Regulation of Drosophila Metamorphosis by Xenobiotic
Huai Deng, Tom K. Kerppola*
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional
responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the
functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes
regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions.
CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of
Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene
chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC
and dKeap1 depletion in the prothoracic gland as well as cncCK6/K6and dKeap1EY5/EY5loss of function mutations in embryos
reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1EY5/EY5loss of function
mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or
dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the
premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely,
constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes
at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription,
and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the
neuroendocrine axis that coordinates insect metamorphosis.
Citation: Deng H, Kerppola TK (2013) Regulation of Drosophila Metamorphosis by Xenobiotic Response Regulators. PLoS Genet 9(2): e1003263. doi:10.1371/
Editor: Eric Rulifson, University of California San Francisco, United States of America
Received June 6, 2012; Accepted December 6, 2012; Published February 7, 2013
Copyright: ? 2013 Deng, Kerppola. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by a grant from the National Institute on Drug Abuse (DA030339) and by a fellowship from the University of Michigan Center for
Organogenesis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Cellular responses to many xenobiotic compounds, including
various toxins and pharmacological agents, are controlled by
mammalian Nrf2 and Keap1, and by the homologous Drosophila
CncC and dKeap1 proteins [1,2,3]. The Nrf2-Keap1 complex has
multiple effects on carcinogenesis. Nrf2-deficient mice have
increased susceptibility to chemical carcinogens, potentially
because of defective activation of cytoprotective genes in response
to carcinogen exposure . Mutations in Nrf2 and Keap1 that are
predicted to disrupt their interactions are found in many human
cancers, suggesting that Nrf2 interactions with Keap1 counteract
cancer progression [1,5]. Conversely, the deletion of Nrf2
suppresses pancreatic and lung tumorigenesis in a mouse model
with constitutively active K-RasG12Dexpression . The mech-
anisms whereby Nrf2 promotes tumorigenesis in conjunction with
K-RasG12Dare not known. Nrf2 and Keap1 are investigated as
potential targets for therapeutic interventions in cancer, neurode-
generative diseases and developmental disorders [1,7].
Nrf2 (NF-E2-Related Factor 2) is a bZIP family transcription
factor that can bind to genes whose transcription is induced by
xenobiotic compounds . Keap1 (Kelch-like ECH-Associated
Protein 1) is a Kelch family protein that can interact with the N-
terminal region of Nrf2, and inhibits the activation of many genes
activated by Nrf2 . Studies in cultured mammalian cells
indicate that Keap1 is predominantly localized to the cytoplasm
, where it promotes Nrf2 degradation and inhibits its
accumulation in the nucleus [8,10,11,12].
Studies of the Drosophila homologues of Nrf2 and Keap1 have
provided insights into the functions of these protein families in
adult flies. The Drosophila cap‘n’collar locus encodes CncC, which
contains a bZIP domain homologous to that of Nrf2 and N-
terminal DLG and ETGE motifs homologous to those that
mediate Nrf2 interaction with Keap1  (Figure 1A). Drosophila
dKeap1 contains Kelch repeats homologous to those that mediate
Keap1 interaction with Nrf2 as well as a sequence motif that is
required for mammalian Keap1 export from the nucleus [3,10].
Overexpression of CncC and depletion of dKeap1 in adult flies
activates the transcription of many genes that protect cells from
xenobiotic compounds, whereas dKeap1 overexpression represses
their transcription, indicating that the functions of these protein
families in the xenobiotic response are conserved between
mammals and Drosophila [2,3].
Several lines of evidence suggest that CncC and dKeap1 also
affect cell proliferation and development. CncC overexpression
and dKeap1 depletion inhibit intestinal stem cell proliferation, and
counteract the proliferative effects of environmental stress in these
cells . Loss of function mutations in cncC and dKeap1 cause
larval lethality [3,15]. The genes regulated by CncC and dKeap1
during larval development had not been established. Elucidation
PLOS Genetics | www.plosgenetics.org1February 2013 | Volume 9 | Issue 2 | e1003263
of the relationship between CncC and dKeap1 functions in
xenobiotic responses and in development is important to define
how the transcription regulatory functions of CncC and dKeap1
are regulated in response to intrinsic and extrinsic stimuli.
In Drosophila and in other holometabolous insects, the onset of
metamorphosis is triggered by an increase in the level of the
endocrine hormone ecdysone [16,17]. Ecdysone is synthesized in
the prothoracic gland (PG) by a series of cytochrome P450 enzymes
. The expression of these ecdysone-biosynthetic genes and the
timing of pupation are regulated by Ras signaling in response to
prothoracicotropic hormone (PTTH) binding to the Torso receptor
[19,20]. Ecdysone facilitates the onset of metamorphosis by
regulating transcription in many tissues, including the salivary
glands where ecdysone-regulated transcription is manifest by puffs
at specific polytene chromosome loci . The transcription factors
that bind to the ecdysone biosynthetic gene promoters and activate
their transcription have remained unknown.
In the work presented here, we found that CncC and dKeap1
occupied the classical ecdysone-regulated puffs on polytene
chromosomes. Depletion of CncC or of dKeap1 in salivary glands
reduced ecdysone-regulated gene transcription. Depletion of
CncC or of dKeap1 in the PG as well as cncC and dKeap1 loss of
function mutations reduced ecdysone biosynthetic gene transcrip-
tion in larvae and in embryos, respectively. The reduced
ecdysteroid levels caused by CncC and by dKeap1 depletion in
the PG delayed pupation and suppressed the premature pupation
caused by constitutive Ras signaling. These observations establish
roles for CncC and dKeap1 in transcriptional programs in
different tissues that coordinate metamorphosis.
Nuclear localization of CncC and dKeap1
To investigate if the subcellular localization of CncC was
regulated by dKeap1 in the manner that has been reported for
mammalian Nrf2 and Keap1, we determined the distributions of
CncC and dKeap1. Both CncC and dKeap1 immunoreactivity
were predominantly nuclear in Drosophila salivary gland cells
(Figure 1B, Figure S1A). Likewise, ectopic CncC and dKeap1
fused to fluorescent proteins were enriched within the nuclei of live
salivary gland cells (Figure 1B, Figure S1A). CncC and dKeap1
were also present in the nuclei of prothoracic gland, imaginal disc
and gut cells, though the proportions that were localized to the
nucleus varied in different tissues (Figure 1C, Figure S1B). The
intensity of anti-dKeap1 immunoreactivity was markedly reduced
in dKeap1EY5/EY5mutant larvae, and the bands corresponding to
endogenous dKeap1 and CncC were not detected by immuno-
blotting of extracts from dKeap1EY5/EY5and cncK6/K6mutant larvae,
demonstrating the specificity of these antibodies (Figure S1C,
S1D). These observations establish that both endogenous as well as
ectopically expressed CncC and dKeap1 were localized to the
nuclei in many different tissues, in contrast to the predominantly
cytoplasmic localization observed for Keap1 and Nrf2 in many
cultured mammalian cell lines.
CncC and dKeap1 occupancy at ecdysone-regulated
puffs on polytene chromatin
To establish if CncC and dKeap1 bound to specific chromatin
loci, we visualized their occupancy on polytene chromosomes by
immunostaining. Anti-CncC and anti-dKeap1 antibodies recog-
nized overlapping sets of loci, including a majority of the classical
ecdysone-regulated early puffs on polytene chromosomes (e.g. 2B,
74EF, 75B, 63F, and 25B) (Figure 1D). Anti-CncC antibodies also
recognized several loci that were not detected by anti-dKeap1
antibodies (e.g. 22B and 97B) and vice versa (e.g. 50C and 94C).
CncC and dKeap1 occupied many non-puff loci, and did not
occupy all puffs, indicating that their occupancy was not controlled
solely by chromatin decondensation. Ectopically expressed CncC
and dKeap1 fusion proteins occupied loci that overlapped those
occupied by endogenous CncC and dKeap1, though they also
occupied additional loci (Figure 1D). Few other sequence-specific
DNA binding proteins have been identified that bind to ecdysone-
regulated puffs [22,23,24]. The overlapping sets of loci occupied
by endogenous and ectopic CncC and dKeap1, as detected by
several different antibodies, corroborate the specificity of CncC
and dKeap1 binding at these loci.
Regulation of ecdysone response genes by CncC and
dKeap1 in salivary glands
To test if CncC and dKeap1 regulated transcription of the early
puff genes that they occupied on polytene chromosomes, we
investigated the effects of CncC as well as dKeap1 depletion in
salivary glands on transcription of ecdysone-regulated genes.
Expression of an shRNA that targets CncC  under the control
of either the 71B-GAL4 or the Sgs3-GAL4 driver reduced the levels
of almost all of the ecdysone-regulated early puff and glue gene
transcripts examined (Figure 2A). In contrast, transcription of most
of the late puff genes that were not prominently occupied by CncC
or dKeap1 was not affected by CncC depletion (Figure 2A). 71B-
GAL4 directs expression throughout salivary gland development
and in imaginal discs ; Transcription directed by Sgs3-GAL4 is
detected only in late 3rdinstar salivary glands , establishing
that the change in transcription of ecdysone-regulated genes was
due to CncC depletion in salivary glands. Expression of a different
shRNA that targets all Cnc isoforms also reduced the levels of all
of the early puff and glue gene transcripts examined (Figure 2A).
The cncC-RNAi transgene had no detectable effects on transcrip-
tion in larvae that lacked a GAL4 driver (Figure S2A).
Expression of an shRNA that targets dKeap1  under the
control of the Sgs3-GAL4 driver also reduced the levels of almost all
of the ecdysone-regulated early puff and glue gene transcripts
examined, but had no effect on most of the late puff gene
Human Nrf2-Keap1 and the fruit fly CncC-dKeap1 protein
complexes function both in response to foreign chemicals
and in development. We found that CncC and dKeap1
control fruit fly development by regulating the production
and actions of the principal hormone that controls the
transformation of larvae into pupae. In hormone-respon-
sive cells, CncC and dKeap1 bound to the genes that are
activated by the hormone. When the amount of CncC or
dKeap1 in these cells was reduced, the genes were not
activated efficiently. When the amount of CncC or dKeap1
was reduced in the organ where the hormone is made, the
genes whose products make the hormone were not
activated efficiently. Because less hormone was made, it
took longer for the larvae to turn into pupae, and the
resulting pupae were bigger. Reduction of the amount of
CncC intercepted previously identified signals for pupa-
tion. Nrf2 is required for the same signals to cause cancer
in mice. The effects of CncC and dKeap1 both on genes
that control hormone production and on genes that are
switched on by the hormone in different organs indicate
that they have multiple roles in the transformation of fruit
fly larvae into pupae.
CncC-dKeap1 Control of Ecdysone Synthesis/Actions
PLOS Genetics | www.plosgenetics.org2February 2013 | Volume 9 | Issue 2 | e1003263
measured in larvae produced by two sub-lines that had been
propagated separately for more than two years (71B.cncC-RNAi-1
and 71B.cncC-RNAi-2, striped and solid bars) and in larvae that
carried the cncC-RNAi transgene, but lacked a GAL4 driver (open
bars). To facilitate comparison of the transcript levels, the level of
each transcript was normalized by the level of the transcript in the
control larvae (71B-GAL4 or w1118). All transcript levels were
normalized by the levels of the Rp49 transcript. The data represent
the means and the standard deviations from two separate
experiments (*, p,0.05). (B). Effects of CncC depletion in the
salivary glands on the level of 20E in the larvae. The levels of 20-
hydroxyecdysone (20E) were measured in the salivary glands of
early wandering third instar larvae of control larvae (71B.) and
transgenic larvae that expressed the shRNA targeting CncC under
the control of the 71B-GAL4 driver (71B.cncC-RNAi). The data
represent the means and standard deviations from two repeats
using 10 larvae in each. Results: We considered the possibility that
the effects of CncC depletion on ecdysone-regulated gene
transcription were caused by changes in ecdysteroid levels in the
larvae. CncC depletion in the salivary glands had no detectable
effect on the level of 20E in the larvae.
ecdysone biosynthetic gene transcription and PG morphology.
CncC and dKeap1 occupancy at the sad gene. (A). Effects of CncC
depletion on the transcription of ecdysone biosynthetic genes in
prothoracic glands. The levels of the transcripts indicated above
the upper graphs were measured in the brain complexes from
control larvae (5015., open bar) and from larvae produced by
two sub-lines expressing the shRNA targeting CncC under the
control of the 5015-GAL4 driver (5015.cncC-RNAi-1 and
5015.cncC-RNAi-2, striped and solid bar). The data represent
the means and standard deviations of two separate experiments (*,
p,0.05). Results: The same changes in ecdysone biosynthetic gene
transcription in the PG were observed in both cncC-RNAi sub-lines,
demonstrating the genetic stability and reproducibility of these
effects. (B). Effects of CncC depletion in the PG on Sad protein
expression. The dissected brain complexes of control larvae
(phm.) and larvae that expressed the shRNA targeting CncC in
the PG (phm.cncC-RNAi) were stained with anti-Sad antibody (red)
and Hoechst (cyan). Results: Expression of the shRNA targeting
CncC in PG under the control of phm-GAL4 driver reduced Sad
immunoreactivity in the PG. The polyploid nuclei of the PG were
identified based on their large size compared to the diploid nuclei
of the brain (right panels). The size and the number of nuclei in the
PG were not altered by expression of the shRNA targeting CncC,
suggesting that CncC depletion did not disrupt the overall
structure of the PG (see also Figure 3C). (C). Effects of dKeap1
depletion on PG morphology. The dissected brain complexes of
larvae that expressed the shRNA targeting dKeap1 in the PG were
stained using Hoechst. Results: The size and the number of nuclei
in the PG were not altered by expression of the shRNA targeting
dKeap1, suggesting that dKeap1 depletion did not disrupt the
overall structure of the PG. (D). CncC and dKeap1 occupancy of
different regions of the sad gene and of the promoter regions of
other ecdysone biosynthetic genes in late embryos. Chromatin
isolated from stage 14–16 embryos was precipitated using anti-
dKeap1 (striped bar), anti-CncC (solid bar) or pre-immune (open
bar) sera. The promoter regions of the genes indicated below the
bars, and regions at different distances from the sad transcription
start site as indicated below the bars were quantified using qPCR.
The data represent the mean values and standard deviations of
replicate qPCR reactions, and are representative of two indepen-
dent experiments. Results: CncC and dKeap1 were enriched near
Effects of CncC and dKeap1 depletion in the PG on
the transcription start site of the sad gene as well as at the
promoter regions of other ecdysone biosynthetic genes.
ecdysteroid level. (A). Expression of the shRNA targeting CncC in
the PG produces giant semi-pupae. Comparison of the pupae
formed by control larvae (phm.) and semi-pupae formed by larvae
that expressed the shRNA targeting CncC under the control of the
phm-GAL4 driver (phm.cncC-RNAi) 7 days after 3rdinstar ecdysis.
Results: A small proportion (2–5%) of the larvae that expressed the
shRNA targeting CncC in the PG did not fully pupate within 7
days after 3rdinstar ecdysis and formed giant semi-pupae. (B).
Effects of expression of the shRNA targeting CncC in the PG on
20E biosynthesis. The levels of 20E were measured in early
wandering control larvae (5015.) and larvae that expressed the
shRNA targeting CncC in the PG (5015.cncC-RNAi-1 and
5015.cncC-RNAi-2) 60 hours after 3rdinstar ecdysis. The data
represent the means and standard deviations from two separate
experiments with 10 larvae each (*, p,0.05).
Effects of CncC depletion in the PG on pupation and
the PG. (A). Effects of the expression of RasV12and the shRNA
targeting CncC separately and together in the PG on pupal
development. The proportion of pupae that were arrested at early
(P1–P9; open bar) and late (P10–P15; striped bar) stages as well as
those that eclosed and produced adults (solid bar) were recorded
for larvae that expressed either RasV12alone (phm.rasV12), RasV12
in combination with the shRNA targeting CncC (phm.rasV12,
cncC-RNAi), or the the shRNA targeting CncC alone (phm.cncC-
RNAi) in the PG. The proportion of animals in each category was
determined in approximately 100 animals of each genotype.
Results: To determine the effects of the genetic interaction
between the expression of RasV12and of the shRNA targeting
CncC in the PG, we examined their effects on pupal development.
When RasV12was expressed alone under the control of the phm-
GAL4 driver, 93% of the pupae were arrested at early stages with
no detectable eye pigmentation or wings. In contrast, the majority
(68%) of pupae formed by larvae that expressed RasV12in
combination with the shRNA targeting CncC under the control of
the phm-GAL4 driver developed to late stages with detectable eye
pigmentation and wings. The genetic interactions between
expression of RasV12and the shRNA targeting CncC suggest that
CncC mediated the regulation of pupation by the Ras signaling
pathway. (B). The transcription of cncC was not affected by
RasV12 expression in the salivary glands. The level of the
transcript was measured in the salivary glands of control larvae
(Sgs3., open bar) and of larvae that expressed RasV12in the
salivary glands (Sgs3.rasV12, solid bar). The transcript level was
normalized by the level of the Rp49 transcript and represents the
mean and standard deviation from two separate experiments.
Genetic interactions between RasV12and CncC in
Primer sequences used to measure the transcript levels
occupancy by ChIP.
Primer sequences used to measure CncC and dKeap1
Plasmid expression vectors. Drosophila stocks. Antisera, polytene
chromosome squash, immunostaining and imaging. Immunoblot-
ting. Quantitation of transcript levels. Chromatin immunoprecip-
Supporting Materials, Methods, and References.
CncC-dKeap1 Control of Ecdysone Synthesis/Actions
PLOS Genetics | www.plosgenetics.org12 February 2013 | Volume 9 | Issue 2 | e1003263
itation (ChIP) analysis. Analysis of the time of pupation.
Measurement of 20E levels. Statistical analyses.
We thank Sergei Avedisov for construction of the CncC, CncB, and
dKeap1 expression vectors; Osamu Shimmi for preparing the Drosophila
lines expressing CncC, CncB, and dKeap1 fusion proteins; Dirk Bohmann
and Michael O’Connor for Drosophila stocks; Zhe Han for sharing
equipment; and members of the Kerppola laboratory for stimulating
Conceived and designed the experiments: HD TKK. Performed the
experiments: HD. Analyzed the data: HD TKK. Contributed reagents/
materials/analysis tools: HD TKK. Wrote the paper: TKK HD.
1. Taguchi K, Motohashi H, Yamamoto M (2011) Molecular mechanisms of the
Keap1-Nrf2 pathway in stress response and cancer evolution. Genes Cells 16:
2. Misra JR, Horner MA, Lam G, Thummel CS (2011) Transcriptional regulation
of xenobiotic detoxification in Drosophila. Genes Dev 25: 1796–1806.
3. Sykiotis GP, Bohmann D (2008) Keap1/Nrf2 signaling regulates oxidative stress
tolerance and lifespan in Drosophila. Dev Cell 14: 76–85.
4. Slocum SL, Kensler TW (2011) Nrf2: control of sensitivity to carcinogens. Arch
Toxicol 85: 273–284.
5. Padmanabhan B, Tong KI, Ohta T, Nakamura Y, Scharlock M, et al. (2006)
Structural basis for defects of Keap1 activity provoked by its point mutations in
lung cancer. Mol Cell 21: 689–700.
6. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, et al. (2011)
Oncogene-induced Nrf2 transcription promotes ROS detoxification and
tumorigenesis. Nature 475: 106–109.
7. Calabrese V, Cornelius C, Dinkova-Kostova AT, Calabrese EJ, Mattson MP
(2010) Cellular stress responses, the hormesis paradigm, and vitagenes: novel
targets for therapeutic intervention in neurodegenerative disorders. Antioxid
Redox Signal 13: 1763–1811.
8. Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, et al. (1999) Keap1
represses nuclear activation of antioxidant responsive elements by Nrf2 through
binding to the amino-terminal Neh2 domain. Genes Dev 13: 76–86.
9. Watai Y, Kobayashi A, Nagase H, Mizukami M, McEvoy J, et al. (2007)
Subcellular localization and cytoplasmic complex status of endogenous Keap1.
Genes Cells 12: 1163–1178.
10. Sun Z, Zhang S, Chan JY, Zhang DD (2007) Keap1 controls postinduction
repression of the Nrf2-mediated antioxidant response by escorting nuclear
export of Nrf2. Mol Cell Biol 27: 6334–6349.
11. Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, et al. (2004) Oxidative
stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate
proteasomal degradation of Nrf2. Mol Cell Biol 24: 7130–7139.
12. Nguyen T, Sherratt PJ, Nioi P, Yang CS, Pickett CB (2005) Nrf2 controls
constitutive and inducible expression of ARE-driven genes through a dynamic
pathway involving nucleocytoplasmic shuttling by Keap1. J Biol Chem 280:
13. McMahon M, Thomas N, Itoh K, Yamamoto M, Hayes JD (2006) Dimerization
of substrate adaptors can facilitate cullin-mediated ubiquitylation of proteins by a
‘‘tethering’’ mechanism: a two-site interaction model for the Nrf2-Keap1
complex. J Biol Chem 281: 24756–24768.
14. Hochmuth CE, Biteau B, Bohmann D, Jasper H (2011) Redox regulation by
Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila. Cell
Stem Cell 8: 188–199.
15. Veraksa A, McGinnis N, Li X, Mohler J, McGinnis W (2000) Cap ‘n’ collar B
cooperates with a small Maf subunit to specify pharyngeal development and
suppress deformed homeotic function in the Drosophila head. Development 127:
16. Thummel CS (2001) Molecular mechanisms of developmental timing in C.
elegans and Drosophila. Dev Cell 1: 453–465.
17. Dubrovsky EB (2005) Hormonal cross talk in insect development. Trends
Endocrinol Metab 16: 6–11.
18. Rewitz KF, Rybczynski R, Warren JT, Gilbert LI (2006) The Halloween genes
code for cytochrome P450 enzymes mediating synthesis of the insect moulting
hormone. Biochem Soc Trans 34: 1256–1260.
19. Rewitz KF, Yamanaka N, Gilbert LI, O’Connor MB (2009) The insect
neuropeptide PTTH activates receptor tyrosine kinase torso to initiate
metamorphosis. Science 326: 1403–1405.
20. Caldwell PE, Walkiewicz M, Stern M (2005) Ras activity in the Drosophila
prothoracic gland regulates body size and developmental rate via ecdysone
release. Curr Biol 15: 1785–1795.
21. Ashburner M (1972) Patterns of puffing activity in the salivary gland
chromosomes of Drosophila. VI. Induction by ecdysone in salivary glands of
D. melanogaster cultured in vitro. Chromosoma 38: 255–281.
22. Cherbas L, Hu X, Zhimulev I, Belyaeva E, Cherbas P (2003) EcR isoforms in
Drosophila: testing tissue-specific requirements by targeted blockade and rescue.
Development 130: 271–284.
23. Hill RJ, Segraves WA, Choi D, Underwood PA, Macavoy E (1993) The reaction
with polytene chromosomes of antibodies raised against Drosophila E75A
protein. Insect Biochem Mol Biol 23: 99–104.
24. Fletcher JC, Thummel CS (1995) The ecdysone-inducible Broad-complex and
E74 early genes interact to regulate target gene transcription and Drosophila
metamorphosis. Genetics 141: 1025–1035.
25. Busson D, Pret AM (2007) GAL4/UAS targeted gene expression for studying
Drosophila Hedgehog signaling. Methods Mol Biol 397: 161–201.
26. Li HM, Buczkowski G, Mittapalli O, Xie J, Wu J, et al. (2008) Transcriptomic
profiles of Drosophila melanogaster third instar larval midgut and responses to
oxidative stress. Insect Mol Biol 17: 325–339.
27. Yoshiyama T, Namiki T, Mita K, Kataoka H, Niwa R (2006) Neverland is an
evolutionally conserved Rieske-domain protein that is essential for ecdysone
synthesis and insect growth. Development 133: 2565–2574.
28. Mirth C, Truman JW, Riddiford LM (2005) The role of the prothoracic gland in
determining critical weight for metamorphosis in Drosophila melanogaster. Curr
Biol 15: 1796–1807.
29. Roth GE, Gierl MS, Vollborn L, Meise M, Lintermann R, et al. (2004) The
Drosophila gene Start1: a putative cholesterol transporter and key regulator of
ecdysteroid synthesis. Proc Natl Acad Sci U S A 101: 1601–1606.
30. Gibbens YY, Warren JT, Gilbert LI, O’Connor MB (2011) Neuroendocrine
regulation of Drosophila metamorphosis requires TGFbeta/Activin signaling.
Development 138: 2693–2703.
31. Layalle S, Arquier N, Leopold P (2008) The TOR pathway couples nutrition
and developmental timing in Drosophila. Dev Cell 15: 568–577.
32. Guengerich FP (2006) Cytochrome P450s and other enzymes in drug
metabolism and toxicity. AAPS J 8: E101–111.
33. Halme A, Cheng M, Hariharan IK (2010) Retinoids regulate a developmental
checkpoint for tissue regeneration in Drosophila. Curr Biol 20: 458–463.
34. Gruntenko NE, Rauschenbach IY (2008) Interplay of JH, 20E and biogenic
amines under normal and stress conditions and its effect on reproduction. J Insect
Physiol 54: 902–908.
35. Zipper LM, Mulcahy RT (2003) Erk activation is required for Nrf2 nuclear
localization during pyrrolidine dithiocarbamate induction of glutamate cysteine
ligase modulatory gene expression in HepG2 cells. Toxicol Sci 73: 124–134.
36. Sun Z, Huang Z, Zhang DD (2009) Phosphorylation of Nrf2 at multiple sites by
MAP kinases has a limited contribution in modulating the Nrf2-dependent
antioxidant response. PLoS ONE 4: e6588. doi:10.1371/journal.pone.0006588
37. Inoue H, Hisamoto N, An JH, Oliveira RP, Nishida E, et al. (2005) The C.
elegans p38 MAPK pathway regulates nuclear localization of the transcription
factor SKN-1 in oxidative stress response. Genes Dev 19: 2278–2283.
38. Chan JY, Kwong M, Lu R, Chang J, Wang B, et al. (1998) Targeted disruption
of the ubiquitous CNC-bZIP transcription factor, Nrf-1, results in anemia and
embryonic lethality in mice. EMBO J 17: 1779–1787.
39. Chan K, Lu R, Chang JC, Kan YW (1996) NRF2, a member of the NFE2
family of transcription factors, is not essential for murine erythropoiesis, growth,
and development. Proc Natl Acad Sci U S A 93: 13943–13948.
40. Chen L, Kwong M, Lu R, Ginzinger D, Lee C, et al. (2003) Nrf1 is critical for
redox balance and survival of liver cells during development. Mol Cell Biol 23:
41. Leung L, Kwong M, Hou S, Lee C, Chan JY (2003) Deficiency of the Nrf1 and
Nrf2 transcription factors results in early embryonic lethality and severe
oxidative stress. J Biol Chem 278: 48021–48029.
42. Malhotra D, Portales-Casamar E, Singh A, Srivastava S, Arenillas D, et al.
(2010) Global mapping of binding sites for Nrf2 identifies novel targets in cell
survival response through ChIP-Seq profiling and network analysis. Nucleic
Acids Res 38: 5718–5734.
43. Chorley BN, Campbell MR, Wang X, Karaca M, Sambandan D, et al. (2012)
Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid
X receptor alpha. Nucleic Acids Res 40: 7416–7429.
44. Bobilev I, Novik V, Levi I, Shpilberg O, Levy J, et al. (2011) The Nrf2
transcription factor is a positive regulator of myeloid differentiation of acute
myeloid leukemia cells. Cancer Biol Ther 11: 317–329.
CncC-dKeap1 Control of Ecdysone Synthesis/Actions
PLOS Genetics | www.plosgenetics.org13 February 2013 | Volume 9 | Issue 2 | e1003263