|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 14 March 2007
doi: 10.1242/dev.000521
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK.
Author for correspondence (e-mail:
helen.white-cooper{at}zoo.ox.ac.uk)
Accepted 7 February 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Spermatogenesis, Transcription, SynMuv, Differentiation, CXC
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) and testis-specific proteasome components
(Ma et al., 2002)], others are
spermatogenesis-specific proteins [e.g. the protamines that replace histones
to tightly package sperm DNA (Jayaramaiah
Raja and Renkawitz-Pohl, 2005)].
In both mammals and insects, germline stem cells divide to produce
spermatogonia. After further mitotic amplification divisions (four in
Drosophila melanogaster), spermatogonia become primary spermatocytes,
committed to differentiation (reviewed by
Fuller, 1993). This
developmental transition results in transcriptional activation in primary
spermatocytes of a large suite of genes required for meiosis and
spermiogenesis. In Drosophila, transcription stops before the meiotic
divisions, so transcripts for late-acting proteins are made pre-meiotically
(Olivieri and Olivieri, 1965).
Meiotic-arrest mutant testes accumulate primary spermatocytes, but lack later
stages of spermatogenesis because mutant primary spermatocytes fail to
initiate transcription of many genes whose products are required after
meiosis. Meiotic-arrest mutants also fail to express some meiotic gene
products; always early (aly)-class gene products differ from
cannonball (can)-class in their regulation of certain cell
cycle genes (Lin et al., 1996;
White-Cooper et al., 1998).
Through their function in controlling production of cell cycle and
differentiation gene products, the meiotic-arrest genes coordinate the
independent processes of meiosis and spermatid morphogenesis.
The aly class of meiotic-arrest genes have a broader target range
than the can class. Four aly-class and five
can-class meiotic-arrest loci have been described
(Ayyar et al., 2003;
Hiller et al., 2004;
Hiller et al., 2001;
Jiang and White-Cooper, 2003;
Perezgazga et al., 2004;
Wang and Mann, 2003;
White-Cooper et al., 2000;
White-Cooper et al., 1998).
aly encodes one of two Drosophila homologues of the C.
elegans synMuvB gene lin-9, the other homologue being
mip130 (Beitel et al.,
2000; White-Cooper et al.,
2000). cookie monster (comr) encodes a novel
protein of unknown function (Jiang and
White-Cooper, 2003). achintya/vismay (achi/vis)
and matotopetli (topi) encode sequence-specific DNA-binding
proteins (Ayyar et al., 2003;
Perezgazga et al., 2004;
Wang and Mann, 2003). Aly,
Comr and Achi/Vis proteins co-immunoprecipitate from testis extracts; Topi was
identified in a yeast two-hybrid screen for Comr interactors
(Perezgazga et al., 2004;
Wang and Mann, 2003). Despite
the interactions between aly-class gene products, the aly
and comr mutant phenotypes are subtly different from those of
topi and achi/vis. Aly and Comr nuclear localisations are
mutually dependent, whereas these proteins require topi and
achi/vis for their concentration on chromatin. aly or
comr (but not topi or achi/vis) mutants display
defects in chromatin organisation. Finally, a small subset of genes are much
more dependent on topi and/or achi/vis than on aly
or comr for their transcription
(Jiang and White-Cooper, 2003;
Perezgazga et al., 2004).
To find further transcriptional regulators in primary spermatocytes, we screened for Aly-binding proteins. We have identified and characterised a new Drosophila meiotic-arrest gene, tombola (tomb), which is expressed specifically in testis. tomb encodes the second Drosophila member of the tesmin/TSO1 CXC-domain protein family, the other being Mip120, a subunit of the same complex as Mip130. We show that Tomb complexes with Aly and Comr. We identify a tomb mutant and show that tomb mutant testes have an aly-class meiotic-arrest phenotype more like that of aly and comr than of topi and achi/vis mutants. Aly and Comr proteins fail to associate with chromatin in the absence of tomb function. Topi protein also localises to chromatin in wild-type and achi/vis primary spermatocytes, but not in aly, comr or tomb mutant cells. Concentration of ectopically expressed EGFP-Tomb on chromatin in the nucleus is normal in achi/vis or can-class mutants, but is altered in aly and comr mutants.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
).
P[GS]12862/CyO was from the Kyoto Drosophila Stock Centre.
Mutant alleles used were aly5, comrZ1340,
achiZ3922visZ3922, topiZ3-2139,
nhtZ2-5946. w1118 or red e were used as
wild-type controls.
Deficiency mapping and P-element excision
Df(2L)cl-h3/SM6b and Df(2L)cl-h1/CyO,
amosRoi-1 (Bloomington Drosophila Stock Centre) were
crossed to P[GS]12862/CyO. Excision of the P[GS]12862
insertion was carried out by crossing w;
P[GS]12862/CyO,
2-3 males to w; Tft/CyO females,
recovering individual white-eyed progeny and back-crossing to w;
Tft/CyO to establish stocks. Excision lines were analysed by testis
squashing and PCR and sequencing of the ORF. Females were crossed to their
balanced brothers to test for fertility; male fertility was tested by crossing
to virgin w1118 females. The testis phenotype was scored
by phase contrast microscopy after dissection and squashing.
Yeast two-hybrid screen and analysis
An Aly(C-terminus)-Gal4-DNA-Binding Domain [Aly(C)-DB] fusion construct was
made by subcloning the ORF (equivalent to amino acids 275 to 534) from a
full-length aly cDNA clone into pGBKT7. We generated and screened a
testis cDNA-Gal4-Activation Domain (AD) fusion protein library using the
Matchmaker Library Construction and Screening Kit (Clontech) as previously
described (Perezgazga et al.,
2004). Colonies were picked from SD -Ade -His -Leu -Trp selection
plates after 7 days. One million independent co-transformants yielded 90
colonies that grew under selective conditions and were blue in the presence of
X-
-Gal. To test for interaction between Tomb and Comr, AH109 yeast
cells were co-transformed with pGADT7-Rec-Tomb and pGBKT7-Comr or
pGBKT7-CG15031 (CG15031 was another clone isolated in the yeast two-hybrid
screen) as a negative control. Transformed cells were plated on SD -Ade -His
-Leu -Trp selection plates containing X--Gal.
Construction of deletion analysis plasmids
PCR products for Tomb deletion derivatives (amino acid residues: 1-73;
1-136; 73-243; 136-243) and full-length Tomb were subcloned into pACT2.
Co-transformation of AH109 yeast cells was with pGBKT7-Aly(C) or
pGBKT7-Kr(Zn-finger) as the negative control
(Perezgazga et al., 2004).
Transformed cells were plated on SD -Ade -His -Leu -Trp/X--Gal
plates.
RT-PCR expression analysis
For semi-quantitative RT-PCR, total RNA was extracted from dissected testes
with Trizol (Invitrogen) and resuspended in RNAse-free water (three
testes-worth per µl). First-strand cDNA was generated from 4 µl of this
sample using oligo-dT primers with the SuperScript II Reverse Transcriptase
System (Invitrogen). cDNA derived from 0.18 testes (0.3 µl of RT reaction)
was used for each RT-PCR reaction and amplified with Taq DNA polymerase
(Qiagen) with 24 amplification cycles. Genomic DNA from wild-type flies was
used as a positive PCR control, and a no-reverse-transcriptase (no-RT)
reaction on wild-type RNA served as a negative control. For RT-PCR from
various developmental stages, total RNA was extracted with Trizol, cDNA
prepared as above, and PCR amplification carried out for 30 cycles. For
re-amplification, 0.5 µl of the first PCR product was used as the template
for a further 30-cycle PCR reaction.
Mapping the 5' and 3' ends of tomb
A 3' RACE Kit was used following the manufacturer's (Invitrogen)
instructions. The RACE products were either directly sequenced, or were
subcloned into pGEM-T-Easy for sequencing. For the 5' end, RT-PCR was
performed using a series of primers upstream of the ATG, paired with a
3' primer within the coding sequence.
Co-expression and co-immunoprecipitation from tissue culture cells and testis extracts
The full-length tomb ORF was subcloned into the mammalian tissue
culture expression vector HA-tagged pCDEF3. The full-length aly ORF
and Kruppel (Kr) zinc-finger region
(Perezgazga et al., 2004) were
similarly subcloned into FLAG-tagged pCDEF3. 293T human kidney cells were
co-transfected with plasmids for expression of HA-Tomb and FLAG-Aly, or
HA-Tomb and FLAG-Kr(Zn-finger), respectively, with lipofectamine 2000 reagent
(Invitrogen). Co-immunoprecipitation was as previously described
(Perezgazga et al., 2004).
Testes dissected from EGFP-Tomb-expressing flies were homogenised in lysis buffer (50 mM Tris-HCl pH 7.5-8.0, 0.5% Triton X-100, 150 mM NaCl, protease inhibitors) (146 testes, 500 µl buffer used), incubated with ethidium bromide (400 µg/ml) for 30 minutes at 4°C, then cleared by centrifugation. 20 µl was retained as the `input' sample, the remainder was pre-cleared with protein G-sepharose, then incubated with mouse anti-GFP (Roche) and precipitated with protein G-sepharose. Beads were washed, then bound proteins were eluted by boiling in SDS sample buffer. Wild-type testes were processed in parallel as a negative control.
GFP fusion construct
The tomb ORF was subcloned in frame into pUAST-EGFP
(Parker et al., 2001).
Numerous independent P-element-mediated insertions were recovered using
standard transformation protocols after injection of w1118
embryos. EGFP-Tomb fusion protein expression was driven using Bam-Gal4-VP16,
which expresses just before the onset of meiotic-arrest gene expression and
functions in all the mutant backgrounds
(Chen and McKearin, 2003).
Bam-GAL4-VP16 (on chromosome 3) was recombined with a third-chromosome
UAS-EGFP-Tomb insertion, and the chromosome was used homozygous to express
tagged protein in testes homozygous for second chromosome male steriles
(tomb, achi/vis, comr, nht). Bam-GAL4-VP16 was recombined with
aly5 to allow expression from a homozygous second
chromosome-linked UAS-EGFP-tomb insertion in this mutant
background.
Generation of the anti-Topi antibody
Anti-peptide antibodies were raised by Moravian-Biotechnology. The
synthesised oligopeptide KNNPTKPIFSDTYL from the Topi C-terminus was coupled
to BSA and used to immunise two rats. The staining patterns for these sera
were indistinguishable.
Microscopy and immunofluorescence
Live testes were dissected, squashed in 2 µg/ml Hoechst 33342 in testis
buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris pH 6.8) and examined by phase
contrast and fluorescence microscopy. Images were captured with a Q-imaging
Retiga 1300 monochrome CCD camera linked to an Olympus BX50 microscope using
Openlab software (Improvision) or on a JVC KY-F75U three-colour CCD camera
with KY-Link software, and imported into Photoshop (Adobe). Aly, Comr and Topi
proteins were visualised by indirect immunofluorescent staining using rabbit
anti-Aly (1:2000), rabbit anti-Comr (1:1000) or rat anti-Topi (1:1000)
antibodies detected with FITC-conjugated secondary antibodies (Jackson), as
described (Jiang and White-Cooper,
2003; White-Cooper et al.,
2000). DNA was co-stained with propidium iodide. Cells were imaged
using a Bio-Rad Radiance Plus confocal microscope mounted on a Nikon E800.
RNA in situ hybridisation
Dig-labelled antisense probes for Cyclin B, Mst87F and
polo were generated as previously described
(White-Cooper et al., 1998).
To synthesise RNA probes for CG3330, CG3927 and CG12907, we
generated 400-600 bp RT-PCR products using total testis RNA as template. For
tomb, the PCR amplified the entire ORF. The 3' PCR primers
included a T3 RNA polymerase promoter site for in vitro transcription of
dig-labelled antisense RNA probes. In situ hybridisation was carried out as
described (White-Cooper et al.,
1998). Primer sequences are available on request.
|
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
)]. To test the in vivo interaction between Aly and Tomb, and
to test whether DNA was implicated in the interaction, we made extracts from
EGFP-Tomb-expressing testes (see below), incubated the extracts with ethidium
bromide, and immunoprecipitated using anti-GFP. We found that Aly
co-immunoprecipitated with EGFP-Tomb, showing that the interaction occurs in
testes, and is DNA independent (Fig.
1B). To define the Aly-interaction region of Tomb, we generated
deletion constructs and tested their ability to interact in the two-hybrid
system. The Aly-interaction domain is found in the C-terminal half of Tomb
(amino acids 136-243). Since aly and comr have identical mutant phenotypes, we suspect that these gene products probably act together in a complex, but we have not detected direct interaction between these proteins. We tested the ability of Tomb to bind Comr by two-hybrid analysis. Yeast co-transformed with Tomb-AD and Comr-DB grew under selective conditions, demonstrating that Comr can interact with Tomb. The Tomb-Aly and Tomb-Comr interactions were specific, as yeast co-transformed with Tomb-AD and CG15031-DB were unable to grow under selective conditions. Co-expression and co-immunoprecipitation experiments in tissue culture cells confirmed that FLAG-Tomb can interact with HA-Comr (data not shown).
The tomb genomic region is complex
(Fig. 2A). The tomb
ORF is embedded within, but in the opposite orientation to, the 3' UTR
of CG31989, which is predicted to encode a conserved protein (Cap-D3)
of unknown function. As no tomb cDNA clones have been sequenced, we
mapped the 5' and 3' ends by RACE and RT-PCR. The 5' end of
tomb overlaps the 3' end of the adjacent gene CG14015.
Translation of the tomb ORF gave a 243 amino acid, 26 kD conceptual
protein with a theoretical pI of 9.4. The predicted Tomb protein contains a
nuclear localisation signal and a CXC motif of the tesmin/TSO1 family
(Fig. 2B). Tesmin has been
described in vertebrates (it also known as Mtl5/MTL5 in mouse and human),
whereas TSO1 is from Arabidopsis, indicating that this domain is
conserved between animals and plants. The only other Drosophila
tesmin/TSO1 CXC-domain protein, Mip120, has been found in a complex with the
second Drosophila lin-9 (aly) homologue, Mip130
(Beall et al., 2002;
Korenjak et al., 2004;
Lewis et al., 2004). A second
tesmin/TSO1 CXC-domain protein, which we refer to as tesmin-like (tesl), was
found in humans and mouse. C. elegans has a single member of this
family, LIN-54 (JC8.6), sea urchin and Ciona each have one homologue.
Including TSO1, the A. thaliana genome has 11 tesmin/TSO1 family
members.
Comparison of the tesmin/TSO1-domain proteins revealed that tomb was unusual in only having a single CXC domain (Fig. 2C). All other family members have either two domains (vertebrate tesl, worm LIN-54 and plant TSO1), or one and a half CXC domains (vertebrate tesmin and plant SOL2). These domains were separated by a conserved, 42 amino acid spacer in animals (50 amino acids in plants). The first and second CXC domains contain several residues in common; however, they are distinguished by characteristic amino acids conserved within repeat 1 or 2, but not between repeats (Fig. 2C). The one and a half CXC-domain proteins lack the N-terminus of the first domain, whereas tomb has only the second CXC domain. E(z) CXC-like domains fall into a separate family.
We also identified a 52 amino acid additional region of homology between
the animal proteins near the C-terminus (31% identity, 50% similarity between
mip120 and human tesmin (hs-tes); 33% identity and 42%
similarity between tomb and hs-tes). Although the primary
sequence conservation is low these sequences are strongly predicted to form a
helix-coil-helix secondary structure (PSIpred)
(McGuffin et al., 2000)
(Fig. 2D). The Aly-interaction
domain of Tomb includes this conserved motif but not the CXC domain.
tomb expression is testis-specific
We investigated the tomb developmental expression profile by
RT-PCR. tomb is entirely included within the CG31989
3' UTR; however, they are encoded on opposite strands. tomb
contains a 62 bp intron, whereas the CG31989 3' UTR lacks
introns, allowing us to distinguish the transcripts. tomb transcript
was detected only in testis (Fig.
3A). Unspliced products, derived from CG31989
transcripts, were not produced from the testis sample, but were found after
re-amplification in gonadectomised adults (both males and females) and embryos
(0-16 hours) (data not shown).
|
|
|
). Inverse
PCR and sequencing of the flanking DNA of P[GS]12862 confirmed that
the element was inserted in codon 174 of tomb. The
P[GS]12862 line was homozygous viable, but male sterile. Homozygous
females were initially semi-sterile; however, this phenotype was later lost
from the stock. The mutant phenotype of P[GS]12862 could be due to disruption of the function of tomb or CG31989 or both, or could be unrelated to the P-insertion. We tested the contribution of CG31989 to the phenotype using P[EY]00456, a P-element insertion in the CG31989 ORF. P[EY]00456 mutant flies were homozygous viable and male and female fertile, as were P[GS]12862/P[EY]00456 trans-heterozygotes, indicating that the phenotype of P[GS]12862 was not due to CG31989 loss-of-function. The male fertility defect of P[GS]12862 was uncovered by both Df(2L)cl-h3 and Df(2L)cl-h1, which delete 25D2-3;26B2-5 and 25D4;25F1-2, respectively (tomb is at 25E5). P[GS]12862/Df females were fully fertile, confirming that the male and female fertility defects of P[GS]12862 were separable. Transposase-mediated excision of P[GS]12862 resulted in full reversion of the mutant phenotype, indicating that the male sterility is caused by the insertion into tomb.
Phase contrast examination of squash preparations of tombGS12862 homozygous or tombGS12862/Df testes revealed that tomb is a meiotic-arrest gene. tomb testes contained morphologically normal stages of spermatogenesis, up to and including mature primary spermatocytes, but no meiotic division or post-meiotic stages (Fig. 4A,B).
Tomb protein is concentrated on chromatin in primary spermatocytes
We expressed an EGFP-Tomb fusion protein in primary spermatocytes using a
Bam-GAL4-VP16 driver, and found that tagged Tomb protein was able to rescue
the meiotic-arrest phenotype of tombGS12862 homozygous
males. This confirmed that the expressed protein is functional, and provided
final confirmation that the meiotic-arrest phenotype is due to loss of
tomb function (Fig.
4C,D). When expressed in a wild-type background, EGFP-tagged Tomb
protein was initially both nuclear and cytoplasmic (at lower levels) in early
primary spermatocytes. In more mature primary spermatocytes, EGFP-Tomb was
restricted to the nucleus and concentrated on chromatin
(Fig. 4E-H): three brightly
labelled major chromosome bivalents apposed to the nuclear membrane were
visible in every nucleus.
tomb is aly-class
aly-class meiotic-arrest mutant primary spermatocytes fail to
express Cyclin B mRNA, whereas can-class mutants express
normal levels of Cyclin B mRNA
(White-Cooper et al., 1998).
tomb mutant testes did not accumulate significant levels of
Cyclin B mRNA (Fig.
5I-L), so tomb is aly-class. RNA in situ
hybridisation confirmed that tomb, like all known meiotic-arrest
genes, is also required for expression of spermatid differentiation genes,
including Mst87F (Fig.
5E-H). tomb mutant testes again resembled other
meiotic-arrest loci in that transcription was not completely blocked; for
example, they accumulated polo transcripts normally
(Fig. 5A-D).
aly-class mutants fall into two subgroups based on primary
spermatocyte DNA morphology (Ayyar et al.,
2003; Jiang and White-Cooper,
2003; Lin et al.,
1996; Perezgazga et al.,
2004). Hoechst 33342 labelling revealed that the tomb DNA
chromosomes were somewhat condensed and fuzzy, like aly or
comr mutants, rather than more condensed and away from the nuclear
envelope as seen in achi/vis or topi mutants
(Fig. 6A-D').
achi/vis and topi also differ slightly from aly and
comr in their target gene specificities
(Perezgazga et al., 2004).
Although all genes that depend on aly or comr for expression
also depend on achi/vis and/or topi, there are a few genes,
including CG3927 and CG12907, whose transcription depends on
achi/vis and topi but not on aly or comr.
Several other genes, including CG3330, depend on all the
aly-class meiotic-arrest genes to some extent for their expression,
but differ between aly or comr and achi/vis or
topi in that their expression is undetectable in testes from the
latter two mutants, but is detected at very low levels in aly or
comr testes. The tomb phenotype was indistinguishable from
that of aly or comr with respect to expression of
CG3927, CG12907 and CG3330
(Fig. 6E-P). Phenotypic
comparison data are summarised in Table
1.
|
|
|
). By contrast, mutation of achi/vis or topi
does not prevent nuclear translocation of Aly and Comr, although these
proteins fail to concentrate on chromatin and show a uniform nuclear
localisation in achi/vis or topi mutant spermatocytes
(Ayyar et al., 2003;
Perezgazga et al., 2004).
Immunofluorescence revealed that in tomb mutant spermatocytes, Aly
and Comr proteins were localised to the nucleus, but were excluded from
chromatin (Fig. 7M-R).
Therefore, tomb function is not required for nuclear import of Aly
and Comr, but is required to load these proteins onto chromatin.
Tomb protein requires Aly and Comr for stability
When expressed in achi/vis
(Fig. 8A,C), or nht (a
can-class meiotic-arrest gene, data not shown) mutant testes,
EGFP-tagged Tomb protein also localised to primary spermatocyte nuclei. The
protein was concentrated on chromatin, but was also found throughout the
nucleoplasm. Therefore, the functions of achi/vis and the
can-class genes are not required to establish or maintain the correct
subcellular localisation of Tomb, although they might be required to enhance
the association of Tomb with chromatin.
By contrast, EGFP-Tomb protein localisation was altered when expressed in comr (Fig. 8B,D) or aly (data not shown) mutant testes. The fusion protein was able to localise to nuclei and chromatin of early primary spermatocytes. However, as spermatocytes matured, the nuclear staining was lost, so that in late primary spermatocytes only very weak, cytoplasmic EGFP fluorescence could be detected. We conclude that aly and comr functions are not required for the localisation of Tomb to the nucleus or chromatin per se, but are required to maintain the nuclear concentration of Tomb by preventing either nuclear export or Tomb degradation.
|
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Parisi et al., 2004). Most
meiotic-arrest genes described to date have been identified through classical
genetics. To find additional gene products that act with those already
isolated, we undertook a reverse genetics approach; we identified
tomb while screening for proteins that could bind Aly in a yeast
two-hybrid system.
Pathway of assembly and localisation of an aly-class meiotic-arrest complex
The tomb predicted protein contains a tesmin/TSO1-family CXC
domain that probably mediates DNA binding. Other tesmin/TSO1-family members
have either two full CXC domains, or one truncated domain and one full domain,
separated by a conserved spacer. Tomb is exceptional in having a single CXC
domain and no spacer sequence. In addition to the CXC domain, we identified a
second region of homology shared between tomb and the other animal
tesmin/TSO1 CXC-domain-containing proteins. This C-terminal domain has
conserved secondary structure, and might be responsible for the Tomb-Aly
interaction.
Direct interactions have been demonstrated between Comr and Topi, whereas
Aly, Comr and Achi/Vis have been found in a complex in vivo
(Perezgazga et al., 2004;
Wang and Mann, 2003). Here, we
additionally show that Aly and Comr can interact with Tomb. In support of our
interaction data, Beall et al. (Beall et
al., 2007) have purified a complex of proteins containing Aly,
Topi, Comr, Tomb and other factors from Drosophila testes extracts
and these components were not detected in ovary-specific extracts. The known
aly-class meiotic-arrest gene products localise primarily on
chromatin in wild-type primary spermatocytes, although Aly and Tomb are also
detected at significant levels in early primary spermatocyte cytoplasm. Only
when all five aly-class gene products are present is full
chromatin-binding activity achieved. There are subtle differences in
aly and comr phenotypes as compared with achi/vis
and topi. Most notably, achi/vis and topi have
broader ranges of target genes than aly and comr
(Perezgazga et al., 2004). We
have previously shown that the nuclear localisations of Aly and Comr are
mutually dependent, i.e. Aly remains cytoplasmic in comr mutants and
vice versa (Jiang and White-Cooper,
2003). We have also shown that topi and achi/vis
act later in the localisation pathway, both gene products being required for
the efficient loading of Aly and Comr onto chromatin
(Ayyar et al., 2003;
Perezgazga et al., 2004). We
can now place tomb into the pathway of complex assembly and activity
(Fig. 9). We propose that Tomb,
Achi/Vis and Topi enter the nucleus independently, whereas Aly and Comr can
only become (or remain) nuclear as a complex. Topi and Achi/Vis probably have
inherent sequence-specific DNA-binding activity, which allows them to localise
independently, albeit inefficiently, to their targets. Like Mip120, Tomb might
also have DNA-binding activity. When in the nucleus, Aly and Comr interact
with Tomb; this complex then promotes Topi and Achi/Vis interactions with
target promoters. Tomb protein is destabilised in the absence of Aly and Comr;
hence, the phenotypes of tomb, aly and comr mutants are
identical with respect to target gene expression levels. DRM, a complex
containing the proteins encoded by the C. elegans aly and
tomb homologues (lin-9 and lin-54), has recently
been described (Harrison et al.,
2006). Formation of the DRM complex was sensitive to loss of
lin-9 or lin-54, just as aly and
tomb are crucial for formation of the aly-class gene product
complex in testis. Mammalian tesmin is cytoplasmic in early pachytene cells,
and normally translocates to the nucleus during late pachytene and diplotene
stages of male meiosis, in a similar manner to fly aly and
tomb (Matsuura et al.,
2002; Sutou et al.,
2003).
|
). mod-null mutants are lethal, but a viable weak
allele is male sterile. Mod was shown to bind sequence elements in certain
testis-specific promoters. Many, but not all, mod target genes are
also meiotic-arrest gene targets. An alternative form of Mod, expressed only
in testis, has an acidic N-terminal domain that probably allows Mod to act as
a transcriptional activator. The can-class meiotic-arrest genes,
which encode testis-specific homologues of the basal transcription factor
complex TFIID (testis TAFs), might activate transcription by
sequestering the polycomb repressor complex away from active chromatin, i.e.
they might activate genes by repressing a repressor
(Chen et al., 2005;
Hiller et al., 2004;
Hiller et al., 2001). In
normal primary spermatocytes, Pc and testis TAFs are primarily nucleolar,
although the proteins are also detected uniformly on chromatin. ChIP analysis
revealed that Sa protein binds promoters of target genes in primary
spermatocytes, suggesting a direct transcriptional activator role for testis
TAFs (Chen et al., 2005). The aly-class meiotic-arrest mutant phenotype is most easily explained in terms of transcriptional activation rather than through the repression of a repressor. The aly-class gene products accumulate on chromatin in primary spermatocytes in transcriptionally active regions, and not in the nucleolus. Their function depends on the chromatin localisation. In addition, lack of testis TAF gene activity results in low (but readily detectable) levels of target gene expression, whereas expression of many target genes in aly-class mutant testes is undetectable.
A testis-specific dREAM/Myb-MuvB complex?
tomb and mip120 (CG6061) are the only
Drosophila tesmin/TSO1 CXC-motif proteins. Likewise, aly and
mip130 (twit, CG3480, EG86E4.4) are the only
Drosophila homologues of lin-9
(White-Cooper et al., 1998).
Mip120 and Mip130 have been described as components of the dREAM/Myb-MuvB
complex found in embryos and tissue culture cells
(Beall et al., 2002;
Korenjak et al., 2004;
Lewis et al., 2004). The dREAM
complex contains, in addition to Mip120 and Mip130, Myb, Caf1p55, Dp, Mip40,
E2F2 and Rbf or Rbf2 (Korenjak et al.,
2004). The MybMuvB complex was purified independently and contains
all the subunits of the dREAM complex as well as several additional proteins
including Rpd3, Lin-52 and l(3)MBT (Lewis
et al., 2004). dREAM/Myb-MuvB regulates DNA replication at chorion
gene amplification origins in ovarian follicle cells
(Beall et al., 2004;
Beall et al., 2002;
Cayirlioglu et al., 2001;
Frolov et al., 2001). In
addition to this role in controlling developmentally regulated DNA
replication, the dREAM/Myb-MuvB complex acts as a transcriptional repressor,
primarily of genes involved in differentiation
(Korenjak et al., 2004;
Lewis et al., 2004). This
transcriptional repressor role is also developmentally regulated as there are
different transcriptional targets for Rbf2 and E2F2 in ovaries, early embryos
and S2 tissue culture cells (Stevaux et
al., 2005).
DRM, a complex containing the C. elegans homologues of the dREAM
subunits has recently been described
(Harrison et al., 2006). The
genes encoding DRM components act together in the SynMuvB genetic pathway that
regulates vulval development redundantly with the SynMuvA and SynMuvC pathways
[see the following studies (Ceol and
Horvitz, 2004; Ceol et al.,
2006; Poulin et al.,
2005) and references therein; see Lipsick
(Lipsick, 2004) for
commentary]. All the dREAM/Myb-MuvB genes are also conserved in mammals, and
recently LIN9, the human homologue of aly/Mip130, has been shown to
have tumour suppressor activity and to work in concert with Rb to promote
differentiation (Gagrica et al.,
2004). LIN9, LIN54 (human Mip120) and hMip40 are all also capable
of binding directly to Rb (Korenjak et
al., 2004).
Drosophila E2f2- and Rbf2-null mutants are viable and
male fertile, but E2F2 females have reduced fertility
(Cayirlioglu et al., 2001;
Frolov et al., 2001;
Stevaux et al., 2005), whereas
Myb-, Dp- and Rbf-null mutants are lethal
(Duronio et al., 1995;
Manak et al., 2002;
Royzman et al., 1997) and
males mutant for weak Dp alleles are sterile
(Duronio et al., 1998) but do
not show a meiotic-arrest phenotype. Thus, the mutant phenotypes of the
DNA-binding subunits dE2F2, Rbf, Rbf2, Dp and Myb are not
consistent with them functioning in testes with aly and tomb
to activate gene expression. Indeed, Rbf2 function in ovaries is
implicated in repression of some testis-specific genes
(Stevaux et al., 2005).
There is remarkable evolutionary conservation of the interaction between dREAM/Myb-MuvB gene products in somatic tissues in mammals, flies and worms. We suggest that gene duplications in Drosophila of lin-54 (tomb/mip120), lin-9 (aly/mip130) and lin-52 (CG12442/lin52) (J.J., K.D. and H.W.-C., unpublished), has led to the evolution of a complex paralogous to the dREAM/MybMuvB complex, but using different DNA-binding subunits, dedicated to testis-specific transcriptional regulation.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Andrews, J., Bouffard, G. G., Cheadle, C., Lu, J. N., Becker, K.
G. and Oliver, B. (2000). Gene discovery using computational
and microarray analysis of transcription in the Drosophila melanogaster
testis. Genome Res. 10,2030
-2043.
Ayyar, S., Jiang, J., Collu, A., White-Cooper, H. and White,
R. (2003). Drosophila TGIF is essential for
developmentally regulated transcription in spermatogenesis.
Development 130,2841
-2852.
Beall, E. L., Manak, J. R., Zhou, S., Bell, M. and Lipsick, J.
S. (2002). Role for a Drosophila Myb-containing
protein complex in site-specific DNA replication.
Nature 420,833
-837.[CrossRef][Medline]
Beall, E. L., Bell, M., Georlette, D. and Botchan, M. R.
(2004). Dm-myb mutant lethality in Drosophila
is dependent on mip130: positive and negative regulation of DNA
replication. Genes Dev.
18,1667
-1680.
Beall, E. L., Lewis, P. W., Bell, M., Rocha, M., Jones, D. L.
and Botchen, M. R. (2007). Discovery of tMAC: a
Drosophila testes-specific meiotic arrest complex paralogous to
Myb-MuvB. Genes Dev. (in press).
Beitel, G. J., Lambie, E. J. and Horvitz, H. R.
(2000). The C. elegans gene lin-9, which acts
in an Rb-related pathway, is required for gonadal sheath cell development and
encodes a novel protein. Gene
254,253
-263.[CrossRef][Medline]
Cayirlioglu, P., Bonnette, P. C., Dickson, M. and Duronio, R.
J. (2001). Drosophila E2f2 promotes the conversion from
genomic DNA replication to gene amplification in ovarian follicle cells.
Development 128,5085
-5098.
Ceol, C. J. and Horvitz, H. R. (2004). A new
class of C. elegans synMuv genes implicates a Tip60/NuA4-like HAT
complex as a negative regulator of Ras signaling. Dev.
Cell 6,563
-576.[CrossRef][Medline]
Ceol, C. J., Stegmeier, F., Harrison, M. and Horvitz, H. R.
(2006). Identification and classification of genes that act
antagonistically to let-60 Ras signaling in Caenorhabditis elegans vulval
development. Genetics
173,709
-726.
Chen, D. and McKearin, D. M. (2003). A discrete
transcriptional silencer in the bam gene determines asymmetric division of the
Drosophila germline stem cell. Development
130,1159
-1170.
Chen, X., Hiller, M. A., Sancak, Y. and Fuller, M. T.
(2005). Tissue specific TAFs counteract Polycomb to turn on
terminal differentiation. Science
310,869
-872.
Duronio, R. J., O'Farrell, P. H., Xie, J.-E., Brook, A. and
Dyson, N. (1995). The transcription factor E2F is required
for S-phase during Drosophila embryogenesis. Genes
Dev. 9,1456
-1468.
Duronio, R. J., Bonnette, P. C. and O'Farrell, P. H.
(1998). Mutations of the Drosophila dDP, dE2F, and cyclin E genes
reveal distinct roles for the E2F-Dp transcription factor and cyclin E during
the G1-S transition. Mol. Cell. Biol.
118,141
-151.
FlyBase Consortium (1999). The FlyBase database
of the Drosophila genome projects and community literature. Nucleic
Acids Res. 27,85
-88.
Frolov, M. H., Huen, D. S., Stevaux, O., Dimova, D.,
Balczarek-Strang, K., Elsdon, M. and Dyson, N. (2001).
Functional antagonism between E2F family members. Genes
Dev. 15,2146
-2160.
Fuller, M. T. (1993). Spermatogenesis. In
The Development of Drosophila. Vol.1
(ed. M. Bate and A. Martinez-Arias), pp.71
-147. Cold Spring Harbor: Cold Spring Harbor
Laboratory Press.
Gagrica, S., Hauser, S., Kolfschoten, I., Osterloh, L., Agami,
R. and Gaubatz, S. (2004). Inhibition of oncogenic
transformation by mammalian Lin-9, a pRB-associated protein. EMBO
J. 23,4627
-4638.[CrossRef][Medline]
Harrison, M., Coel, C. J., Lu, X. and Horvitz, H. R.
(2006). Some C. elegans class B synthetic multivulva proteins
encode a conserved LIN-35 Rb-containing complex distinct from a NuRD-like
complex. Proc. Natl. Acad. Sci. USA
103,16782
-16787.
Hiller, M. A., Lin, T.-Y., Wood, C. and Fuller, M. T.
(2001). Developmental regulation of transcription by a
tissue-specific TAF homolog. Genes Dev.
15,1021
-1030.
Hiller, M. A., Chen, X., Pringle, M. J., Suchorolski, M.,
Sancak, Y., Viswanathan, S., Bolival, B., Lin, T.-Y., Marino, S. and Fuller,
M. T. (2004). Testis-specific TAF homologs collaborate to
control a tissue-specific transcription program.
Development 131,5297
-5308.
Jayaramaiah Raja, S. and Renkawitz-Pohl, R.
(2005). Replacement by Drosophila melanogaster protamines and
Mst77F of histones during chromatin condensation in late spermatids and role
of sesame in the removal of these proteins from the male pronucleus.
Mol. Cell. Biol. 25,6165
-6177.
Jiang, J. and White-Cooper, H. (2003).
Transcriptional activation in Drosophila spermatogenesis involves the mutually
dependent function of aly and a novel meiotic arrest gene cookie monster.
Development 130,563
-573.
Kemphues, K. J., Raff, R. A., Kaufman, T. C. and Raff, E. C.
(1979). Mutation in a structural gene for a ß-tubulin
specific to testis in Drosophila melanogaster. Proc. Natl. Acad.
Sci. USA 76,3991
-3995.
Korenjak, M., Taylor-Harding, B., Binne, U. K., Satterlee, J.
S., Stevaux, O., Aasland, R., White-Cooper, H., Dyson, N. and Brehm, A.
(2004). Native E2F/RBF complexes contain Myb-interacting proteins
and repress transcription of developmentally controlled E2F target genes.
Cell 119,181
-193.[CrossRef][Medline]
Lewis, P. W., Beall, E. L., Fleischer, T. C., Georlette, D.,
Link, A. J. and Botchan, M. (2004). Identification of a
Drosophila Myb-E2F2/RBF transcriptional repressor complex.
Genes Dev. 18,2929
-2940.
Lin, T.-Y., Viswanathan, S., Wood, C., Wilson, P. G., Wolf, N.
and Fuller, M. T. (1996). Coordinate developmental control of
the meiotic cell cycle and spermatid differentiation in Drosophila
males. Development 122,1331
-1341.[Abstract]
Lipsick, J. S. (2004). synMuv verite - Myb
comes into focus. Genes Dev.
18,2837
-2844.
Ma, J., Katz, E. and Belote, J. M. (2002).
Expression of proteasome subunit isoforms during spermatogenesis in Drosophila
melanogaster. Insect Mol. Biol.
11,627
-639.[CrossRef][Medline]
Manak, J. R., Mitiku, N. and Lipsick, J. S.
(2002). Mutation of the Drosophila homologue of the Myb
protooncogene causes genomic instability. Proc. Natl. Acad. Sci.
USA 99,7438
-7443.
Matsuura, T., Kawasaki, Y., Miwa, K., Sutou, S., Ohinata, Y.,
Yoshida, F. and Mitsui, Y. (2002). Germ cell-specific
nucleocytoplasmic shuttling protein, tesmin, responsive to heavy metal stress
in mouse testes. J. Inorg. Biochem.
88,183
-191.[CrossRef][Medline]
McGuffin, L. J., Bryson, K. and Jones, D. T.
(2000). The PSIPRED protein structure prediction server.
Bioinformatics 16,404
-405.
Mikhaylova, L., Boutaneav, A. and Nurminsky, D. I.
(2006). Transcriptional regulation by Modulo integrates meiosis
and spermatid differentiation in male germ line. Proc. Natl. Acad.
Sci. USA 103,11975
-11980.
Olivieri, G. and Olivieri, A. (1965).
Autoradiographic study of nucleic acid synthesis during spermatogenesis in
Drosophila melanogaster. Mutat. Res.
2, 366-380.[Medline]
Parisi, M., Nuttall, R., Edwards, P., Minor, J., Naiman, D., Lu,
J., Doctolero, M., Vainer, M., Chan, C., Malley, J. et al.
(2004). A survey of ovary-, testis-, and soma-biased gene
expression in Drosophila melanogaster adults. Genome
Biol. 5,R40
.[CrossRef][Medline]
Parker, L., Gross, S. and Alphey, L. (2001).
Vectors for the expression of tagged proteins in Drosophila.
Biotechniques 31,1280
-1286.[Medline]
Perezgazga, L., Jiang, J., Bolival, B., Hiller, M. A., Benson,
E., Fuller, M. T. and White-Cooper, H. (2004). Regulation of
transcription of meiotic cell cycle and terminal differentiation genes by the
testis-specific Zn finger protein matotopetli.Development 131,1691
-1702.
Poulin, G., Dong, Y., Fraser, A., Hopper, N. and Ahringer,
J. (2005). Chromatin regulation and sumoylation in the
inhibition of Ras-induced vulval development in Caenorhabditis elegans.
EMBO J. 24,2613
-2623.[CrossRef][Medline]
Royzman, I., Whittaker, A. J. and Orr-Weaver, T. L.
(1997). Mutations in Drosophila DP and E2F distinguish
G1-S progression from an associated transcriptional program. Genes
Dev. 11,1999
-2011.
Stevaux, O., Dimova, D., Ji, J.-Y., Moon, N. S., Frolov, M. and
Dyson, N. (2005). Retinoblastoma family 2 is required in vivo
for the tissue-specific repression of dE2F2 target genes. Cell
Cycle 4,1272
-1280.[Medline]
Sutou, S., Miwa, K., Matsuura, T., Kawasaki, Y., Ohinata, Y. and
Mitsui, Y. (2003). Native tesmin is a 60-kilodalton protein
that undergoes dynamic changes in its localisation during spermatogenesis in
mice. Biol. Reprod. 68,1861
-1869.
Toba, G., Ohsako, T., Miyata, N., Ohtsuka, T., Seong, K. H. and
Aigaki, T. (1999). The gene search system: a method for
efficient detection and rapid molecular identification of genes in Drosophila
melanogaster. Genetics
151,725
-737.
Wang, Z. and Mann, R. S. (2003). Requirement
for two nearly identical TGIF-related homeobox genes in Drosophila
spermatogensis. Development
130,2853
-2865.
White-Cooper, H., Schafer, M. A., Alphey, L. S. and Fuller, M.
T. (1998). Transcriptional and post-transcriptional control
mechanisms coordinate the onset of spermatid differentiation with meiosis I in
Drosophila. Development
125,125
-134.[Abstract]
White-Cooper, H., Leroy, D., MacQueen, A. and Fuller, M. T.
(2000). Transcription of meiotic cell cycle and terminal
differentiation genes depends on a conserved chromatin-associated protein,
whose nuclear localisation is regulated. Development
127,5463
-5473.[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  E.L. Davies and M.T. Fuller Regulation of Self-renewal and Differentiation in Adult Stem Cell Lineages: Lessons from the Drosophila Male Germ Line Cold Spring Harb Symp Quant Biol, March 27, 2009; (2009) sqb.2008.73.063v1. [Abstract] [PDF]
|
|
|
|
 |
|
 P. G. Engstrom, S. J. Ho Sui, O. Drivenes, T. S. Becker, and B. Lenhard Genomic regulatory blocks underlie extensive microsynteny conservation in insects Genome Res., December 1, 2007; 17(12): 1898 - 1908. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
residues conserved within CXC(1) or CXC(2), but which differ between the
domains. The E(z) Cys-rich region is shown as an outgroup. (D)
Alignment and predicted secondary structure (beneath) of the animal
tesmin-family protein C-termini. Predictions of secondary structure are shown
in the same order as the sequences (i.e. the first line is the Tomb secondary
structure prediction). Black lines indicate high confidence helix predictions;
the intervening region (grey line) has either no strong structural prediction
(Tomb), or a strong coil prediction (Mip120, Hs-tes, Hs-tesl).
