.52 Da, suggest that the bound
molecule may be cholesterol (MW, 386.6 Da).
To further verify the identity of the molecule bound to
the DHR96 LBD, purified receptor was extracted with
chloroform–methanol and a portion of the extract was
derivatized and analyzed by gas chromatography/MS
(GC/MS) with electron ionization (EI). A single major
peak at 19 min was observed in the GC/MS chromato-
gram (Fig. 1C). The retention time of this peak was iden-
tical to that of a derivatized cholesterol standard (Fig. 1E).
Moreover, the major peak of the DHR96 sample on GC/
MS produced an EI spectrum (Fig. 1D) that matched
a previously described spectrum for derivatized choles-
terol (Fig. 1F; Bitsch et al. 2003). Based on these two inde-
pendent modes of MS—ESI/MS and GC/MS—we con-
clude that the DHR96 LBD is capable of binding a single
molecule of cholesterol. GC/MS analysis of several other
Drosophila nuclear receptors indicated that this interac-
tion is specific to DHR96 (Supplemental Fig. S1). In
addition, although this interaction is stable through three
rounds of protein chromatography, the partial dissocia-
tion of ligands at 10 V and complete dissociation at 50 V
suggests that the bound cholesterol is exchangeable.
DHR96 regulates the transcriptional
response to cholesterol
The observation that DHR96 binds cholesterol raises the
possibility that it may mediate transcriptional responses
to this compound. To test this possibility, control and
DHR96 mutants were grown on low-cholesterol medium
in the absence or presence of 0.03% cholesterol, and were
subjected to microarray analysis. From this analysis, 117
genes were identified that are up-regulated at least 1.4-
fold in response to cholesterol in wild-type larvae, along
with 270 genes that are down-regulated (Fig. 2A,B;
SupplementalTable S1).Thisresponseappears toberapid,
occurring within 1–2 h of cholesterol treatment, and
displays similar cholesterol dose response profiles (Sup-
plemental Fig. S2). We also see a response to sitosterol
treatment, indicating that it is not specific to cholesterol
(Supplemental Fig. S3). Interestingly, a number of the
cholesterol-regulated genes are predicted to play central
roles in cholesterol metabolism and transport. These
include CG32186, which encodes a predicted ABCA3-
like transporter; CG6472, which encodes a lipoprotein
lipase (LPL) homolog; and CG8112, which encodes acyl-
CoA:cholesterol acetyltransferase (ACAT) (Supplemental
Table S5). This enzyme plays a critical role in esterifying
cholesterol, which is the primary stored form of intracel-
lular cholesterol (Ikonen 2008). Four Drosophila homo-
logs of NPC genes are also regulated by cholesterol, in-
cluding npc1b and three NPC2 homologs: npc2b, npc2c,
and npc2d (Huang et al. 2007).
Importantly, this transcriptional response is almost
entirely dependent on DHR96 function. Only 13% of
the genes that are up-regulated by cholesterol in wild-
type larvae (15 genes), and 10% of the genes that are
down-regulated (27 genes), display a similar profile of
expression in DHR96 mutants treated with cholesterol
(Fig. 2A,B; Supplemental Fig. S4). Moreover, many addi-
tional genes become responsive to cholesterol in DHR96
mutants, with 355 genes up-regulated and 446 genes
down-regulated at least 1.4-fold, indicating that DHR96
normally plays a key role in suppressing this transcrip-
tional program (Supplemental Fig. S4; Supplemental
Table S2). These DHR96-regulated genes include four of
the remaining five NPC2 family members: npc2e, npc2f,
npc2g, and npc2h (Fig. 2C; Supplemental Table S5).
DHR96 also regulates Lip3 (CG8823), which encodes
a predicted cholesterol ester hydrolase; CG9663, which
encodes an ABCG1 homolog; CG11162, which encodes
a sterol-C4-methyl oxidase; and several genes that encode
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).
Mass spectrometry identifies cholesterol bound to the
Horner et al.
2712 GENES & DEVELOPMENT
predicted stearoyl-CoA-desaturases. Many genes in-
volved in other aspects of lipid metabolism are also
misregulated in DHR96 mutants (Supplemental Table
S5). In addition, DHR96 itself is down-regulated approx-
imately twofold by cholesterol in wild-type larvae, and
this response fails to occur in DHR96 mutants, suggest-
ing that there is autoregulation. Further examination of
the cholesterol-regulated genes in both wild-type and
DHR96 mutant larvae revealed that many of these genes
are expressed in the midgut, consistent with the critical
role of dietary cholesterol uptake in a cholesterol auxo-
troph such as Drosophila (Supplemental Tables S5, S6).
Northern blot hybridizations confirmed that genes
such as npc2c and CG14745 (which encodes a predicted
peptidoglycan recognition protein) are induced by cho-
lesterol in wild-type larvae, while genes such as npc1b,
CG5932 (which encodes a gastric lipase), and CG31148
(which encodes a predicted enzyme in sphingolipid me-
tabolism) are repressed by cholesterol (Fig. 2C). Other
genes, such as npc2e, are not responsive to cholesterol in
wild-type larvae. All of these genes, however, are mis-
regulated in DHR96 mutants.
DHR96 mutants are unable to survive
on a low-cholesterol diet
The central role of DHR96 in mediating transcriptional
responses to cholesterol suggests that it may contribute
to the regulation of cholesterol homeostasis. As an initial
test of this possibility, we examined how control and
DHR961mutants respond to growth on a low-cholesterol
diet. Whereas most wild-type larvae grown on this me-
dium develop through to adulthood, DHR96 mutants
arrest their development primarily as second instar larvae
and die within several days (Fig. 3A). Supplementation
with a complete nutrient source, yeast, was sufficient to
rescue this lethality (Fig. 3B). Efficient rescue was also
achieved bysupplementing with cholesterol, demonstrat-
ing that the lack of this essential nutrient is a cause of the
lethality (Fig. 3C). Widespread heat-induced expression of
a wild-type DHR96 transgene, or specific expression of
DHR96 in the midgut, is also sufficient to rescue the
lethality of DHR96 mutants grown on the low-choles-
terol diet (Fig. 3D,E). Specific expression of DHR96 in the
fat body of DHR96 mutants had no effect. Taken together,
these observations indicate that DHR96 mutants are
unable to survive under limiting cholesterol conditions,
and that this phenotype is due to a specific loss of DHR96
function in the midgut. Similar results were obtained
when control and DHR96 mutants were raised on a
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 DHR961mutants, 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 DHR961mutant 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.
Most cholesterol-regulated genes depend on DHR96 for
mutants arrest development on a low-cholesterol medium. CanS
control and DHR961mutant larvae were maintained on a low-
cholesterol medium without supplementation (?yeast) (A), supple-
mented with yeast (+yeast) (B), or supplemented with 0.03% choles-
terol (+chol) (C), and scored for the percent of adults that eclosed. (D)
DHR961mutants carrying a heat-inducible wild-type DHR96 trans-
gene (+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 DHR961mutant 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 with-
out 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 DHR961mutant larvae
were grown on the low-cholesterol medium either without supple-
mentation (?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
DHR961mutant larvae, maintained either without any transgenes
(?), with a wild-type DHR96 genomic construct (P+), or with
Mex-GAL4;UAS-DHR96 (Mex > DHR96) were grown in the pres-
ence 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 6SE. (*) P < 0.05; (**) P < 5 3 10?4.
DHR96 regulates cholesterol homeostasis. (A–C) DHR96
DHR96 regulates cholesterol homeostasis
GENES & DEVELOPMENT 2713
chloroform-extracted medium that is deprived of all
sterols (data not shown). A dose response study on the
low-cholesterol diet revealed that 0.03% cholesterol is
the ideal concentration for rescue (Supplemental Fig. S5).
This amount is identical to the optimal amount required
for Drosophila larval survival on a minimal defined me-
dium (Sang 1956). A number of other sterols are able to
substitute for cholesterol in these experiments, including
7-dehydrocholesterol, ergosterol, dehydroergosterol, sitos-
terol, and stigmasterol (data not shown), consistent with
their ability to support normal growth of wild-type Dro-
sophila (Cooke and Sang 1970). Supplementing the low-
cholesterol medium with other lipids, however, including
triacylglycerol (TAG) and oleic acid, had no effect, in-
dicating that the rescue is specific to sterols (data not
metabolism is essential for larval development. Drosoph-
ila larvae grown in the absence of sterols arrest develop-
ment at the first or second instar, similar to the lethal
phase of DHR96 mutants grown under low-cholesterol
conditions (Cooke and Sang 1970). Similarly, mutants for
npc1a die as first instar larvae due to defects in intracel-
lular cholesterol trafficking and reduced production of the
molting hormone 20E (Huang et al. 2005; Fluegel et al.
2006). Double mutants for npc2a and npc2b also fail to
progress through development and can be rescued by
feeding 20E (Huang et al. 2007). DHR96 mutants grown
on a high-cholesterol diet fail to show any clear defects in
intracellular cholesterol localization, as detected by filipin
staining (Supplemental Fig. S6). In addition, feeding 20E to
DHR96 mutants maintained on the low-cholesterol diet
does not rescue their lethality (data not shown). These
observations do not support the hypothesis that the
lethality in DHR96 mutants is due to a defect in choles-
terol trafficking that affects 20E production. Rather, this
phenotype is likely to arise from other defects, such as
inefficient cholesterol utilization, changes in cholesterol
storage, or disruption of other lipid metabolic pathways.
DHR96 mutants accumulate cholesterol
To determine whether DHR96 mutants display defects in
cholesterol homeostasis, we measured total cholesterol
levels in control and DHR961mutant larvae grown on the
low-cholesterol medium, in either the absence or pres-
ence of 0.03% cholesterol. DHR96 mutants have the
same level of cholesterol as wild-type larvae when
propagated without cholesterol supplementation (Fig.
3F). In the presence of added cholesterol, however,
DHR96 mutants display cholesterol levels that are sig-
nificantly higher than the 10%–20% increase seen in
control larvae (Fig. 3G). This cholesterol accumulation
defect can be rescued by either a wild-type genomic
DHR96 transgene or expression of wild-type DHR96 in
the midgut of mutant larvae, indicating that it arises from
a specific loss of DHR96 function in this tissue (Fig. 3H).
An npc1b mutation rescues the cholesterol
accumulation defect in DHR96 mutants
Expression of npc1b, which is required for dietary cho-
lesterol absorption, is down-regulated when wild-type
larvae are treated with cholesterol (Fig. 2C). The obser-
vation that this switch fails to occur in DHR96 mutants
could explain why these animals accumulate excess
cholesterol when grown on a high-cholesterol diet. Con-
sistent with this, we found that DHR96 is expressed in
the region of the larval midgut, where npc1b exerts its
functions (Fig. 4A; King-Jones et al. 2006; Voght et al.
2007). This overlap in DHR96 and npc1b expression also
appears to be functionally significant because specific
expression of wild-type DHR96 in the midgut of DHR96
mutants is sufficient to restore appropriate npc1b tran-
scriptional repression in response to cholesterol (Fig.
4B). Importantly, the accumulation of cholesterol in
DHR96 mutants is dependent on npc1b function because
npc1b;DHR96 double mutants have wild-type levels of
cholesterol (Fig. 4C). This is consistent with the normal
notype 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 DHR961mutant 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 expres-
sion 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
npc1b1;DHR961double 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;DHR961double 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, DHR961mutants, npc1b1mu-
tants, and npc1b1;DHR961double mutants were grown on a low-
cholesterol medium supplemented with either
radioactive lipid were normalized to the
presented as normalized to a wild-type level of 100%. Error bars
are SE. (*) P < 0.05; (**) P < 0.01; (***) P < 1 3 10?4.
npc1b contributes to the cholesterol accumulation phe-
mutant larvae, and
3H-oleic acid along with
14C-glucose. Levels of
14C-glucose and are
Horner et al.
2714 GENES & DEVELOPMENT
cholesterol levels seen in npc1b mutants at this stage of
development, which likely derive from maternal loading
of cholesterol during oogenesis (Voght et al. 2007).
We tested whether DHR96 mutants require npc1b
function for their ability to absorb dietary cholesterol.
Control larvae, DHR961mutants, npc1b1mutants, and
npc1b1;DHR961double mutants were grown on the low-
cholesterol medium in the presence of
Radioactive glucose was also added to the food in order to
normalize the levels of cholesterol absorption and to
control for different feeding rates (Voght et al. 2007).
level of cholesterol uptake as wild-type larvae (Fig. 4D).
This result is consistent with the wild-type levels of total
cholesterol seen in DHR96 mutants grown on a low-
cholesterol diet (Fig. 3F). In contrast, both npc1b mutants
and npc1b;DHR96 double mutants display a dramatic
reduction in cholesterol absorption (Fig. 4D). This result
agrees with the original study of npc1b mutants, and
indicates that DHR96 mutants require NPC1b to take up
dietary cholesterol (Voght et al. 2007). A similar result
was seen with a key plant sterol,
that this pathway mediates general sterol absorption (Fig.
4D). Moreover, the npc1b mutation has no effect on the
ability of DHR96 mutants to absorb oleic acid, consistent
with its specific role in sterol uptake (Fig. 4D). Taken
together, these observations indicate that the misregula-
tion of npc1b expression in DHR96 mutants is sufficient
to explain their inability to maintain proper cholesterol
homeostasis when grown on a high-cholesterol diet.
It is important to note, however, that the cholesterol
accumulation defect seen in DHR96 mutants does not
appear to be related to the lethality that is observed when
the mutant is grown on a low-cholesterol diet. DHR96
mutants that arrest their development when maintained
on a low-cholesterol diet display normal levels of choles-
terol (Fig. 3F). In addition, the npc1b;DHR96 double
mutants are small and die earlier than npc1b mutants
alone when raised in either the presence or absence of
cholesterol, in spite of their wild-type cholesterol levels
(M Horner and CS Thummel, unpubl.). Thus, it is likely
that the DHR96 mutants suffer from additional meta-
bolic defects beyond their inability to properly regulate
npc1b transcription. This conclusion is supported by the
widespread effects of the DHR96 mutation on the expres-
sion of genes involved in lipid metabolism and midgut
physiology, as well as the expression of DHR96 in tissues
outside the midgut (King-Jones et al. 2006). Further
studies of DHR96 mutants should help to uncover its
other key metabolic activities.
DHR96 coordinates the uptake of dietary lipids
The studies reported here define essential functions for
DHR96 in maintaining cholesterol homeostasis during
larval development. A parallel set of studies in our
laboratory has also identified nonessential functions for
DHR96 in lipid metabolism during adult stages due to
their reduced levels of stored energy in the form of TAG
(Sieber and Thummel 2010). Interestingly, microarray
analysis of DHR96 mutant adults raised on normal
growth medium revealed widespread effects on the tran-
scription of genes expressed in the midgut, many of which
are identical to the DHR96 regulatory targets described in
this study. Among this core set of DHR96 target genes is
CG5932, which encodes a gastric lipase that is required
in the midgut is sufficient to explain the defects that we
observed in TAG homeostasis in DHR96 mutants, anal-
ogous to the important role of DHR96 in regulating npc1b
expression to maintain proper cholesterol homeostasis.
Interestingly, CG5932 transcription is also regulated by
cholesterol, raising the possibility that this dietary sterol,
acting through DHR96, may coordinate dietary TAG
breakdown with cholesterol absorption (Fig. 2C). This
convergence of our results points to a central role for
DHR96 in midgut physiology and provides a strong foun-
functions in maintaining lipid homeostasis.
DHR96 is a nuclear protein, in the presence or absence
regulator of transcription (Fig. 4A; data not shown). It has
a unique P-box sequence within its DNA-binding domain
(DBD), which determines its DNA-binding specificity.
This sequence is shared by only three Caenorhabditis
elegans nuclear receptors: DAF-12, NHR-48, and NHR-8.
Although a preferred binding site has been identified for
DAF-12, it not known whether DAF-12 contacts this
sequence as a homodimer or a heterodimer with another
nuclear receptor (Shostak et al. 2004). Similarly, it is
unclear whether DHR96 binds DNA as a monomer,
homodimer, or heterodimer with the Drosophila retinoid
X receptor (RXR) ortholog, like its mammalian homologs.
In addition, the observation that DHR96 target genes
display all possible combinations of regulatory responses
to cholesterol and the DHR96 mutation indicates that
there is further complexity in this pathway. Studies are
under way to define the molecular mechanisms by which
DHR96 regulates target gene transcription.
DHR96 represents an ancestral regulator
of cholesterol metabolism
Ligands have been identified for only two of the 18
canonical nuclear receptors in Drosophila: ecdysone re-
ceptor (EcR) and E75 (Koelle et al. 1991; Reinking et al.
2005). This study adds a third such adopted orphan
receptor to the list: DHR96. The identification of choles-
terol as a DHR96 ligand fits with its membership in the
NR1I subfamily of nuclear receptors (Nuclear Receptors
Nomenclature Committee 1999). The closest mamma-
lian and C. elegans homologs of DHR96—pregnane X
receptor (PXR), constitutive androstane receptor (CAR),
vitamin D3 receptor (VDR), and DAF-12—are all tran-
scriptionally responsive to cholesterol derivatives. Similar
responses to sterol derivatives are seen for members of the
next most closely related group of receptors, the NR1H
group, which includes LXR, farnesoid X receptor (FXR),
and EcR. These observations support the proposal that
the NR1H and NR1I groups arose from an ancestral
progenitor that acted as a sterol receptor in primitive
It is important, however, to note that although choles-
terol copurifies with the DHR96 LBD, it may not be the
natural ligand for this receptor. In our efforts to address
this issue, we were unable to detect changes in DHR96
activity in response to exogenous cholesterol, dietary
factors, or genetic backgrounds that disrupt cholesterol
absorption or intracellular trafficking (data not shown).
Further studies are required to determine whether cho-
lesterol or a related metabolite acts as a regulatory ligand
to modulate the activation status of DHR96.
DHR96 regulates cholesterol homeostasis
GENES & DEVELOPMENT 2715
Although the sequence of DHR96 is most closely
related to mammalian PXR and CAR, it shares significant
functional characteristics with its more distant cousin,
LXRa. LXRa binds oxysterols and, similar to DHR96
mutants, LXRa mutant mice fail to respond properly to
dietary cholesterol and accumulate hepatic cholesterol
when maintained on a high-cholesterol diet (Peet et al.
1998). LXRs also play a central role in the transcriptional
response to dietary cholesterol in mice, and directly or
indirectly control a number of genes that are related to
DHR96-regulated genes, including ABCG1, LPL, steroyl-
CoA desaturase, NPC1, and NPC2 (Maxwell et al. 2003;
Rigamonti et al. 2005; Kalaany and Mangelsdorf 2006).
Finally, like DHR96, LXRs repress NPC1L1 expression,
although it is not clear whether this regulation is direct
(Duval et al. 2006; Valasek et al. 2007). Taken together,
these results indicate that DHR96 and mammalian LXRs
act through similar regulatory pathways to control cho-
lesterol homeostasis. This work establishes a new frame-
work for understanding how cholesterol levels are sensed
in Drosophila, and the molecular mechanisms by which
cholesterol homeostasis is maintained.
Materials and methods
All studies were performed using Canton-S (CanS) wild-type flies and
DHR961-null mutants that had been crossed to CanS for nine generations
(King-Jones et al. 2006). The following stocks were used in this study:
npc1a1(from M. Scott), npc1b1and NPC1b-GAL4 (both from L. Pallanck),
CG-Gal4 (Hennig et al. 2006), and Mex-Gal4 (Phillips and Thomas 2006).
Flies were maintained on standard Bloomington Stock Center medium
with malt at 25°C.
Total cholesterol levels were determined using an Amplex Red Choles-
terol Assay Kit (Invitrogen). Embryos staged at 0–3 h were collected and
allowed to hatch overnight in the absence of food at 25°C. First instar
larvae were transferred to low-cholesterol medium with either vehicle
alone or 0.03% cholesterol and grown for 2 d at 25°C. After 2 d, 30 larvae
per sample were collected and homogenized in 100 mL of 13 buffer
included in the kit. The homogenate was centrifuged at 5000 rpm for
5 min and the supernatant was transferred to new tubes. A 50-mL aliquot
(15 larvae equivalents) was assayed according to the kit instructions and
measured in a Molecular Devices SpectraMax M2 fluorometer. Choles-
terol levels were normalized to protein in each homogenate using
a Bradford assay (Bio-Rad). Samples were prepared in triplicate, each
experiment was repeated at least three times, and the resulting data was
pooled. Data was analyzed using an unpaired two-tailed Student’s t-test
with unequal variance. All data are reported as the mean with 13 SEM.
See the Supplemental Material for additional materials and methods.
We thank M. Scott and L. Pallanck for stocks, A.-F. Ruaud and J. Metherall
for helpful discussions, B. Milash for advice on microarray analysis, M.
Carvalho and S. Eaton for the sterol-depleted food protocol, A. Kukis for
advice on lipid extraction and small molecule MS, and A.-F. Ruaud, K.
Schuske, M. Sieber, and J. Tennessen for comments on the manuscript.
This research was supported by NIH grant 1R01DK075607.
Bitsch F, Aichholz R, Kallen J, Geisse S, Fournier B, Schlaeppi JM. 2003.
Identification of natural ligands of retinoic acid receptor-related
orphan receptor a ligand-binding domain expressed in Sf9 cells–a mass
spectrometry approach. Anal Biochem 323: 139–149.
Brown MS, Goldstein JL. 1997. The SREBP pathway: Regulation of
cholesterol metabolism by proteolysis of a membrane-bound tran-
scription factor. Cell 89: 331–340.
Cooke J, Sang J. 1970. Utilization of sterols by larvae of Drosophila
melanogaster. J Insect Physiol 16: 801–812.
Davis HR Jr, Basso F, Hoos LM, Tetzloff G, Lally SM, Altmann SW. 2008.
Cholesterol homeostasis by the intestine: Lessons from Niemann-
Pick C1 Like 1 (NPC1L1). Atheroscler Suppl 9: 77–81.
Duval C, Touche V, Tailleux A, Fruchart JC, Fievet C, Clavey V, Staels B,
Lestavel S. 2006. Niemann-Pick C1 like 1 gene expression is down-
regulated by LXR activators in the intestine. Biochem Biophys Res
Commun 340: 1259–1263.
Fluegel ML, Parker TJ, Pallanck LJ. 2006. Mutations of a Drosophila NPC1
gene confer sterol and ecdysone metabolic defects. Genetics 172: 185–196.
Ge L, Wang J, Qi W, Miao HH, Cao J, Qu YX, Li BL, Song BL. 2008. The
cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-
induced internalization of NPC1L1. Cell Metab 7: 508–519.
Hennig KM, Colombani J, Neufeld TP. 2006. TOR coordinates bulk and
targeted endocytosis in the Drosophila melanogaster fat body to
regulate cell growth. J Cell Biol 173: 963–974.
Huang X, Suyama K, Buchanan J, Zhu AJ, Scott MP. 2005. A Drosophila
model of the Niemann-Pick type C lysosome storage disease: dnpc1a
is required for molting and sterol homeostasis. Development 132:
Huang X, Warren JT, Buchanan J, Gilbert LI, Scott MP. 2007. Drosophila
Niemann-Pick type C-2 genes control sterol homeostasis and steroid
biosynthesis: A model of human neurodegenerative disease. Devel-
opment 134: 3733–3742.
Ikonen E. 2008. Cellular cholesterol trafficking and compartmentaliza-
tion. Nat Rev Mol Cell Biol 9: 125–138.
Kalaany N, Mangelsdorf D. 2006. LXRS and FXR: The yin and yang of
cholesterol and fat metabolism. Annu Rev Physiol 68: 159–191.
King-Jones K, Horner MA, Lam G, Thummel CS. 2006. The DHR96
nuclear receptor regulates xenobiotic responses in Drosophila. Cell
Metab 4: 37–48.
Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P, Hogness DS.
1991. The Drosophila EcR gene encodes an ecdysone receptor, a new
member of the steroid receptor superfamily. Cell 67: 59–77.
Maxwell KN, Soccio RE, Duncan EM, Sehayek E, Breslow JL. 2003. Novel
putative SREBP and LXR target genes identified by microarray
analysis in liver of cholesterol-fed mice. J Lipid Res 44: 2109–2119.
Nuclear Receptors Nomenclature Committee. 1999. A unified nomen-
clature system for the nuclear receptor superfamily. Cell 97: 161–163.
Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE,
Mangelsdorf DJ. 1998. Cholesterol and bile acid metabolism are
impaired in mice lacking the nuclear oxysterol receptor LXR a. Cell
Phillips MD, Thomas GH. 2006. Brush border spectrin is required for
early endosome recycling in Drosophila. J Cell Sci 119: 1361–1370.
Reinking J, Lam MM, Pardee K, Samson HM, Liu S, Yang P, Williams S,
White W, Lajoie G, Edwards A, et al. 2005. The Drosophila nuclear
receptor E75 contains heme and is gas responsive. Cell 122: 195–207.
Rigamonti E, Helen L, Lestavel S, Mutka AL, Lepore M, Fontaine C,
Bouhel MA, Bultel S, Fruchart JC, Ikonen E, et al. 2005. Liver X
receptor activation controls intracellular cholesterol trafficking and
esterification in human macrophages. Circ Res 97: 682–689.
Sang J. 1956. The quantitative nutritional requirements of Drosophila
melanogaster. J Exp Biol 33: 45–72.
Shostak Y, Van Gilst MR, Antebi A, Yamamoto KR. 2004. Identification
of C. elegans DAF-12-binding sites, response elements, and target
genes. Genes & Dev 18: 2529–2544.
Sieber MH, Thummel CS. 2010. The DHR96 nuclear receptor controls
triacylglycerol homeostasis in Drosophila. Cell Metab (in press).
Va’nt Hoog EG. 1936. Aseptic culture of insects in vitamin research
(continued). Zt. Vitaminforsch. 5: 118–126.
Valasek MA, Clarke SL, Repa JJ. 2007. Fenofibrate reduces intestinal
cholesterol absorption via PPARa-dependent modulation of NPC1L1
expression in mouse. J Lipid Res 48: 2725–2735.
Voght SP, Fluegel ML, Andrews LA, Pallanck LJ. 2007. Drosophila NPC1b
promotes an early step in sterol absorption from the midgut epithe-
lium. Cell Metab 5: 195–205.
Wang DQ. 2007. Regulation of intestinal cholesterol absorption. Annu
Rev Physiol 69: 221–248.
Horner et al.
2716GENES & DEVELOPMENT