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mutants are unable to initiate metamorphosis because low levels of
Npc1a
result in reduced uptake of cholesterol and consequently insufficient ecdy-
sone production. However,
broad
mutants could not be rescued with
Npc1a
overexpression, indicating that the reason for the lethality is not just due to
the lack of NPC1a. The authors also report the identification of a 220-bp
DNA region within the
Npc1a
regulatory region that is necessary and suf-
ficient to drive the ring gland-specific expression of a reporter gene, which
constitutes the first
cis
-regulatory module (CRM) reported for the ring
gland. Studies in vertebrates have shown that the regulatory regions of
the human, porcine, and mouse
NPC1
genes harbor a conserved consensus
element recognized by sterol-regulatory element binding proteins
(SREBPs) (
Gevry, Schoonjans, Guay, & Murphy, 2008
). SREBP proteins
induce
NPC1
expression in steroidogenic cells under low intracellular cho-
lesterol concentrations. While this is an intriguing parallel,
Drosophila
SREBP (HLH106) has not been linked to ecdysone or cholesterol pathways
in prothoracic gland cells, but rather plays a role in regulating fatty acid bio-
synthesis (
Kunte, Matthews, & Rawson, 2006
).
The authors further suggest that the Z4 isoform of Broad directly binds
to a conserved 20-bp region within the ring gland CRM, based on published
binding data for the Z4 isoform we mentioned earlier. Experimentally, they
show that
broad
npr-3
, an allele of
broad
with defects in Z4, has very low
Npc1a
transcript levels, and that overexpression of Z4, but not Z1-Z3 leads to a
concomitant increase of
Npc1a
transcript levels. While these data convinc-
ingly tie BR-Z4 to the regulation of
Npc1a
, more experiments are needed to
show that the Z4 isoform is indeed binding the proposed ring gland CRM.
As the authors point out,
broad
expression is not restricted to the ring gland,
while the identified enhancer activity appears to be restricted to this tissue.
It is possible that other ring gland-specific transcription factors are required
to facilitate BR-Z4 binding to the ring gland CRM, but more data, such as
chromatin IP, are needed to demonstrate direct binding of BR-Z4 to this
DNA region.
Finally, a range of genes with known ring gland expression was severely
downregulated in a
broad
npr-3
mutant background, including several Hallow-
een genes and the PTTH receptor Torso (
Fig. 2.4
B). This raises the inter-
esting question as to whether
broad
acts as a general regulator of these critical
ecdysone biosynthetic genes, or whether this observation reflects a second-
ary response to the loss of
Npc1a
. It seems plausible that the reduced avail-
ability of cholesterol caused by the loss of functional NPC1a in turn triggers a
widespread feedback response that downregulates ecdysone production by
