|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online January 23, 2009
doi: 10.1242/10.1242/dev.027656
1 Centre de Regulació Genòmica (CRG-UPF), Gene Regulation
Programme, Dr Aiguader 88, 08003 Barcelona, Spain.
2 Departamento de Genética, Facultad Ciencias Biológicas,
Universitad de Valencia, Dr Moliner 50, 46100 Burjasot, Spain.
* Author for correspondence (e-mail: fatima.gebauer{at}crg.es)
Accepted 15 December 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: UNR, Dosage compensation, DCC, MSL, Translation, roX1/2
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
2-fold (reviewed by
Gilfillan et al., 2004
;
Lucchesi et al., 2005;
Straub and Becker, 2007;
Mendjan and Akhtar, 2007).
Hyper-transcription requires the binding of the dosage compensation complex
(DCC) to hundreds of sites along the male X chromosome. The DCC is composed of
five proteins (MSL-1, MSL-2, MSL-3, MLE and MOF), the mutation of which causes
male-specific lethality, and for this reason the DCC is also known as the
male-specific lethal (MSL) complex. The DCC also contains two non-coding RNAs
(roX1 and roX2) that appear to have redundant functions
(Meller et al., 1997;
Franke and Baker, 1999). MSL-2
is a limiting RING finger protein that, together with MSL-1, nucleates the
assembly of the DCC (Kelley et al.,
1995; Bashaw and Baker,
1995). MLE (Maleless) is a helicase thought to be required for
stable integration of roX RNAs into the DCC
(Meller et al., 2000), whereas
MSL-3 is a chromodomain protein, and MOF (Males absent on the first) is an
acetyl-transferase that promotes the acetylation of histone H4 on lysine 16
(H4K16), a modification that specifically marks the compensated X chromosome
(Akhtar and Becker, 2000;
Smith et al., 2000). Other
proteins, in addition to the DCC components, have been implicated in dosage
compensation, including the H3S10 kinase JIL-1
(Wang et al., 2001), the DNA
supercoiling factor (SCF) (Furuhashi et
al., 2006), the chromatin-binding protein SU(VAR)3-7
(Spierer et al., 2005), and
the nuclear pore components Mtor and NUP153
(Mendjan et al., 2006).
In female flies, dosage compensation is inhibited because the expression of
msl-2 is repressed by the female-specific RNA-binding protein Sex
lethal (SXL). Enforced expression of MSL-2 leads to the assembly of the DCC on
both female X chromosomes and to lethality
(Kelley et al., 1995). SXL
binds to both untranslated regions (UTRs) of msl-2 pre-mRNA and
inhibits first the splicing of a facultative intron in the 5' UTR of the
transcript, and then its translation in the cytoplasm
(Bashaw and Baker, 1997;
Kelley et al., 1997;
Gebauer et al., 1998).
Translational repression of msl-2 by SXL occurs by a double-block
mechanism whereby SXL bound to the 3' UTR inhibits the recruitment of
the small ribosomal subunit, and SXL bound to the 5' UTR inhibits the
scanning of those subunits that presumably have escaped the 3'-mediated
control (Gebauer et al., 2003;
Beckmann et al., 2005). Studies
performed in cell-free translation extracts and cultured cells have shown that
translational repression requires the recruitment of the co-repressor Upstream
of N-ras (UNR) to sequences adjacent to the SXL binding sites in the 3'
UTR (Abaza et al., 2006;
Duncan et al., 2006). UNR is
an evolutionarily conserved RNA-binding protein that contains five cold-shock
domains (CSDs) and two glutamine (Q)-rich regions. The first CSD (CSD1)
mediates interactions with SXL and msl-2 mRNA, whereas the N-terminal
third of the protein carries most of the translational repression function in
vitro (Abaza and Gebauer,
2008). Although UNR is a ubiquitous, primarily cytoplasmic protein
that is present in both males and females, it binds to msl-2 only in
females because its association depends on SXL. Thus, SXL provides a
sex-specific function to UNR.
To gain insight into the roles of UNR in development, we have analyzed hypomorphic mutant flies that lack the C-terminal half of UNR, as well as flies that overexpress full-length UNR or a fragment containing CSDs 1 and 2. In Unr hypomorphic mutant females, the DCC was detected on a limited set of high-affinity sites on the X chromosomes, indicating that, as predicted from translation studies, UNR represses DCC formation in females. Unexpectedly, Unr mutant males showed decreased DCC recruitment to the X chromosome. Consistent with this, UNR knockdown in male Drosophila SL2 cells abrogated DCC binding without affecting the levels of DCC components or their nucleocytoplasmic distribution. In addition, flies overexpressing UNR showed preferential male lethality and DCC recruitment defects, and the X chromosome of both mutant and transgenic Unr males exhibited an altered morphology. Importantly, roX1 and roX2 RNAs co-immunoprecipitated with UNR in males, suggesting that UNR might function by targeting these non-coding RNAs. These results uncover new roles for UNR in the regulation of dosage compensation in males by a mechanism that is independent of msl-2 translation.
|
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). The
insertion was precisely mapped by PCR from genomic DNA using
Unr-specific primer UNR2658 (5'-CATAGAAGGTTTCTCTAGA-3')
and PiggyBac-specific primer 3F1 (5'-CCTCGATATACCGATAAAAC-3').
FLUNR and UNR1+2 transgenic flies were obtained by standard
P-element transformation of Oregon R w1118 flies
with the pUASp-FLUNRGFP and pUASp-UNR1+2GFP constructs, respectively. To
generate these constructs, the coding sequences for full-length
Drosophila UNR (amino acids 1-1039) or a fragment containing CSDs 1+2
(amino acids 197-372) were obtained by PCR amplification and subcloned into
pEGFP-N1 (Clontech). The UNR-GFP fusions generated in this manner were then
cloned into the KpnI/XbaI sites of pUASp
(Rorth, 1998). At least three
homozygote viable lines with insertions in the second and third chromosomes
were recovered for FLUNR, and one on the X chromosome for UNR1+2.
FLUNR and UNR1+2 homozygous flies were crossed with
Gal4 driver lines to induce expression before gastrulation
(Act5C-Gal4; FlyBase ID: FBtp0001406), at early gastrulation
(en-Gal4), or in the salivary glands of third-instar larvae
(Sgs3-Gal4; FlyBase ID: FBtp0016397).
Cell culture and RNAi treatment
Drosophila SL2 and Kc cells were grown at 25°C in Schneider
medium (Gibco) supplemented with 100 units/ml penicillin/streptomycin and 10%
FCS. RNAi against Unr was performed by incubating
2x106 SL2 cells with 22.5 µg of double-stranded (ds) RNAs
corresponding to either the UNR coding region (nt 2139-2691, relative to the
start codon) or the 3' UTR (nt 3120 to 3530 from the start codon) in 1.5
ml Schneider medium without serum. After a 40-minute incubation at 25°C,
the volume was doubled with Schneider medium supplemented with 20% FCS and the
cells were plated in p35 dishes. Cells treated similarly but without
double-stranded (ds) RNA addition were carried in parallel as control. Cells
were recovered 6-9 days after plating, and the efficiency of UNR depletion
tested by western blot. Both dsRNAs depleted UNR efficiently. No deleterious
effect of RNAi treatment on cell viability was detected.
Western blot and immunoprecipitation
Adult and larval Drosophila extracts were prepared as described
(Wilhelm et al., 2000) using a
DIAX 900 homogenizer (Heidolph) at 4°C. Total protein extracts from SL2
and Kc cells were prepared in RIPA buffer [150 mM NaCl, 10 mM Tris-HCl pH 7.5,
0.1% SDS, 1% deoxycholic acid (DOC), 5 mM EDTA, 1% Triton X-100] supplemented
with 1x Complete protease inhibitor cocktail (Roche). Briefly, cell
pellets were resuspended in RIPA buffer, incubated on ice for 20 minutes and
centrifuged to recover the supernatant. Cytoplasmic and nuclear protein
preparations were obtained by first incubating cells in hypotonic buffer [10
mM HEPES pH 7.6, 10 mM KOAc, 0.5 mM Mg(OAc)2, 5 mM DTT, 1% Triton
X-100, 1x Complete protease inhibitor cocktail] for 5 minutes on ice.
The cells were then homogenized, centrifuged and the supernatant recovered as
the cytoplasmic fraction. The pellet was further washed in PBS, resuspended in
RIPA buffer and processed as described above to obtain the nuclear
fraction.
Protein extracts were resolved by SDS-PAGE, transferred to PVDF membranes
and blocked with 5% dried milk powder in PBS. Incubation with primary
antibodies was performed overnight at 4°C. Anti-MSL-2 antibodies were
provided by P. Becker (Adolf-Butenandt-Institute, Munich, Germany) and used at
1:10 dilution. Antibodies to MSL-1 (1:1000), MLE (1:3000), MOF (1:500), MSL-3
(1:5000), Mtor (1:1000) and NUP153 (1:1000) were kindly provided by A. Akhtar
(Mendjan et al., 2006) and
used at the indicated dilutions. Anti-UNR
(Abaza et al., 2006) and
anti-tubulin (Sigma) were used at 1:500 and 1:10,000, respectively. Secondary
detection was with protein A-HRP (Invitrogen) followed by chemiluminescence
(ECL, Amersham) and autoradiography for UNR, MOF, NUP153 and tubulin, and with
Alexa 660-conjugated anti-rat antibodies followed by detection with the
Odyssey system (Molecular Probes) for all other antibodies.
Adult Drosophila extracts for immunoprecipitation were prepared by homogenizing frozen fly powder with one volume of 10 mM HEPES-KOH pH 7.4, 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 1x Complete protease inhibitor cocktail and 40 U/ml RNasin. Sixteen milligrams of extract were cross-linked by incubation for 10 minutes at 4°C with 1% formaldehyde and the reaction stopped with 250 mM glycine pH 7.0 for 5 minutes. One volume of RIPA buffer was added and the extract processed as described above for SL2 cells. Immunoprecipitation was performed with magnetic Dynabeads coupled to protein A, pre-blocked with 100 ng/µl yeast tRNA. After immunoprecipitation, beads were washed with RIPA buffer, and eluates obtained by incubation with 50 mM Tris-HCl pH 7.4, 5 mM EDTA, 10 mM DTT, 1% SDS. Eluates were treated with proteinase K and incubated at 70°C for 1 hour to reverse the cross-linking. RNA was extracted with Trizol (Invitrogen) followed by Turbo DNase treatment, and reverse transcription was performed with oligo dT20 followed by PCR with specific oligos for roX1, roX2, Dad and LIMK1.
Immunostainings
Preparation and immunostaining of polytene chromosomes was performed as
described
(http://www.igh.cnrs.fr/equip/cavalli/Lab%20Protocols/Immunostaining.pdf).
Immunostaining on SL2 cells was performed as described
(Akhtar et al., 2000). Salivary
glands were dissected in PBS, fixed in Brower Fix buffer (150 mM PIPES pH 6.9,
3 mM MgSO4, 1.5 mM EGTA, 1.5% NP40) supplemented with 1.3%
formaldehyde and stained as described for polytene chromosomes. Anti-MSL-2
antibodies used in immunostainings were kindly provided by P. Becker and used
at 1:500. All other antibodies against DCC components were provided by A.
Akhtar and used at the following dilutions: MSL-1 (1:500), MLE (1:500), MOF
(1:500), MSL-3 (1:250), Mtor (1:500). Anti-histone H3 antibodies (Abcam) were
used at 1:250. Secondary detection was with Cy3-conjugated anti-rat (Jackson
Lab.) and Alexa 488-conjugated anti-rabbit (Molecular Probes) antibodies.
Polytene chromosomes, salivary gland and SL2 cell images were captured with a
Leica DFC350FX or Leica DMI 6000B digital camera, and TCS SPE and TCS SP2
microscopes with HCX PL APO CS 40x/1.25-0.75 or 63x/1.40-0.60
oil-immersion objectives. Images were taken using appropriate filter
combinations and arranged using PhotoShop (Adobe).
Staining of eye imaginal discs was performed in 0.1 M phosphate buffer, 0.3% Triton X-100 and 10% normal goat serum. Anti-MSL-2 was used at 1:500 and FITC-conjugated anti-rabbit (Calbiochem) was used as secondary antibody. All image acquisitions were performed using a Leica TCS-NT confocal laser-scanning microscope and Leica LCS software.
Fluorescent signal quantification
Fluorescent signal quantification of eye imaginal discs was performed on
maximum projections of the same number of confocal sections taken with the
same exposure time from a region posterior to the morphogenetic furrow, using
the Leica LCS software. Conditions for image capture were set using eye discs
from w1118 females as negative controls. Student's
t-test was used to analyze the differences in signal intensity
between samples. Fluorescent signal quantification of polytene chromosomes was
performed on images taken with the same exposure time using ImageJ
software.
Quantitative RT-PCR
Total RNA was isolated with Trizol from either SL2 cells or larvae, treated
with DNase and further purified using Nucleospin columns (Macherey-Nagel).
First-strand cDNAs were synthesized from 1 µg of total RNA with AMV-RT
(Promega). The reaction mixture was serially diluted and amplified by
quantitative PCR using the LightCycler DNA Master SYBR Green I Kit (Roche) and
the following gene-specific primers: roX1,
5'-ACAAATGAACCCAAAGCGTC-3' and
5'-GTATCATTGTCTCGCTCGCA-3'; roX2,
5'-TCGATTTAGAGCGAGATGACAA-3' and
5'-TAAAAGCATCTCGAATTTCCGT-3'; and rp49,
5'-CCACCAGTCGGATCGATATG-3' and
5'-CACGTTGTGCACCAGGAACT-3'. qPCR was performed on a LightCycler
480 (Roche) and the amplification curves were analyzed using the associated
software. Appropriate dilutions and efficiencies of amplification were set for
each primer. Quantitative values were normalized to the internal standard
rp49 (RpL32 - FlyBase).
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). To
investigate the roles of UNR in vivo, we attempted to interfere with UNR
function by generating transgenic flies that overexpress full-length UNR
(FLUNR) or a fragment containing CSDs 1 and 2 (UNR1+2). The
proteins were fused to GFP (Fig.
1A) and their expression was driven by a GAL4-responsive promoter.
Three independent lines were obtained for FLUNR and one for
UNR1+2 (Table 1).
Homozygous transgenic flies were crossed with flies carrying the Gal4
gene driven by promoters of increasing strength. Because the DCC is formed at
the early blastoderm stage (Franke et al.,
1996) and UNR is expressed early in embryogenesis
(Abaza et al., 2006), we used
the engrailed (en) and Actin5C (Act5C)
promoters to drive GAL4 expression and specifically increase UNR levels at an
early stage of development. Even limited UNR overexpression with the
en-Gal4 driver dramatically decreased the viability of transgenic
flies as compared with control siblings of the same progeny in which the
transgene was not expressed (Fig.
1B, left panel). Lethality occurred before the third-instar larvae
stage. Analysis of the male-to-female ratio showed that lethality
preferentially affected males (see numbers above the bars in the left panel of
Fig. 1B). These effects were
dose-dependent because increased UNR expression at 29°C with the
en-Gal4 driver yielded no male survivors
(Fig. 1B and
Table 1). Stronger
overexpression of FLUNR with the Act5C-Gal4 driver resulted in
complete lethality for both sexes (Fig.
1B, left panel). Significantly, maximal overexpression yielded
levels of UNR that were similar to those of the endogenous protein (see Fig.
S1A in the supplementary material), indicating that the amount of UNR must be
finely tuned during development. Lethality from UNR1+2 expression was only
observed under the Act5C-Gal4 driver
(Fig. 1B, right panel;
Table 1). Similar to the effect
of FLUNR, expression of UNR1+2 resulted in preferential male lethality. These
data suggest that UNR plays a crucial role during embryogenesis, particularly
in male flies.
|
|
UNR inhibits DCC formation in female flies
To further examine the roles of UNR in development, we analyzed a
hypomorphic Unr mutant obtained from the FlyBase collection. The
Pbac{PB}UNR[c01923] line derives from a general gene disruption
screening by insertion of a unique PiggyBac element
(Thibault et al., 2004). We
mapped the precise insertion of the PiggyBac element by PCR of genomic DNA
followed by sequencing, and found that the insertion disrupted the UNR open
reading frame yielding a truncated product of 638 amino acids that lacked the
C-terminal Q-rich region and CSDs 4 and 5
(Fig. 3A). Western blot
analysis confirmed the presence of the truncated UNR protein in both
heterozygous and homozygous flies (Fig.
3B). Sixty percent of the homozygous flies survived beyond the
pupae stage and died shortly after eclosion with no sex-specific bias,
indicating that the N-terminal half of UNR suffices for development.
To check whether truncation of UNR resulted in derepression of
msl-2 translation and yielded significant DCC formation, we first
stained eye imaginal discs of female mutants with anti-MSL-2 antibodies.
Indeed, compared with wild-type females, increased staining of the X
chromosome was observed in mutant females, visualized as a localized signal
within the nuclei of the imaginal disc cells
(Fig. 4Aa-f). Quantification of
the MSL-2 signal confirmed these observations
(Fig. 4A, bar chart). MSL-2
staining in mutant females did not reach the levels of wild-type males,
suggesting that the N-terminal half of UNR supports strong inhibition of
msl-2 (Fig. 4Ac,f and
bar chart). These results were corroborated by anti-MSL-2 staining of polytene
chromosomes (Fig. 4Ag-k).
Whereas no MSL-2 was observed on the X chromosomes of wild-type females
(Fig. 4Ag,j), MSL-2 was
detected on a few X chromosome sites in mutant females
(Fig. 4Ah,k). Other DCC
components, such as MSL-1, MLE and MSL-3, were recruited to these sites,
suggesting stable assembly of the DCC (Fig.
4B and data not shown). The sites closely mapped to previously
identified high-affinity DCC binding sites
(Dahlsveen et al., 2006;
Lyman et al., 1997;
Demakova et al., 2003)
(Table 2). These results
indicate that UNR inhibits DCC formation in female flies.
|
|
).
Significantly, autosomal MLE staining was less affected, suggesting that the
effect of UNR is specific to the X chromosome
(Fig. 5C, asterisks). Histone
H3 staining was similar in wild-type and mutant male X chromosomes, suggesting
that the observed decrease in DCC staining is not a consequence of general
chromatin defects (Fig. 5C).
These results reveal a new function for Drosophila UNR in the
efficient assembly or recruitment of the DCC to the male X chromosome.
UNR affects DCC binding without altering the levels or nucleocytoplasmic distribution of DCC components
To further examine the role of UNR in DCC formation, we knocked-down UNR
from Drosophila male SL2 cells. Western blot analysis indicated that
UNR was efficiently depleted after RNAi treatment (see
Fig. 6B, UNR panel). Staining
of untreated SL2 cells with anti-MSL-2 antibodies showed a strong localized
signal within the nucleus corresponding to the X chromosome territory
(Fig. 6A). Consistent with the
results in flies, UNR depletion resulted in a less intense MSL-2 signal, which
was dispersed within the nucleoplasm (Fig.
6A). Importantly, both the level and nucleocytoplasmic
distribution of MSL-2 were unaffected, indicating that UNR is required for
correct DCC assembly or targeting in males independently of changes in MSL-2
expression or localization (Fig.
6B, MSL-2 panel).
Previous studies have reported reduced DCC targeting after RNAi treatment
of SL2 cells against the nuclear pore components Mtor and NUP153
(Mendjan et al., 2006), or
after alteration of the balance between DCC components
(Demakova et al., 2003). We
checked whether the levels and distribution of these proteins were affected by
the depletion of UNR. Staining for Mtor and NUP153 showed no significant
differences between untreated and UNR RNAi-treated cells
(Fig. 6A and data not shown).
In addition, western blot analysis indicated that both the amount and
distribution of all DCC protein components, and of Mtor and NUP153, remained
unchanged (Fig. 6B). These data
indicate that UNR promotes DCC binding to the male X chromosome by a mechanism
that is independent of translation of DCC components.
Loss of DCC staining on the X has also been observed in roX
mutants, which lack the RNA component of the DCC
(Franke and Baker, 1999;
Meller et al., 2000;
Meller and Rattner, 2002). We
therefore tested whether depletion of UNR resulted in loss of roX2.
No loss of roX2 was detected by qRT-PCR of total RNA from
UNR-depleted cells (see Fig. S2 in the supplementary material). Similarly, the
levels of roX1 and roX2 in Unr mutant males were
nearly identical to those in wild-type males (see Fig. S2 in the supplementary
material). Thus, UNR does not affect the expression of roX RNAs.
|
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Duncan et al., 2006). In this
work, we have analyzed the in vivo roles of UNR by characterizing a
hypomorphic Unr mutant and testing the effect of overexpressing UNR
in flies. Our results uncover dual, mechanistically distinct roles for UNR in
the regulation of dosage compensation in males and females.
Specific recruitment of UNR to the 3' UTR of msl-2 mRNA by
SXL is required for repression of msl-2 translation both in vitro and
in cell culture (Abaza et al.,
2006; Duncan et al.,
2006). A prediction from these results is that UNR represses
dosage compensation in female flies. Indeed, in hypomorphic mutant females
lacking the C-terminal half of UNR, the DCC assembles on a set of X
chromosomal sites (Fig. 4).
These sites map closely with positions previously described as being
high-affinity sites, which are occupied by the DCC in conditions of low
complex concentration (e.g. Demakova et
al., 2003). These observations suggest partial derepression of
msl-2 translation in mutant females. Two of the high-affinity sites
correspond to the loci for roX1 and roX2 RNAs
(Table 2, cytological positions
3F and 10C, respectively). Expression of these RNAs requires MSL-2 and their
stability depends on their association to the DCC complex
(Meller et al., 2000;
Rattner and Meller, 2004). The
fact that roX levels were similarly low in mutant and wild-type
females supports the notion that msl-2 translational derepression in
the mutant is only partial (see Fig. S2 in the supplementary material). These
results indicate that the N-terminal half of UNR exerts strong translational
inhibition in vivo, and are consistent with in vitro data showing that amino
acids 1-397 of UNR are sufficient for translational repression in functional
tethering assays (Abaza and Gebauer,
2008). Appropriate UNR levels are essential for viability and
development because moderate (2-fold) overexpression of UNR results in
complete lethality early in development for both males and females
(Fig. 1). Accordingly, keeping
the correct stoichiometry between UNR and SXL is important for translational
control of msl-2, and might be necessary for the regulation of other
substrates (Abaza and Gebauer,
2008).
Unexpectedly, Unr mutant males showed decreased MSL-2 staining on
the X chromosome, and UNR-depleted SL2 cells showed MSL-2 delocalization from
the X chromosome and redistribution in the nucleoplasm (Figs
5 and
6). Reduced MSL-2 targeting to
the X chromosome correlated with defective recruitment of other DCC components
(Fig. 5). These effects were
independent of variations in MSL-2 levels, consistent with the observation
that UNR does not bind to msl-2 mRNA in males
(Abaza et al., 2006). Because
DCC targeting defects have been observed under conditions of unbalanced
concentrations of MSL proteins or disturbed MSL/roX ratios, we
reasoned that UNR might regulate the levels of other DCC constituents in males
(Demakova et al., 2003;
Dahlsveen et al., 2006;
Meller et al., 2000;
Oh et al., 2003). Strikingly,
however, the levels and nucleocytoplasmic distribution of all DCC protein
components remained unaltered in UNR-depleted cells
(Fig. 6). Similarly, the levels
of roX RNAs in Unr mutant flies or UNR-depleted cells were
indistinguishable from those in the wild type (see Fig. S2 in the
supplementary material). We conclude that UNR does not interfere with the
expression or localization of DCC components.
|
|
). In
addition, RNase treatment of polytene chromosomes removes MLE from the DCC,
suggesting that MLE recruitment to the X chromosome requires roX RNAs
(Richter et al., 1996).
Conversely, MLE is an RNA helicase necessary for roX incorporation
into the DCC and its helicase activity is necessary for spreading of the DCC
along the X chromosome (Meller et al.,
2000; Morra et al.,
2008). Thus, the binding of MLE and of roX RNAs to the X
chromosome appear to be interdependent. A possible explanation for the role of
UNR in males is that UNR affects the function of these DCC components. UNR is
a CSD-containing protein and, in bacteria, CSD proteins associate with RNA
helicases to modify the structure of RNA and regulate gene expression
(reviewed by Horn et al.,
2007). Indeed, mammalian UNR binds to the IRES of Apaf1
mRNA and modifies its conformation
(Mitchell et al., 2003).
Therefore, UNR might associate with MLE in order to promote the appropriate
structure of the roX RNAs for incorporation into the DCC or for
subsequent spreading along the X chromosome. In support of this hypothesis,
UNR specifically binds to both roX1 and roX2 RNAs in males
(Fig. 7). In addition, as
previously observed in blastoderm embryos, a fraction of UNR localizes to the
nucleus of SL2 and salivary gland cells, where both MLE and roX
concentrate (see Fig. S3 in the supplementary material)
(Abaza et al., 2006).
UNR could also function indirectly, via the regulation of chromatin
structure, to promote DCC recruitment to the X chromosome. The Unr
hypomorphic mutant and the transgenic Unr flies show abnormal
packaging of the male X chromosome, consisting of bloated or knotted
chromatin. The observation that staining of histone H3 appears normal suggests
that the first level of chromatin compaction remains unaltered in Unr
mutants (Fig. 5C). In order to
regulate chromatin structure, UNR could interact with chromatin remodeling
factors. For example, a member of the trithorax group, ALL-1 (MLL - Human Gene
Nomenclature Database), was found to interact with human UNR (CSDE1) in a
yeast two-hybrid assay (Leshkowtz et al., 1996). Alternatively, UNR could
control the expression of chromatin regulators that influence X chromosome
morphology, such as ISWI, NURF, JIL-1 or SU(VAR)3-7
(Deng et al., 2005;
Corona et al., 2007;
Carré et al., 2008). It
is interesting to note that although mutations of most of these factors do not
concur with loss of DCC binding, null mutations of Su(var)3-7 result
in both a bloated X chromosome and depletion of the DCC from the X chromosome
(Spierer et al., 2008). Thus,
UNR could regulate the expression of SU(VAR)3-7 - or of other regulators with
similar functions - in order to modulate DCC recruitment. In summary, at this
point our results do not allow us to conclude whether the chromatin-packaging
and DCC-binding defects observed in males are dissociable events.
Nevertheless, the fact that UNR binds to roX RNAs implicates a direct
role of UNR in DCC recruitment. Further studies are necessary to clarify the
relationship between the multiple nuclear functions of UNR.
Our results show that UNR performs opposing functions in the regulation of
dosage compensation in males and females. Dosage compensation is
evolutionarily linked to sex determination. In D. melanogaster, a
single master protein regulates both processes: SXL determines the female
sexual fate and represses dosage compensation. However, SXL is not
sex-specifically expressed in other distant species of Diptera, raising the
possibility that the use of SXL for sex determination is a recent adaptation
of the Drosophila genus
(Pomiankowski et al., 2004).
Perhaps, SXL made use of an existing regulator of dosage compensation, namely
UNR, and adapted its function to a new role in females. Further genetic
studies and biochemical analyses will help to identify the interactors and
substrates that mediate the diverse roles of UNR.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/689/DC1
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Abaza, I. and Gebauer, F. (2008). Functional
domains of Drosophila UNR in translational control.
RNA 14,482
-490.
Abaza, I., Coll, O., Patalano, S. and Gebauer, F.
(2006). Drosophila UNR is required for translational repression
of male-specific lethal 2 mRNA during regulation of X-chromosome dosage
compensation. Genes Dev.
20,380
-389.
Akhtar, A. and Becker, P. B. (2000). Activation
of transcription through histone H4 acetylation by MOF, an acetyltransferase
essential for dosage compensation in Drosophila. Mol.
Cell 5,367
-375.[CrossRef][Medline]
Akhtar, A., Zink, D. and Becker, P. B. (2000).
Chromodomains are protein-RNA interaction modules.
Nature 407,405
-409.[CrossRef][Medline]
Bashaw, G. J. and Baker, B. S. (1995). The
msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding
protein whose expression is sex specifically regulated by Sex-lethal.
Development 121,3245
-3258.[Abstract]
Bashaw, G. J. and Baker, B. S. (1997). The
regulation of the Drosophila msl-2 gene reveals a function for Sex-lethal in
translational control. Cell
89,789
-798.[CrossRef][Medline]
Beckmann, K., Grskovic, M., Gebauer, F. and Hentze, M. W.
(2005). A dual inhibitory mechanism restricts msl-2 mRNA
translation for dosage compensation in Drosophila.
Cell 122,529
-540.[CrossRef][Medline]
Carré, C., Ciurciu, A., Komonyi, O., Jacquier, C.,
Fagegaltier, D., Pidoux, J., Tricoire, H., Tora, L., Boros, I. M. and
Antoniewski, C. (2008). The Drosophila NURF remodelling and
the ATAC histone acetylase complexes functionally interact and are required
for global chromosome organization. EMBO Rep.
9, 187-192.[CrossRef][Medline]
Corona, D. F., Siriaco, G., Armstrong, J. A., Snarskaya, N.,
McClymont, S. A., Scott, M. P. and Tamkun, J. W. (2007). ISWI
regulates higher-order chromatin structure and histone H1 assembly in vivo.
PLoS Biol. 5,e232
.[CrossRef][Medline]
Dahlsveen, I. K., Gilfillan, G. D., Shelest, V. I., Lamm, R. and
Becker, P. B. (2006). Targeting determinants of dosage
compensation in Drosophila. PLoS Genet.
2, e5.[CrossRef][Medline]
Demakova, O. V., Kotlikova, I. V., Gordadze, P. R., Alekseyenko,
A. A., Kuroda, M. I. and Zhimulev, I. F. (2003). The MSL
complex levels are critical for its correct targeting to the chromosomes in
Drosophila melanogaster. Chromosoma
112,103
-115.[CrossRef][Medline]
Deng, H., Zhang, W., Bao, X., Martin, J. N., Girton, J.,
Johansen, J. and Johansen, K. M. (2005). The JIL-1 kinase
regulates the structure of Drosophila polytene chromosomes.
Chromosoma 114,173
-182.[CrossRef][Medline]
Duncan, K., Grskovic, M., Strein, C., Beckmann, K., Niggeweg,
R., Abaza, I., Gebauer, F., Wilm, M. and Hentze, M. W.
(2006). Sex-lethal imparts a sex-specific function to UNR by
recruiting it to the msl-2 mRNA 3' UTR: translational repression for
dosage compensation. Genes Dev.
20,368
-379.
Franke, A. and Baker, B. S. (1999). The rox1
and rox2 RNAs are essential components of the compensasome, which mediates
dosage compensation in Drosophila. Mol. Cell
4, 117-122.[CrossRef][Medline]
Franke, A., Dernburg, A., Bashaw, G. J. and Baker, B. S.
(1996). Evidence that MSL-mediated dosage compensation in
Drosophila begins at blastoderm. Development
122,2751
-2760.[Abstract]
Furuhashi, H., Nakajima, M. and Hirose, S.
(2006). DNA supercoiling factor contributes to dosage
compensation in Drosophila. Development
133,4475
-4483.
Gebauer, F., Merendino, L., Hentze, M. W. and Valcarcel, J.
(1998). The Drosophila splicing regulator sex-lethal directly
inhibits translation of male-specific-lethal 2 mRNA.
RNA 4,142
-150.[Abstract]
Gebauer, F., Grskovic, M. and Hentze, M. W.
(2003). Drosophila sex-lethal inhibits the stable association of
the 40S ribosomal subunit with msl-2 mRNA. Mol. Cell
11,1397
-1404.[CrossRef][Medline]
Gilfillan, G. D., Dahlsveen, I. K. and Becker, P. B.
(2004). Lifting a chromosome: dosage compensation in Drosophila
melanogaster. FEBS Lett.
567, 8-14.[CrossRef][Medline]
Horn, G., Hofweber, R., Kremer, W. and Kalbitzer, H. R.
(2007). Structure and function of bacterial cold shock proteins.
Cell Mol. Life Sci. 64,1457
-1470.[CrossRef][Medline]
Kelley, R. L., Solovyeva, I., Lyman, L. M., Richman, R.,
Solovyev, V. and Kuroda, M. I. (1995). Expression of msl-2
causes assembly of dosage compensation regulators on the X chromosomes and
female lethality in Drosophila. Cell
81,867
-877.[CrossRef][Medline]
Kelley, R. L., Wang, J., Bell, L. and Kuroda, M. I.
(1997). Sex lethal controls dosage compensation in Drosophila by
a non-splicing mechanism. Nature
387,195
-199.[CrossRef][Medline]
Kotlikova, I. V., Demakova, O. V., Semeshin,V. F., Shloma, V.
V., Boldyreva, L. V., Kuroda, M. I. and Zhimulev, I. F.
(2006). The Drosophila dosage compensation complex binds to
polytene chromosomes independently of developmental changes in transcription.
Genetics 172,963
-974.
Leshkowitz, D., Rozenblatt, O., Nakamura, T., Yano, T., Dautry,
F., Croce, C. M. and Canaani, E. (1996). ALL-1 interacts with
unr, a protein containing multiple cold shock domains.
Oncogene 13,2027
-2031.[Medline]
Lucchesi, J. C., Kelly, W. G. and Panning, B.
(2005). Chromatin remodeling in dosage compensation.
Annu. Rev. Genet. 39,615
-651.[CrossRef][Medline]
Lyman, L. M., Copps, K., Rastelli, L., Kelley, R. L. and Kuroda,
M. I. (1997). Drosophila male-specific lethal-2 protein:
structure/function analysis and dependence on MSL-1 for chromosome
association. Genetics
147,1743
-1753.[Abstract]
Meller, V. H. and Rattner, B. P. (2002). The
roX genes encode redundant male-specific lethal transcripts required for
targeting of the MSL complex. EMBO J.
21,1084
-1091.[CrossRef][Medline]
Meller, V. H., Wu, K. H., Roman, G., Kuroda, M. I. and Davis, R.
L. (1997). roX1 RNA paints the X chromosome of male
Drosophila and is regulated by the dosage compensation system.
Cell 88,445
-457.[CrossRef][Medline]
Meller, V. H., Gordadze, P. R., Park, Y., Chu, X., Stuckenholz,
C., Kelley, R. L. and Kuroda, M. I. (2000). Ordered assembly
of roX RNAs into MSL complexes on the dosage-compensated X chromosome in
Drosophila. Curr. Biol.
10,136
-143.[CrossRef][Medline]
Mendjan, S. and Akhtar, A. (2007). The right
dose for every sex. Chromosoma
116,95
-106.[CrossRef][Medline]
Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P.,
Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J. et al.
(2006). Nuclear pore components are involved in the
transcriptional regulation of dosage compensation in Drosophila.
Mol. Cell 21,811
-823.[CrossRef][Medline]
Mitchell, S. A., Spriggs, K. A., Coldwell, M. J., Jackson, R. J.
and Willis, A. E. (2003). The Apaf-1 internal ribosome entry
segment attains the correct structural conformation for function via
interactions with PTB and unr. Mol. Cell
11,757
-771.[CrossRef][Medline]
Morra, R., Smith, E. R., Yokoyama, R. and Lucchesi, J. C.
(2008). The MLE subunit of the Drosophila MSL complex uses its
ATPase activity for dosage compensation and its helicase activity for
targeting. Mol. Cell. Biol.
28,958
-966.
Oh, H., Park, Y. and Kuroda, M. I. (2003).
Local spreading of MSL complexes from roX genes on the Drosophila X
chromosome. Genes Dev.
17,1334
-1339.
Pomiankowski, A., Nöthiger, R. and Wilkins, A.
(2004). The evolution of the Drosophila sex determination
pathway. Genetics 166,1761
-1773.
Rattner, B. P. and Meller, V. H. (2004).
Drosophila male-specific lethal 2 protein controls sex-specific expression of
the roX genes. Genetics
166,1825
-1832.
Richter, L., Bone, J. R. and Kuroda, M. I.
(1996). RNA-dependent association of the Drosophila maleless
protein with the male X chromosome. Genes Cells
1, 325-336.[Abstract]
Rorth, P. (1998). Gal4 in the Drosophila female
germline. Mech. Dev. 78,113
-118.[CrossRef][Medline]
Smith, E. R., Pannuti, A., Gu, W., Steurnagel, A., Cook, R. G.,
Allis, C. D. and Lucchesi, J. C. (2000). The drosophila MSL
complex acetylates histone H4 at lysine 16, a chromatin modification linked to
dosage compensation. Mol. Cell. Biol.
20,312
-318.
Spierer, A., Seum, C., Delattre, M. and Spierer, P.
(2005). Loss of the modifiers of variegation Su(var)3-7 or HP1
impacts male X polytene chromosome morphology and dosage compensation.
J. Cell Sci. 118,5047
-5057.
Spierer, A., Begeot, F., Spierer, P. and Delattre, M.
(2008). SU(VAR)3-7 links heterochromatin and dosage compensation
in Drosophila. PLoS Genet.
4,e1000066
.[CrossRef][Medline]
Straub, T. and Becker, P. B. (2007). Dosage
compensation: the beginning and end of generalization. Nat. Rev.
Genet. 8,47
-57.[CrossRef][Medline]
Thibault, S. T., Singer, M. A., Miyazaki, W. Y., Milash, B.,
Dompe, N. A., Singh, C. M., Buchholz, R., Demsky, M., Fawcett, R.,
Francis-Lang, H. L. et al. (2004). A complementary transposon
tool kit for Drosophila melanogaster using P and piggyBac. Nat.
Genet. 36,283
-287.[CrossRef][Medline]
Wang, Y., Zhang, W., Jin, Y., Johansen, J. and Johansen, K.
M. (2001). The JIL-1 tandem kinase mediates histone H3
phosphorylation and is required for maintenance of chromatin structure in
Drosophila. Cell 105,433
-443.[CrossRef][Medline]
Wilhelm, J. E., Mansfield, J., Hom-Booher, N., Wang, S., Turck,
C. W., Hazelrigg, T. and Vale, R. D. (2000). Isolation of a
ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes.
J. Cell Biol. 148,427
-440.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  M. E. Gelbart and M. I. Kuroda Drosophila dosage compensation: a complex voyage to the X chromosome Development, May 1, 2009; 136(9): 1399 - 1410. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
