Institut de Génétique et de Biologie Moléculaire et Cellulaire, Department of Developmental Biology, CNRS/INSERM/ULP, Boite Postale 10142, 67404 Illkirch Cedex, France
* Author for correspondence (e-mail: phr{at}igbmc.u-strasbg.fr)
Accepted 28 July 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Toutatis, Pnr, Chip, Iswi, Neural development, achaete-scute, Enhancer-promoter communication, Drosophila melanogaster
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The patterning of the large sensory bristles (macrochaetae) on the thorax
of Drosophila melanogaster is a powerful model to study how enhancers
communicate with promoters during regulation of gene expression. Each
macrochaeta derives from a precursor cell selected from a group of equivalent
ac-sc-expressing cells, the proneural cluster
(Gomez-Skarmeta et al., 2003).
ac and sc encode basic helix-loop-helix proteins (bHLH) that
heterodimerize with Daughterless (Da) to activate expression of downstream
genes required for neural fate. Transcription of ac and sc
in the different sites of the imaginal disc is initiated by enhancers of the
ac-sc complex (Gomez-Skarmeta et
al., 1995
; Garcia-Garcia et
al., 1999
) and the expression is maintained throughout development
by autoregulation mediated by the (Ac-Sc)-Da heterodimers binding to E boxes
within the ac-sc promoters
(Martinez and Modolell, 1991
;
Van Doren et al., 1991
;
Van Doren et al., 1992
). Each
enhancer interacts with specific transcription factors that are expressed in
broader domains than the proneural clusters and define the bristle prepattern
(Gomez-Skarmeta et al., 2003
).
Thus, the GATA factor Pannier (Pnr)
(Ramain et al., 1993
;
Heitzler et al., 1996
) binds
to the dorsocentral (DC) enhancer
(Garcia-Garcia et al., 1999
)
and activates proneural expression to promote development of DC sensory
organs. The Drosophila LIM-domain-binding protein 1 (Ldb1), Chip
(Morcillo et al., 1997
)
physically interacts both with Pnr and the (Ac-Sc)-Da heterodimer to give a
multiprotein proneural complex which facilitates the enhancer-promoter
communication (Dorsett, 1999
;
Ramain et al., 2000
).
Chromatin plays a crucial role in control of eukaryotic gene expression and
is a highly dynamic structure at promoters
(Näär et al., 2001).
In Drosophila, the polycomb (Pc) group and the trithorax (Trx) group
proteins are chromatin components that maintain stable states of gene
expression and are involved in various complexes
(Simon and Tamkun, 2002
). The
Pc group proteins are required to maintain repression of homeotic genes such
as Ultrabithorax, presumably by inducing a repressive chromatin
structure. Members of the Trx group were identified by their ability to
suppress dominant Polycomb phenotypes. We recently provided evidence that
enhancer-promoter communication during Pnr-driven proneural development is
negatively regulated by the Brahma (Brm) chromatin remodelling complex
(Heitzler et al., 2003
;
Treisman et al., 1997
;
Collins et al., 1999
),
homologous to the yeast SWI/SNF complex.
Here, we present Toutatis (Tou), a protein that associates both with Pnr
and Chip and that positively regulates activity of the proneural complex
encompassing Pnr and Chip during enhancer-promoter communication. Tou has been
previously identified in a genetic screen for dominant modifiers of the
extra-sex-combs phenotype displayed by mutant of polyhomeotic
(ph), a member of the Pc group in Drosophila
(Fauvarque et al., 2001). Tou
shares functional domains with Acf1, a subunit of both the human and
Drosophila ACF (ATP-utilizing chromatin assembly and remodelling
factor) and CHRAC (chromatin accessibility complex)
(Ito et al., 1999
), and with
TIP5 of NoRC (nucleolar remodelling complex)
(Strohner et al., 2001
).
Hence, Tou regulates activity of the proneural complex during
enhancer-promoter communication, possibly through chromatin remodelling.
Moreover, we show that Iswi (Deuring et
al., 2000
), a highly conserved member of the SWI2/SNF2 family of
ATPases, is also necessary for activation of ac-sc and neural
development. Since Iswi is shown to physically interact with Tou, Pnr and
Chip, we suggest that a complex encompassing Tou and Iswi directly regulates
activity of the proneural complex during enhancer-promoter communication,
possibly through chromatin remodelling.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ß-Galactosidase assays on transformants of the L40 yeast strain were
carried out as described previously
(Seipel et al., 1992).
VP16AD and LexADBD fusion proteins expressed throughout
the current study did not activate the ß-galactosidase reporter in L40
cells. Expression of LexA and VP16 fusion proteins were analysed by western
blot using anti-LexA- or anti-VP16-specific antibodies.
Plasmid constructions
Sequences encoding the Tou domains (TouA: Thr546-Met1415, TouB:
Met1-Ala600, TouC: Thr546-Glu1120, TouD: Ala1089-Met1415, TouE:
Pro1303-Iso1866, TouF: Pro1826-Val2086, TouG: Asp2059-Asn2615, TouH:
Pro2446-Leu2857, TouI: His2831-Ser3116, TouJ: Thr546-Glu953, TouK:
Leu954-Glu1120, TouL: Ala1089-Pro1238, TouM: Gly1239-Asn1307 and TouN:
Pro1308-Met1415) were PCR amplified and inserted in frame with
VP16AD into pASV4. The fragment encoding TouA was also inserted
into pXJB, which allows expression in transfected Cos cells of tagged proteins
carrying the B epitope of the oestrogen receptor at their N terminus
(Ramain et al., 2000). The
expression vector pXJPnr, encoding the truncated wild-type Pnr has been
described previously (Haenlin et al.,
1997
). The expression vector encoding the full-length Chip
carrying a N-terminal Flag epitope (F-Chip) has also been described previously
(Ramain et al., 2000
).
pXJF-Iswi encodes a full-length Iswi carrying an N-terminal Flag epitope. The
expression vectors encoding the N-terminal domain of Chip
(B10-NTChip) and the C-terminal domain of Chip
(B10-CTChip) carrying at their N terminus the B epitope of the
oestrogen receptor were described by Ramain et al.
(Ramain et al., 2000
).
Sequences encoding the NT Chip, the CT Chip and Iswi
were also inserted in frame with the LexADBD into pBTM116.
Fly stocks and genetics
The following mutant stocks were used: pnrD1,
pnrVX1 (Ramain et al.,
1993), ChipE
(Ramain et al., 2000
) and
osa616 (Treisman et
al., 1997
). tou1 and tou2
(Fauvarque et al., 2001
).
touE44.1 was generated by imprecise excision of
EY08961 (Bellen et al.,
2004
). Misexpression of full-length transgenes was achieved using
the UAS/Gal4 system, using the following stocks: EP622, an EP target element
from the collection of P. Rorth (Rorth,
1996
) allowing overexpression of tou,
UAS-IswiK159R (Deuring et
al., 2000
). Thirty hemithoraces were examined for each of the
genotypes presented in Fig. 2.
For each genotype, the phenotype was found to be remarkably similar from fly
to fly.
DNA tranfections in Cos cells, immunoprecipitations
Cell transfections, protein extracts preparations, immunoprecipitations and
western blot analysis follow the method of Haenlin et al.
(Haenlin et al., 1997).
GST pull-down assays
The method of Ramain et al. (Ramain et
al., 2000) was used for GST pull-down assays.
Staining for ß-galactosidase activity
Wing discs were stained using the method of Cubadda et al.
(Cubadda et al., 1997). A101
contains a lacZ gene inserted into the neuralized locus
(Boulianne et al., 1991
) and is
found by specific staining in all sensory organs precursors
(Huang et al., 1991
). The
transgenic strain DC-aclacZ has been described previously
(Garcia-Garcia et al., 1999
).
In Fig. 6, each staining was
for one hour at 22°C and a representative imaginal disc is shown for each
genotype. The stainings were done at different stages of the study but they
were performed using the same batch of reagents. In addition, control
stainings of imaginal discs from wild-type larvae were included in each
experiment to allow comparisons between the different genotypes.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Toutatis function is required for neural development
Pnr promotes development of DC sensory organs
(Garcia-Garcia et al., 1999;
Ramain et al., 2000
). Tou
physically interacts with Pnr in yeast and tou mRNA was found to be
ubiquitously expressed in the wing imaginal disc (data not shown).
tou mRNA is expressed in the wing pouch, in agreement with the wing
defects associated with tou alleles
(Fauvarque et al., 2001
) and
in the dorsal-most region of the thorax, covering the site of appearance of
the DC bristles and the domain of pnr expression. Hence, we asked
whether Tou is also required during development of DC sensory organs.
tou1 corresponds to a PlacW transposon insertion within
the large intron of tou at 836 bases pairs (bp) upstream of the
second exon of tou that contains the ATG translational start
(Fig. 1A) (Fauvarque et al., 2001).
tou2 was generated by imprecise excision of the
tou1 insertion, in which 200 bp of the intron was replaced
by a 7 kb sequence from the P-element. Oregon wild-type flies have 2.00 DC
bristles per heminotum (Fig.
2A) We found that both tou1 and
tou2 give rise to homozygous escapers lacking DC bristles
(Fig. 2B) (1.92±0.05 and
1.4±0.05 DC bristles/heminotum, respectively). It suggests that
tou is necessary for DC neural development. tou1
and tou2 are associated with molecular lesions affecting
intronic sequences and display an abnormal bristle phenotype, suggesting that
tou1 and tou2 disrupt functioning of
an important regulatory element. Accordingly, the level of tou
expression is strongly reduced in tou2 thoracic discs
(data not shown), suggesting that tou2 is likely to be a
loss of function allele. This hypothesis is reinforced by the fact that the
loss of DC bristles is more severe for tou1/Df(2R)en-SFX31
and tou2/Df(2R)en-SFX31 (1.23±0.09 and
1.15±0.06 DC bristles/heminotum, respectively) than for
tou1 and tou2 (1.92±0.05 and
1.4±0.05 DC bristles/heminotum, respectively). Df(2R)en-SFX31
is a deficiency that spans tou and other genes between 48A1 and 48B5.
Moreover, the requirement for tou to promote DC neural development is
further demonstrated by analysis of touE44.1.
touE44.1 was generated by imprecise excision of the transposon
insertion in EY08961 (Fig.
1A) (Bellen et al.,
2004
) and encodes a mutant protein lacking the C terminus,
containing the PHD fingers and the bromodomain. Since the
touE44.1 flies lack DC bristles (1.57±0.13 DC
bristles per heminotum) and because PHD fingers and the bromodomain are
believed to play important functions, we conclude that
touE44.1 is a loss-of-function allele and that Tou is
required to promote neural development.
|
We also analysed the effects of overexpressed tou on neural
development. We made use of the UAS/Gal4 system and tou was
overexpressed in the dorsal compartment of the disc
Gal4-apMD544
(Rincon-Limas et al., 1999).
Gal4-apMD544/EP622 flies have excess DC
sensory organs (2.78±0.12 DC bristles/heminotum)
(Fig. 2G). Moreover, the loss
of DC bristles associated with ChipE is suppressed when
Tou is overexpressed (Fig. 2H).
Indeed, ChipE apGal4/ChipE EP622
flies have 2.23±0.08 DC bristles/heminotum.
|
Toutatis interacts both with the DBD and the C terminus of Pannier
TouA physically interacts with the DBD of Pnr in yeast. Since the C
terminus of Pnr also mediates physical interactions with cofactors
(Ramain et al., 2000), we then
investigated whether TouA interacts with the C terminus of Pnr in yeast
(Fig. 3A). Expression vectors
encoding either the C terminus of Pnr fused to the LexA DBD
(LexADBDPnrCT) or the unfused LexA DBD
(LexADBD) were introduced into the L40 yeast strain together with
the vector for the unfused VP16 activation domain (VP16AD) or for
TouA fused to VP16AD (VP16ADTouA). L40 cells contain a
lacZ reporter driven by eight LexA binding sites. Protein extracts
were prepared from L40 transformants grown in liquid medium and were assayed
for ß-galactosidase activity (Fig.
3B). No reporter activity was observed in extracts made from yeast
expressing LexADBDPnrCT/VP16AD,
LexADBD/VP16ADTouA or
LexADBD/VP16AD. In contrast, a robust activation of the
reporter was detected with yeast expressing
LexADBDPnrCT/VP16ADTouA, indicating that TouA
associates with the C terminus of Pnr (Fig.
3B). Then, both the DBD and the C terminus of Pnr physically
interact with TouA. Accordingly, we found that TouA interacts with wild-type
Pnr (PnrWT) in which both the DBD and the C terminus of Pnr are
present and with PnrVX1, a truncated Pnr lacking the C terminus
(Fig. 3B). TouA also associates
with PnrD1, a mutated Pnr in which the structure of the DBD is
probably disrupted since a coordinating cysteine of the N terminal zinc finger
has been replaced by a tyrosine (Fig.
3B).
|
To investigate the interactions between Tou and Pnr in more detail, we constructed several expression vectors encoding in yeast contiguous domains of Tou (Fig. 3A,F; TouB to TouK). The various segments of Tou were fused with VP16AD and introduced into yeast together with unfused LexADBD or LexADBDPnrWT. We observed activation of the reporter only in yeast expressing LexADBDPnrWT/VP16ADTouC and in yeast expressing LexADBDPnrWT/VP16ADTouJ, showing that the amino terminus of the MBD domain of Tou mediates physical interactions with Pnr in yeast.
Toutatis also interacts with Chip
Chip is an essential cofactor of Pnr
(Ramain et al., 2000) since it
facilitates enhancer/promoter communication necessary for ac-sc
expression during Pnr-driven neural development. Since TouA associates with
Pnr in yeast, we made use of the yeast two-hybrid system to ask whether TouA
also associates with Chip. Chip was fused to LexADBD and assayed
for interaction with VP16ADTouA. We observed a strong increase of
ß-galactosidase activity in extracts made from yeast expressing
LexADBDChip/VP16ADTouA above the background level seen
with extracts made from yeast expressing
LexADBD/VP16ADTouA or
LexADBDChip/VP16AD
(Fig. 4B; data not shown),
indicating that TouA associates with Chip
(Fig. 4B).
Sequence comparison between the Ldb proteins from various species reveals
two conserved functional domains, involved in protein-protein interactions
(Matthews and Visvader, 2003).
Thus, Chip (Fig. 4A) contains a
N-terminal homodimerization domain, also involved in interactions with Pnr
(Ramain et al., 2000
) and Osa
(Heitzler et al., 2003
) and a
C-terminal LIM interacting domain (LID) mediating heterodimerization with
LIM-homeodomain proteins (LIM-HD)
(Fernandez-Funez et al., 1998
)
and basic helix-loop-helix proteins (bHLH)
(Ramain et al., 2000
).
We next examined the interaction between TouA and Chip. Assays for ß-galactosidase activity revealed that the interaction between TouA and Chip is mediated by the N-terminal homodimerization domain (Fig. 4B). We also investigated whether TouA can interact with Chip in a cultured cell line by immunoprecipitating proteins extracts of Cos cells transfected with expression vectors encoding TouA and Chip, the N-terminal domain of Chip (NT Chip) or the C-terminal domain of Chip (CT Chip) (Fig. 4C). We found that TouA physically interacts with Chip and the interaction is mediated by the N-terminal domain of Chip. Finally, we further found that in vitro translated 35S-labelled TouA interacted with GST Chip attached to glutathione-bearing beads (Fig. 4D) but did not bind GST control beads, whereas GST Chip did not bind the negative luciferase input.
We next investigated the interaction between Tou and Chip in more detail. The segments of Tou (Fig. 3A: TouB to TouI; Fig. 4E: TouL to TouN) were fused with the VP16AD and introduced into yeast together with unfused LexADBD or LexADBDChip. We found that only TouD containing the DDT domain associates with Chip (Fig. 4E). Moreover, the physical interaction was shown to be mediated by the DDT domain itself (Fig. 4E; TouM).
Tou functionally cooperates with Pnr and Chip during neural development.
Since Tou physically interacts with Pnr and Chip, we then asked whether a
ternary complex containing Tou, Pnr and Chip can exist in living cells. We
performed double immunoprecipitations of extracts made from Cos cells
containing Pnr, Tou and Chip (Fig.
5). The extract was first immunoprecipitated with the M2 antibody
recognizing the flagged TouA (Fig.
5B; IP 1) and the precipitated proteins were recovered by elution
with the Flag peptide. The eluate was then immunoprecipitated with the B10
antibody (Fig. 5B: IP 2)
directed against the tagged Chip. We observed that Pnr coimmunoprecipitates
with Chip and TouA (Fig. 5B),
indicating that the trimer Chip-Tou-Pnr can be formed. Moreover, this suggests
that Chip and Pnr act together to recruit Tou and to target its activity to
the ac-sc promoter sequences. This observation is
reminiscent to what was previously observed on the role of Osa during neural
development (Heitzler et al.,
2003). Osa belongs to the Brm complex and it was suggested that
Pnr and Chip cooperate to recruit Osa and to target activity of the Brm
complex to the ac-sc promoter sequences, leading to negative
regulation of enhancer-promoter communication. However, Tou and Osa display
antagonistic activities during regulation of ac-sc and may probably
define distinct chromatin remodelling complexes since the loss of DC bristles
associated with reduced tou function (tou2 flies
have 1.4±0.05 DC bristles/heminotum) is suppressed by lowering the
dosage of osa. Indeed, tou2; osa616/+
flies have 1.97±0.07 DC bristles/heminotum
(Fig. 2K).
Toutatis is required to activate proneural expression
To investigate whether the interactions between Pnr, Chip and Tou function
in vivo, we examined effects of both loss-o-function and gain-of-function of
tou on expression of a lacZ reporter driven by a minimal
ac promoter fused to the DC enhancer [transgenic line
DC:ac-lacZ (Garcia-Garcia et al.,
1999)]. We found that expression is strongly impaired in
tou2 flies and in touE44.1 flies
(Fig. 6B,C), in agreement with
the lack of DC bristles. In contrast, overexpressed Tou
(apGal4/EP622) leads to increased lacZ expression
at the DC site (Fig. 6G,H),
consistent with the excess of DC bristles
(Fig. 2D). The excess of DC
bristles in pnrD1 correlates with expanded ac-sc
expression at the DC site (Fig.
6D). Since loss of tou function suppresses the excess DC
bristles associated with pnrD1, we addressed the
consequence of loss of tou function on expression of the
lacZ reporter in pnrD1/+ flies. We found that
expanded lacZ expression associated with pnrD1 is
decreased when tou function is simultaneously reduced
(Fig. 6A,D,E). We conclude that
Pnr and Tou cooperate during neural development and regulate ac-sc
expression through the DC enhancer.
|
We first investigated genetic interactions between Iswi and
pnr. We made use of Iswi1 and
Iswi2, which are both characterized by a point mutation
that introduces a premature stop codon and are consequently predicted to
encode truncated Iswi. However, Iswi1 and
Iswi2 behave as null alleles since truncated
Iswi1 and Iswi2 are not detected in western analyses,
suggesting that the C terminus is required to stabilize Iswi
(Deuring et al., 2000). The
Iswi2/+ flies are wild type (2.00 DC bristle/heminotum)
whereas the pnrD1/+ flies have excess DC bristles
(3.34±0.1 DC bristle/heminotum). However, this excess is lowered when
Iswi function is simultaneously reduced
[Iswi2/+; pnrD1/+ flies display
2.84±0.18 DC bristles/heminotum]
(Fig. 2C,L. Conversely, the
lack of DC bristles associated with pnrVX1
(1.86±0.03 DC bristles/heminotum) is aggravated when Iswi
function is simultaneously reduced (1.61±0.07 DC bristles/heminotum).
We also observed that the loss of DC bristles associated with
ChipE (1.6±0.07 DC bristles/heminotum) is
accentuated when Iswi function is simultaneously reduced (1.29±0.07 DC
bristles/heminotum) (data not shown). Thus, loss-of-function Iswi
alleles behave like loss-of-function tou alleles, implying that Iswi
is also required during neural development and suggesting that Iswi and Tou
may act as subunits of a multiprotein complex.
|
Overexpression of IswiK159R in the precursor cells using the
scaGal4 driver provokes a loss of multiple sensory
bristles (Deuring et al.,
2000) and it has been proposed that Iswi has a late function
during neural development, essential for either viability or division of the
precursor cells. We then induced widespread expression of IswiK159R
in the dorsal compartment of the imaginal wing disc using
Gal4-apMD544. Overexpression of
IswiK159R is therefore induced early during development and results
in a lack of sensory organs, including a frequent loss of DC bristles
(Fig. 2M).
Tou and Iswi promote DC development and could be subunits of a multiprotein complex. However, we can also hypothesize that Tou can substitute for Iswi function and vice versa. To address this issue, we overexpressed IswiK159R in either a wild-type genetic background or in conditions of loss of tou function. Overexpressed IswiK159R in the domain of pnr expression reduces DC bristles (1.56±0.11 bristle/heminotum) (Fig. 2N). The loss of DC bristles is aggravated when the tou function is simultaneously reduced. Indeed, tou2/+; pnrGal4/UASIswiK159R and tou2; pnrGal4/UASIswiK159R flies have 1.14±0.12 bristle/heminotum (data not shown) and 0.75±0.11 bristle/heminotum (Fig. 2O), respectively. This observation reinforces the hypothesis that Tou and Iswi could be subunits of a complex during neural development.
We next investigated whether overexpressed IswiK159R affects the
activity of the DC enhancer. We found that overexpression of
IswiK159R leads to (Fig.
6I) reduced expression of the lacZ reporter driven by the
ac minimal promoter fused to the DC enhancer (line DC:
aclacZ) (Garcia-Garcia et al.,
1999). We next examined the consequences of loss of Iswi
function associated with the Iswi1/Iswi2
transheterozygous combination, which dies during late larval stages
(Deuring et al., 2000
). We
observed that loss of Iswi function leads to decreased lacZ
expression (Fig. 6F). These
observations indicate that Iswi is also necessary for activation of
ac-sc expression at the DC site, although we cannot rule out the
possibility that this interaction is indirect.
|
We next addressed whether Iswi can interact with Chip and Pnr, by testing the abilities of in vitro translated 35S-labelled Iswi to bind to GST-Chip attached to glutathione-bearing beads. We found that Iswi associates with full-length Chip. Similarly, we observed that Iswi can also associate both with the DBD and the C terminus of Pnr (Fig. 7C,D). Since Pnr and Chip directly regulate ac-sc expression at the DC site, our findings suggest that Iswi and Tou may belong to a complex, which, in vivo, regulates the activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The activity of the Drosophila complex is antagonized by
dimerization with various transcription factors. Pnr associates with U-shaped
(Ush), a protein structurally related to Friend of Gata, since they both
contain multitype zinc fingers (Cubbada et al., 1997;
Haenlin et al., 1997;
Tsang et al., 1997
). Moreover,
regulation of GATA factors by Ush and Fog appears remarkably conserved during
evolution since they contain nine zinc fingers, four of which (zfs1, 5, 6 and
9) mediate interaction with the N-terminal zinc finger of the DBD of GATA
factors (Fox et al., 1999
).
The activity of the proneural complex is also antagonized by dimerization of
Chip with the LIM-HD Apterous and by dimerization of the proneural bHLH Ac-Sc
with the bHLH Extra macrochaetae (Emc). Chip is an essential cofactor for Pnr
and Apterous and neural development relies on a competition between Pnr and Ap
to associate with the common Chip cofactor
(Ramain et al., 2000
). Emc
lacks the basic domain required for DNA binding and it is believed that it
sequesters Ac and Sc and prevents them from associating with Da to give the
(Ac-Sc)-Da heterodimer required for activation of proneural expression
(Ellis et al., 1990
;
Garrell and Modolell,
1990
).
|
Does Toutatis regulate proneural expression through chromatin remodelling?
Enhancer-promoter communication is of fundamental importance, as in most
cases the activities of enhancers play a determining role in turning on and
off specific genes in a temporally and spatially regulated manner. Models to
explain long range activation of gene expression invoke enhancer-promoter
communication either through protein-protein interactions resulting in
formation of loops, the free sliding of proteins recruited by a remote
enhancer along DNA or the establishment of modified chromatin structure by
facilitator factors which generate a chain of higher order complexes along the
chromatin fibre (Bulger and Goudine,
1999; Dorsett,
1999
).
In Drosophila, Chip was postulated to be a such facilitator
required both for activity of a wing specific enhancer of the cut
locus (Morcillo et al., 1996;
Morcillo et al., 1997
) and for
activity of the DC enhancer of the ac-sc complex
(Ramain et al., 2000
).
Enhancer-promoter communication at the ac-sc complex is negatively
regulated by the Brm complex whose activity is targeted to the ac-sc
promoter sequences through dimerization of the Osa subunit with both Pnr and
Chip (Heitzler et al., 2003
).
The Brm complex is thought to remodel chromatin in a way that represses
transcription.
We describe, here, the role of Tou and Iswi which may act together as
subunits of a multiprotein complex to positively regulate activity of Pnr and
Chip during enhancer-promoter communication. Tou and Iswi therefore display
opposite activity to that of the Brm complex, raising questions about their
molecular function during neural development. Tou shares essential functional
domains with members of the WAL family of chromatin remodelling proteins,
including Acf1 of ACF and CHRAC (Ito et
al., 1999) and also TIP5 of NoRC (nucleolar remodelling complex),
involved in repression of the rDNA
(Santoro et al., 2002
).
Importantly, Acf1 and TIP5 associate in vivo with Iswi
(Ito et al., 1999
;
Strohner et al., 2001
),
showing that Iswi can mediate both activation and repression of gene
expression. Tou positively regulates Pnr/Chip function during the period of
ac-sc expression in neural development, and it associates with Iswi.
Since Iswi also positively regulates Pnr/Chip function, we hypothesize that a
complex encompassing Tou and Iswi acts during long-range activation of
proneural expression, possibly through chromatin remodelling. Further studies
will help to resolve this issue.
Interestingly, Chip and Pnr seem to play similar roles both during recruitment of the Brm complex and recruitment of Tou and Iswi, since they dimerize with Osa, Tou and Iswi. In addition, Pnr and Chip apparently cooperate to strengthen the physical association with Osa and Tou. However, Osa, on the one hand, and Tou and Iswi, on the other, display antagonistic activities during neural development. Since they are ubiquitously expressed, accurate regulation of ac-sc expression would require a strict control of the stoichiometry between Osa, Tou and Iswi. It remains to be investigated whether the functional antagonism between Osa and Tou/Iswi relies on a molecular competition for association with Pnr and Chip. Determination of this would require a complete molecular definition of the putative complex encompassing Tou and Iswi, together with a full understanding of how this complex and the Brm complex molecularly interact with the proneural complex to regulate enhancer-promoter communication during development.
Iswi is directly required for activation of proneural expression
Biochemical analysis of Iswi and Iswi-containing complexes, together with
genetic studies of Iswi and associated proteins in flies and in budding yeast,
has revealed roles for Iswi in a wide variety of nuclear processes, including
transcriptional regulation, chromosome organization and DNA replication
(Corona and Tamkun, 2004).
Accordingly, Iswi was found to be a subunit of various complexes, including
NURF (nucleosome remodelling factor)
(Badenhorst et al., 2002
), ACF
and CHRAC. Iswi-containing complexes were primarily recognized as factors that
facilitate in vitro transcription from chromatin templates
(Mizuguchi et al., 1997
;
Mizuguchi et al., 2001
; Okada
et al., 1998). However, genetic analysis in Drosophila and in
Saccharomyces cerevisiae have provided evidence that Iswi-containing
complexes are involved in both transcriptional activation and repression in
vivo. For example, immunostaining of Drosophila polytene chromosomes
of salivary glands showed that Iswi is associated with hundreds of euchromatic
sites in a pattern that is non-overlapping with RNA polymerase II
(Deuring et al., 2000
). It
suggests that Iswi may play a general role in transcriptional repression. In
contrast, it was also demonstrated that expression of engrailed and
Ultrabithorax are severely compromised in Iswi-mutant
Drosophila larvae (Deuring et
al., 2000
). Recent studies have also shown that a mouse
Iswi-containing complex, NoRC, plays an essential role during repression of
transcription of the rDNA locus by RNA polymerase I
(Strohner et al., 2001
;
Santoro et al., 2002
;
Zhou et al., 2002
). Here, we
present Tou, a protein that is structurally related to the TIP5 subunit of
NoRC (Santoro et al., 2002
).
Tou positively regulates enhancer-promoter communication during Pnr-driven
proneural development and its activity is targeted to the ac-sc
promoter sequences through dimerization with Pnr and Chip. We also provide
evidence that Iswi is required during neural development. Overexpression of
IswiK159R in the precursor cells of the sensory organs
using the scaGal4 driver
(Deuring et al., 2000
) leads
to flies lacking multiple bristles, suggesting that Iswi functions late during
neural development, essential for either cell viability or division of the
precursor cell. Using the Iswi1/Iswi2
transheterozygous combination and individuals overexpressing
IswiK159R in earlier stages of development and in less
restricted patterns, we show that Iswi also regulates ac-sc
expression. Interestingly, the regulation is probably direct since Iswi
associates with the transcription factors Pnr and Chip, known to promote
ac-sc expression at the DC site
(Garcia-Garcia et al., 1999
;
Ramain et al., 2000
). Since
Iswi interacts with Tou, we propose that Tou and Iswi may positively regulate
activity of Pnr and Chip during enhancer-promoter communication, possibly as
subunits of a multiprotein complex involved in chromatin remodelling.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Badenhorst, P., Voas, M., Rebay, I. and Wu, C.
(2002). Biological functions of the Iswi chromatin remodelling
complex NURF. Genes Dev.
16,3186
-3198.
Beckstead, R., Ortiz, J. A., Sanchez, C., Prokopenko, S. N., Chambon, P., Losson, R. and Bellen, H. J. (2001). Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit ßFTZ-F1 dependent transcription. Mol. Cell 7,753 -765.[CrossRef][Medline]
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W.,
Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M.,
Hoskins, R. A. and Spradling, A. C. (2004). The BDGP gene
disruption project: single transposon insertions associated with 40% of
Drosophila genes. Genetics
167,761
-781.
Boulianne, G. L., de la Concha, A., Campos-Ortega, J., Jan, L. Y. and Jan, Y. N. (1991). The Drosophila neurogenic gene neuralised encodes a novel protein and is expressed in precursors of larval and adult neurons. EMBO J. 10,2975 -2983.[Abstract]
Bulger, M. and Groudine, M. (1999). Looping
versus linking: toward a model for long distance gene activation.
Genes Dev. 13,2465
-2477.
Cantor, A. B. and Orkin, S. H. (2002). Transcriptional regulation of erythropoiesis: an affair involving multiple partners. Oncogene 21,3368 -3376.[CrossRef][Medline]
Collins, R. T., Furukawa, T., Tanese, N. and Treisman, J. E.
(1999). Osa associates with the Brahma chromatin remodelling
complex and promotes the activation of some target genes. EMBO
J. 18,7029
-7040.
Corona, D. F. V. and Tamkun, J. W. (2004). Multiple roles for Iswi in transcription, chromosome organization and DNA replication. Biochim. Biophys. Acta 1677,113 -119.[Medline]
Cubadda, Y., Heitzler, P., Ray, R., Bourouis, M., Ramain, P.,
Gelbart, W., Simpson, P. and Haenlin, M. (1997).
u-shaped encodes a zinc finger protein that regulates the proneural
genes achaete and scute during formation of bristles in
Drosophila. Genes Dev.
11,3083
-3095.
Deuring, R., Fanti, L., Armstrong, J. A., Sarte, M., Papoulas, O., Prestel, M., Daubresse, G., Verardo, M., Moseley, S. L., Berloco, M., Tsukiyama, T., Wu, C., Pimpinelli, S. and Tamkun, J. W. (2000). The Iswi chromatin-remodelling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Mol. Cell 5,355 -365.[CrossRef][Medline]
Dorsett, D. (1999). Distant liaisons: long-range enhancer-promoter interactions in Drosophila. Curr. Opin. Genet. Dev. 9, 505-514.[CrossRef][Medline]
Ellis, H. M., Spann, D. R. and Posakony, J. W. (1990). extramachrochaetae, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins. Cell 61, 27-38.[CrossRef][Medline]
Fauvarque, M. O., Laurenti, P., Boivin, A., Bloyer, S., Griffin-Shea, R., Bourbon, H. M. and Dura, J. M. (2001). Dominant modifiers of the polyhomeotic extra-sex-combs phenotype induced by marked P element insertional mutagenesis in Drosophila. Genet. Res. 78,137 -148.[Medline]
Fernandez-Funez, P., Lu, C. H., Rincon-Limas, D. E.,
Garcia-Bellido, A. and Botas, J. (1998). The relative
expression amounts of apterous and its co-factor dLdb/Chi
are critical for dorso-ventral compartmentalization in the Drosophila
wing. EMBO J. 17,6846
-6853.
Fox, A. H, Liew, C., Holmes, M., Kowalski, K., Mackay, J. and
Crossley, M. (1999). Transcriptional cofactors of the FOG
family interact with GATA proteins by means of multiple zinc fingers.
EMBO J. 18,2812
-2822.
Fyodorov, D. V. and Kadonaga, J. T. (2002).
Binding of Acf1 to DNA involves a WAC motif and is important for ACF-mediated
chromatin assembly. Mol. Cell. Biol.
22,6344
-6353.
Garcia-Garcia, M. J., Ramain, P., Simpson, P. and Modolell,
J. (1999). Different contributions of pannier and
wingless to the patterning of the dorsal mesothorax of
Drosophila. Development
126,3523
-3532.
Garrell, J. and Modolell, J. (1990). The Drosophila extramachrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell 61,39 -48.[CrossRef][Medline]
Gomez-Skarmeta, J. L., Rodriguez, I., Martinez, C., Culi, J., Ferres-Marco, D., Beamonte, D. and Modolell, J. (1995). Cis-regulation of achaete and scute shared enhancer-like elements drive their coexpression in proneural clusters of the imaginal discs. Genes Dev. 9,1869 -1882.[Abstract]
Gomez-Skarmeta, J. L., Campuzano, S. and Modolell, J. (2003). Half a century of neural prepatterning: the story of a few bristles and many genes. Nature 4, 587-598.
Haenlin, M., Cubadda, Y., Blondeau, F., Heitzler, P., Lutz, Y.,
Simpson, P. and Ramain, P. (1997). Transcriptional activity
of Pannier is regulated negatively by heterodimerization of the GATA-DNA
binding domain with a cofactor encoded by the u-shaped gene of
Drosophila. Genes Dev.
11,3096
-3108.
Heitzler, P., Haenlin, M., Ramain, P., Calleja, M. and Simpson,
P. (1996). A genetic analysis of pannier, a gene
necessary for viability of dorsal tissues and bristle positioning in
Drosophila. Genetics
143,1271
-1287.
Heitzler, P., Vanolst, L., Biryukova, I. and Ramain, P.
(2003). Enhancer-promoter communication mediated by Chip during
Pannier-driven proneural patterning is regulated by Osa. Genes
Dev. 17,591
-596.
Huang, F., Dambly-Chaudière, C. and Ghysen, A. (1991). The emergence of sense organs in the wing disc of Drosophila. Development 111,1087 -1095.[Abstract]
Ito, T., Levenstein, M. E., Fyodorov, D. V., Kutach, A. K.,
Kobayashi, R. and Kadonaga, J. T. (1999). ACF consists of two
subunits, Acf1 and Iswi, that function cooperatively in the ATP-dependent
catalysis pf chromatin assembly. Genes Dev.
13,1529
-1539.
Le Douarin, B., Heery, D. M., Gaudon, C., vom Baur E. and Losson, R. (2001). Yeast two-hybrid screening for proteins that interact with nuclear hormone receptors. Methods Mol. Biol. 176,227 -248.[Medline]
Lu, X., Meng, X., Morris, C. A. and Keating, M. T. (1998). A novel human gene, WSTF, is deleted in Williams syndrome. Genomics 54,241 -249.[CrossRef][Medline]
Martinez, C. and Modolell, J. (1991). Cross-regulatory interactions between the proneural achaete and scute genes of Drosophila. Science 251,1485 -1487.[Medline]
Matthews, J. M. and Visvader, J. E. (2003).
LIM-domain-binding protein 1, a multifunctional cofactor that interacts
with diverse proteins. EMBO Rep.
4,1132
-1137.
Mizuguchi, G., Tsukiyama, T., Wisniewski, J. and Wu, C. (1997). Role of nucleosome remodelling factor NURF in transcriptional activation of chromatin. Moll. Cell 1, 141-150.[CrossRef]
Mizuguchi, G., Vassilev, A., Tsukiyama, T., Nakatani, Y. and Wu,
C. (2001). ATP-dependent nucleosome remodelling and histone
hyperacetylation synergistically facilitate transcription of chromatin.
J. Biol. Chem. 276,14773
-14783.
Morcillo, P., Rosen, C. and Dorsett, D. (1996).
Genes regulating the remote wing margin enhancer in the Drosophila cut locus.
Genetics 144,1143
-1154.
Morcillo, P., Rosen, C., Baylies, M. K. and Dorsett, D.
(1997). Chip, a widely expressed chromosomal protein required for
segmentation and activity of a remote wing margin enhancer in
Drosophila. Genes Dev.
11,2729
-2740.
Näär, A. M., Lemon, B. D. and Tjian, R. (2001). Transcriptional coactivator complexes. Annu. Rev. Biochem. 70,475 -501.[CrossRef][Medline]
Okada, M. and Hirose, S. (1998). Chromatin
remodelling mediated by Drosophila GAGA factor and Iswi activates
fushi tarazu gene transcription in vitro. Mol. Cell.
Biol. 18,2455
-2461.
Ramain, P., Heitzler, P., Haenlin, M. and Simpson, P.
(1993). pannier, a negative regulator of
achaete and scute in Drosophila, encodes a zinc
finger protein with homology to the vertebrate transcription factor GATA-1.
Development 119,1277
-1291.
Ramain, P., Khechumian, R., Khechumian, K., Arbogast, N., Ackermann, C. and Heitzler, P. (2000). Interactions between Chip and the Achaete/Scute-Daughterless heterodimers are required for Pannier-driven proneural patterning. Mol. Cell 6, 781-790.[CrossRef][Medline]
Rincon-Limas, D. E., Lu, C. H., Canal., I., Calleja, M.,
Rodriguez- Esteban, C., Izpisua-Belmonte, J. C. and Botas, J.
(1999). Conservation of the expression and function of
apterous orthologs in Drosophila and mammals.
Proc. Natl. Acad. Sci. USA
96,2165
-2170.
Rorth, P. (1996). A modular misexpression
screen in Drosophila detecting tissue specific phenotype.
Proc. Natl. Acad. Sci. USA
93,12418
-12422.
Santoro, R., Li, J. and Grummt, I. (2002). The nucleolar remodelling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription. Nat. Genet. 32,393 -396.[CrossRef][Medline]
Seipel, K., Georgiev, O. and Schaffner, W. (1992). Different activation domains stimulate transcription from remote and proximal positions. EMBO J. 13,4961 -4968.
Simon, J. A. and Tamkun, J. W. (2002). Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes. Curr. Opin. Genet. Dev. 12,210 -218.[CrossRef][Medline]
Strohner, R., Nemeth, A., Jansa, P., Hofmann-Rohrer, U.,
Santoro, R., Längst, G. and Grummt, I. (2001). NoRC-a
novel member of mammalian Iswi-containing chromatin remodelling machines.
EMBO J. 20,4892
-4900.
Treisman, J., Luk, A., Rubin, G. M. and Heberlein, U.
(1997). eyelid antagonizes wingless signaling
during Drosophila development and has homology to the Bright family
of DNA-binding proteins. Genes Dev.
11,1949
-1962.
Tsang, A. P., Visvader, J. E., Turner, C. A., Fujiwara, Y., Yu, C., Weiss, M. J., Crossley, M. and Orkin, S. H. (1997). FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 90,109 -119.[CrossRef][Medline]
Van Doren, M., Powell, P. A., Pasternak, D., Singson, A. and Posakony, J. W. (1991). The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA-binding by daughterless/achaete-scute protein complexes. Development 113,245 -255.[Abstract]
Van Doren, M., Ellis, H. M. and Posakony, J. W. (1992). Spatial regulation of proneural gene activity: auto- and cross-activation of achaete is antagonized by extramacrochaetae. Genes Dev. 6,2592 -2605.[Abstract]
Wadman, I. A., Osada, H., Grütz, G. G., Agulnick, A. D.,
Westphal, H., Forster, A. and Rabbitts, T. H. (1997). The
LIM-only protein Lmo2 is a bridging molecule assembling an erythroid
DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI
proteins. EMBO J. 16,3145
-3157.
West, A. G., Gaszner, M. and Felsenfeld, G.
(2002). Insulators: many functions, many mechanisms.
Genes Dev. 16,271
-288.
Zhou, Y., Santoro, R. and Grummt, I. (2002).
The chromatin remodelling complex NoRC targets HDAC1 to the ribosomal gene
promoter and represses RNA polymerase I transcription. EMBO
J. 21,4632
-4640.
Related articles in Development:
|