Department of Zoology, Oxford University, South Parks Rd, Oxford, OX1 3PS, UK
* Author for correspondence (e-mail: helen.white-cooper{at}zoo.ox.ac.uk)
Accepted 31 October 2002
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SUMMARY |
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Key words: Drosophila, Meiosis, Transcription, Spermatogenesis, Nuclear localisation
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INTRODUCTION |
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In Drosophila spermatogenesis, transcription is essentially shut
off upon entry into the meiotic divisions, therefore all transcripts required
at later stages must be made in primary spermatocytes
(Olivieri and Olivieri, 1965).
Four mitotic divisions of a spermatogonial cell produce a cyst of 16 primary
spermatocytes, which immediately undergo premeiotic S phase. They then enter
an extended G2 period, characterised by high transcriptional activity and cell
growth. The meiotic divisions result in a cyst of 64 round spermatids, which
use stored mRNAs to produce proteins needed for their dramatic morphological
changes during elongation, before finally individualising to form motile sperm
(reviewed by Fuller,
1993
).
Although entry into spermatid differentiation is independent of progression
through the meiotic divisions, these processes are subject to coordinate
control, mediated by the `meiotic arrest' class of genes, including aly,
can, mia and sa (Lin et al.,
1996), and several other unpublished mutations (M. Fuller,
personal communication). The meiotic arrest genes are essential for the
transcription of many mRNAs involved in spermatid differentiation, and thus
are required for spermatid differentiation. The meiotic arrest genes also
control accumulation of proteins involved in the meiotic divisions, e.g. the
cdc25 homologue Twine, and thus link differentiation to the cell
cycle (White-Cooper et al.,
1998
). The meiotic arrest genes of Drosophila have been
split into two classes, based on the mechanism by which they control
accumulation of Twine. The can class (including can, mia and
sa) post-transcriptionally regulate Twine production. By contrast
aly regulates transcription of twine. Two other meiotic
regulators, cyclin B and boule, are also transcriptional
targets of aly, but not can, mia or sa.
Three meiotic arrest genes have been cloned to date, aly, can and
nht (Hiller et al.,
2001; White-Cooper et al.,
2000
). can and nht encode testis-specific
homologues of TAFII80 and TAFII110, respectively. These
are subunits of the basal transcription factor TFIID, whose role is
to facilitate RNA polymerase II binding to the proximal promoter region. The
aly (always early) gene discussed in this paper should not
be confused with the regulator of RNA export Aly
(Zhou et al., 2000
).
aly encodes a homologue of a C. elegans negative regulator
of vulval induction, the SynMuvB gene lin-9. The SynMuv B pathway
includes several genes whose products form a complex (NURD) that regulates
chromatin structure (Lu and Horvitz,
1998
; Solari and Ahringer,
2000
).
The mechanism by which lin-9 regulates the NURD complex is not
understood. LIN-9 has not been shown to be a component of the NURD complex
itself, so may therefore act upstream. The subcellular localisation of LIN-9
in C. elegans has not been determined. We previously showed that Aly
protein localised to chromatin in maturing Drosophila primary
spermatocytes. This localisation suggests that the lin-9 homologue,
Aly, may act in close concert with a NURD complex on chromatin
(White-Cooper et al., 2000).
The nuclear localisation of Aly protein is both regulated and essential for
the normal function of the protein, as the protein produced by several mutant
alleles remains cytoplasmic, despite the presence of a nuclear localisation
signal.
We report the identification and cloning of cookie monster (comr), a novel aly-class Drosophila meiotic arrest gene. Like aly, comr transcription is testis specific in males, but low levels of transcript were detected at earlier stages of development. We show that Comr protein is localised to the nucleus of primary spermatocytes, and concentrated on decondensed regions of chromatin. The Comr pattern is similar but not identical to that of Aly. We show that the nuclear localisation of these two proteins is mutually dependent. Finally, we show that active RNA polymerase II is limited to discrete regions of the nuclei of primary spermatocytes. These regions of high transcriptional activity are a subset of the Comr localisation domain, but the level of Comr protein does not predict the level of active RNA polymerase II.
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MATERIALS AND METHODS |
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Isolation of a comr mutant allele
A large scale mutagenesis screen to isolate new viable mutants induced by
EMS on second chromosomes marked with cn and bw, was
conducted by Charles Zuker and colleagues (E. Koundakjian, R. Hardy, D. Cowen
and C. Zuker, personal communication). All the lines generated were tested for
male sterility by Barbara Wakimoto and Dan Lindsley, who then conducted a
preliminary morphological examination by phase-contrast microscopy to
determine the defect in each male sterile line. These lines were re-screened
in Margaret Fuller's laboratory, and those with a meiotic arrest phenotype
were tested inter se for allelism. comr corresponds to line
Z2-1340, and was represented by a single mutant allele. Mutant
phenotypes were scored by squashing cut testes in testis buffer (183 mM KCl,
47 mM NaCl, 10 mM Tris pH 6.8) and observing with phase contrast optics.
Hoechst 33342 (4 µg/ml) was included in the dissection buffer to visualise
chromosomes in live spermatocytes.
PCR from flies
Df(2R)Egfr3/Df(2R)X58-7 and Df(2R)XE2900/Df(2R)X58-7
males were selected from crosses based on the lack of the balancer chromosome.
Testes of some mutant males were dissected to confirm the mutant phenotypes,
the remaining mutant flies were used for genomic DNA extraction. Genomic DNA
was simultaneously extracted from wild-type male flies, and from flies
homozygous for a different mutation generated in the same mutagenesis screen.
These DNAs were used as PCR templates to amplify the following candidate genes
predicted by the genome sequencing and annotation projects: CG9284, CG4386,
CG13492, CG4363, CG4377, CG4372, CG9294, CG13493 and PpN58A
(Adams et al., 2000).
Sequencing of candidate genes
PCR primers were designed to amplify all of the predicted ORF from
candidate genes, CG4363, CG4377, CG4372, CG9294 and CG13493 as sets of
overlapping products. The amplified fragments were sequenced from both ends
using BigDye terminator cycle sequencing reagent (ABI), reactions were run on
a ABI 377 automated DNA sequencing system. Sequence alignments were carried
out using Sequencher 3.1 (GeneCodes Corp).
RT-PCR of comr
Total RNA was prepared using TRIzol reagent (Roche) from testes of
wild-type male flies, the whole bodies of wild-type males and females
respectively, and from wild-type male carcasses (testes were removed by
dissection). The total RNA was treated by DNAse I to remove possible
contamination of genomic DNA. RT-PCR was carried out using Superscript
Preamplificaion System (Invitrogen). A pair of oligo primers
(5'-GATTACCAGGGTATGCAGGA and 5'-GGCTTTGCTTTAAACCTGGT) was used to
amplify a 979 bp cDNA fragment from the open reading frame of comr.
Lack of DNA contamination was confirmed by RT-PCR of the ubiquitously
expressed gene PP1-87B using primers that spanned an intron.
In situ hybridisation
The 979 bp comr RT-PCR product was subcloned into pBluescript
(Stratagene). pBluescript clones containing cyclin B, cyclin A, Mst87F,
boule and polo were prepared as previously described
(White-Cooper et al., 1998).
Antisense dig-labelled RNA probes were made using the Roche Dig-RNA labelling
mix, according to the manufacturer's instructions. Hydrolysis was for 15
minutes per 500 bp. Hybridisation was carried out as previously described
(White-Cooper et al.,
1998
).
Antibody production
Anti-peptide antibodies were raised by Moravian-Biotechnology. The
synthesised oligo-peptide, KSRRYDLRNSKRNPR (amino acids 586-600) at the C
terminus of the predicted Comr protein, was coupled to BSA and used to
immunise a rabbit. The serum was preadsorbed against fixed Drosophila
ovaries to deplete non-specific or low affinity antibodies. Rabbit anti-Comr
was used at a dilution of 1:5000 for western blotting.
Western blotting
Testes were dissected from young males in testis buffer and transferred to
an Eppendorf tube and snap frozen in liquid nitrogen. They were thawed,
5xSDS sample buffer added and the samples were boiled for 5 minutes then
spun in a microfuge for 5 minutes. Typically 20 testes were loaded on each
lane of a 10% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose
membrane, which was then blocked in 5% fat-free powdered milk in PBS+0.1%
Tween-20. Primary and HRP-conjugated secondary antibodies were diluted in
blocking solution and incubated with the blot overnight at 4°C and room
temperature for 1 hour, respectively. Signals were detected using Pierce
SuperSignal chemi-luminescence reagent and Kodak MBX blue sensitive X-ray
film.
Immunohistochemistry and Immunofluorescence
For immunohistochemistry, testes were dissected from 0 to 1-day-old males
in testis buffer and fixed in 4% paraformaldehyde in HEPES buffer (0.1 M HEPES
pH 6.9, 2 mM MgSO4, 1 mM EGTA) for 30 minutes. The testes were
rinsed once then transferred to cell culture inserts (Falcon) in a 24-well
tissue culture plate, washed three times for 20 minutes each in PBS+0.1%
Triton-X100 (PBSTx), blocked for 30 minutes in PBSTx+5%FCS, then incubated
overnight at 4°C or 4 hours at room temperature in primary antibody
diluted in this blocking buffer. After four 15 minute washes in PBSTx the
testes were incubated 2 hours at room temperature or overnight at 4°C with
Universal biotinylated secondary antibody (Vector), preadsorbed against
Drosophila embryos and diluted 1:1000 in blocking buffer. After four
15 minute washes in PBSTx, the testes were incubated for 1 hour at room
temperature with ExtrAvidin-HRP (Sigma), diluted 1:1000 in PBSTx. Testes were
washed three times for 15 minutes each in PBSTx, rinsed once in PBS then
stained with diamino-benzidine (0.5 mg/ml) + 0.001%H2O2
in PBS. The colour reaction was monitored periodically and stopped by washing
with PBS. Testes were mounted in 85% glycerol and observed with an Olympus
BX50 microscope equipped with DIC optics. Rabbit anti-Aly was diluted 1:4000
and rabbit anti-Comr was preadsorbed against ovaries and diluted 1:1000.
For immunofluorescence, testes were dissected from 0-1 day old males in testis buffer, about five males were used per slide. The testes were transferred to a 25 µl drop of testis buffer on a poly-l-lysine-treated slide and cut open with tungsten needles. Paraformaldehyde (25 µl of 4% solution) in HEPES buffer was added and the testes were left to fix for 12 minutes at room temperature. Testes were squashed by adding a coverslip, the slide was dunked in liquid nitrogen and the coverslip was removed with a scalpel. Testes were then stored in PBS+0.1% Tween-20 (PBSTw) until all the samples had been prepared. Samples were blocked for 30 minutes with PBSTw+ 5%FCS, then incubated with primary antibody diluted in blocking solution overnight at 4°C. Testes were rinsed once then washed four times for 10 minutes each in PBSTw. Testes were incubated with secondary antibodies diluted in blocking solution for 2 hours at room temperature. When DNA was to be stained with propidium iodide, RNAse A (0.5 mg/ml) was included with the secondary antibody incubation. Samples were washed as before and mounted in 85% glycerol + 2.5% n-propyl gallate. For DNA staining propidium iodide (1µg/ml) was included in the mounting medium. Coverslips were sealed with nail varnish and the samples were imaged using a BioRad Radiance Plus confocal microscope.
The monoclonal antibody against RNA polymerase II phospho-C-terminal domain (P-CTD) (Clone H5) was obtained from Covance/BAbCO and used at a dilution of 1:100. Pre-adsorbed anti-Comr antibody was used at 1:1000. Secondary antibodies coupled to Cy3 or FITC were used at 1:1000 (Jackson).
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RESULTS |
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All of the four previously described meiotic arrest loci show defects in
transcription in primary spermatocytes of many genes required for spermatid
differentiation, including Mst87F and fzo. The meiotic
arrest genes were subdivided into aly class and can class
because certain cell cycle genes, namely cyclin B, twine and
boule were transcribed in can-class mutants (can,
mia and sa) but not in aly
(White-Cooper et al., 1998). A
set of diagnostic RNA in situ hybridisation experiments revealed that
comr resembled aly rather than the can-class mutant
mia (Fig. 2).
comr mutant testes transcribed polo, but failed to
transcribe both boule and Mst87F.
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The phenotype of comr also resembled aly rather than the can-class genes in terms of the chromosome morphology in mutant primary spermatocytes, after staining with vital Hoechst (33342). can-class mutants had apparently normal chromosome morphology whereas aly had defects in chromatin structure. The major chromosome bivalents of wild-type primary spermatocytes formed three discrete, clearly delineated domains within each nucleus (Fig. 1C). By contrast the chromosome bivalents of comrZ1340 mutant primary spermatocytes were fuzzy, with indistinct boundaries (Fig. 1D). comr mutant spermatocytes therefore resemble aly rather than can based on their failure to express mRNA for certain cell cycle control genes and their aberrant chromosome morphology. Thus, comr is the second member of the aly-class meiotic arrest genes of Drosophila.
Cloning of comr
Meiotic recombination mapping localised comr genetically to
96.0±5.7, between c and px on the right arm of
chromosome 2. Complementation tests with deficiency chromosomes placed the
comr locus in the physical region 57F-58A
(Fig. 3). The left-hand end of
the region is defined by the proximal break point of deficiency
Df(2R)X58-7, which is reported as 58A1-2
(Kerrebrock et al., 1995). The
right-hand end is defined by the distal breakpoint of Df(2R)Egfr3,
reported as 57F5-11 (Price et al.,
1989
). Both these deletions uncovered comr, suggesting
that they overlap. Although Df(2R)Egfr3/Df(2R)X58-7 flies were viable
and female fertile they were male sterile, indicating that they do indeed
overlap. The testes of transheterozygous males had a phenotype
indistinguishable in squash preparations from
comrZ1340/comrZ1340 or
comrZ1340/Df. The boundaries of the overlapping deleted
region were identified by PCR of predicted genes from transheterozygous
Df(2R)Egfr3/Df(2R)X58-7 males. PCR products were generated for
CG4386, CG9284 and PpN58A from this template DNA, indicating that these
genomic regions were not disrupted in at least one of the two deletion lines.
By contrast, no PCR products were generated for the predicted genes CG13492,
CG4363, CG4377, CG4372, CG9294 and CG13493, indicating that these loci are
disrupted by both deletions. Clearly none of these six loci is required for
viability or female fertility, but at least one is required for male
fertility. A third deletion, Df(2R)XE-2900, which deletes from
57F2-58A1 also uncovered comrZ1340. Additionally
Df(2R)X58-7/Df (2R)XE-2900 flies were viable, but the males had a
phenotype indistinguishable in squash preparations from
comrZ1340/comrZ1340. PCR from these
transheterozygotes revealed that CG13493 was deleted in
Df(2R)XE-2900, but none of the other candidate genes was deleted,
making CG13493 the strongest candidate for the comr gene. The open
reading frame of CG13493 was sequenced from wild-type,
comrZ1340 and another mutant derived from the Zuker screen
to represent the background chromosome. comrZ1340
contained a single base change (C-T) in CG13493, which was not present in the
background chromosome. This mutated codon 12 (CAG) to a stop codon (TAG),
presumably resulting in a null allele. No coding region mutations were found
in any of the other candidate genes sequenced. Thus CG13493 is
comr.
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The comr ORF, based on genome project predictions and RT-PCR (see
below) encodes a novel 600 amino acid protein, with a predicted molecular
weight of 68.4 kDa, and a predicted pI of 5.1
(Fig. 4A). The Comr protein
showed no significant homology to any protein in the protein or translated EST
databases. The predicted protein contained an acidic domain in the C terminus
of the protein (amino acids 518-570), and a predicted nuclear localisation
sequence (NLS) (amino acids 583-589). In addition, a region that may represent
a very divergent PB1 domain (amino acids 348-431) was identified
(Fig. 4B). PB1 domains have
been found in several signal transduction proteins, including kinase C iota
(KPCI), and have been shown to mediate protein-protein interactions
(Ito et al., 2001;
Ponting et al., 2002
).
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Expression of comr
comr was represented only once in the Drosophila EST
database (of 246620 EST sequences), this cDNA (AT27686) is derived from a
testis library and contains a 65 bp 5'UTR. The developmental expression
profile of comr was determined by RT-PCR. The transcript was testis
specific in adult males, being undetectable in gonadectomised males
(Fig. 5A), consistent with the
fully viable but male sterile phenotype of the mutant. Low levels of the
transcript were also detected in whole females
(Fig. 5A), embryos and larvae
(data not shown). RNA in situ hybridisation revealed a uniform transcript
distribution in embryos (data not shown).
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In testes, the comr transcript was expressed at highest levels in
early primary spermatocytes, as revealed by RNA in situ hybridisation. The
transcript level decreased as the spermatocytes grew, and became undetectable
as the primary spermatocytes entered the meiotic divisions
(Fig. 5B). This expression
pattern of comr is essentially identical to the pattern we have
previously reported for aly
(White-Cooper et al., 2000).
The earliest defect we have detected in comr or aly mutant
testes is failure to initiate transcription of target genes in very early
primary spermatocytes. The strong expression of comr transcript in
early primary spermatocytes is consistent with this phenotype.
Subcellular localisation of Comr protein
An antibody raised against a peptide from the C terminus of Comr protein
recognised a 100 kDa protein in wild-type testes that was absent from
comrZ1340 mutant testes
(Fig. 6A). Although this
protein was significantly larger than the predicted size for Comr (68 kDa) the
low pI of the protein could affect its mobility in SDS-PAGE. We expressed Comr
with an N-terminal Flag tag in mammalian tissue culture cells. Western blot
analysis of these transiently transfected cells using anti-flag antibodies and
the anti Comr antibody showed that ectopically expressed tagged Comr protein
migrated as a single band with an apparent molecular weight of 100 kDa (data
not shown). The anti-Comr antibody also crossreacts with a 120 kDa protein
present in wild type, Df(2R)Egfr3/Df(2R)X58-7 and
comrZ1340 mutant testes (upper band in
Fig. 6A).
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In wild-type testes the anti-Comr antibody recognised the nuclei of young and maturing primary spermatocytes (Fig. 6B). Whole-mount immunohistochemistry revealed staining throughout the primary spermatocytes nuclei, as well as a single more concentrated spot of staining within each nucleus. No staining was detected in cells undergoing the meiotic divisions, or at any later stage. When comrZ1340 mutant testes were stained under the same conditions, the antibody still recognised the single spot within each nucleus, but the general nuclear staining was absent (Fig. 6C). Therefore Comr protein appeared to localise throughout the nucleus in primary spermatocytes. The concentrated spot in the nucleus is likely to be due to crossreactivity of the antibody to the 120 kDa protein.
To explore the relationship between Comr localisation and chromatin in primary spermatocytes, we carried out indirect immunofluorescent staining of Comr double labelled with the DNA dye propidium iodide. Staining with the anti-Comr antibody was restricted to the nuclei of primary spermatocytes. Meiotic and post-meiotic cells did not stain with the antibody. Additionally no staining of any somatic cell type (e.g. cyst cells, sheath or accessory gland) was observed, showing that the protein is germ cell specific. Within primary spermatocyte nuclei the anti-Comr antibody showed two distinct types of staining pattern. A brightly stained `stringy' region (arrow in Fig. 6E) probably corresponds to the darkly stained spot seen in the immunohistochemistry. This may be the Y-loops, a very decondensed region of the Y chromosome, which is transcribed in primary spermatocytes. Weaker, somewhat spotty, staining was found throughout the nucleus, concentrated near the condensed regions of chromatin (arrowheads in Fig. 6D,E). Again, to determine which component(s) of this pattern were attributable to the crossreacting antigen, we stained comrZ1340 mutant testes. The stringy staining in a small region of the nucleus was still detected in comr mutant spermatocytes (Fig. 6H, arrow). However the general nuclear staining was absent from these cells (Fig. 6G,H, arrowheads), confirming our findings from the immunohistochemistry. We conclude that Comr protein is expressed only in primary spermatocytes in the testis, that it is nuclear and that is associated with regions of chromatin.
The nuclear localisations of Comr and Aly are mutually dependent
The identical mutant phenotypes of comr and aly suggest
that they act in the same pathway; we were interested in examining whether
comr regulated aly or vice versa. To determine how
comr interacts with known meiotic arrest loci, we first investigated
the expression of comr and aly in various mutant
backgrounds. comr transcript was detected by in situ hybridisation
and RT-PCT at a level similar to wild type in aly mutant testes
(Fig. 5C, and data not shown).
Transcription of comr was also detected in testes of
can3 (Fig.
5D). Comr protein was detected by western blotting in testis
extracts from aly mutant males, as well as in testis extracts from
males mutant for the can-class meiotic arrest gene, mia
(Fig. 6A). Therefore,
transcription and translation of comr is upstream of the action of
any known meiotic arrest mutant. The level of Comr protein in aly
mutant testes was lower than in mia mutant testes, suggesting a
potential role for aly in ensuring the accumulation of Comr protein
to normal levels. The crossreacting 120 kDa antigen also appeared to be less
abundant in all the mutant genotypes compared with wild type. aly
transcript and protein levels were similar to wild type in
comrZ1340 mutant testes (data not shown).
The similar localisation pattern of Comr and Aly proteins, combined with their identical mutant phenotypes, suggested that the proteins may interact directly or indirectly. To dissect this relationship, we examined the subcellular localisation pattern of one protein in a background mutant for the other. As both aly and comr are expected to act upstream of the can-class genes, staining of mia mutant testes was used to control for nonspecific effects of the developmental arrest characteristic of all the meiotic arrest mutants. In wild-type testes Aly protein was localised to the nuclei of maturing primary spermatocytes (Fig. 7A). Nuclear staining of primary spermatocytes in cysts gave a spotty appearance to the apical region of the testis. By contrast, Aly protein showed a honeycomb distribution pattern in comrZ1340 mutant spermatocytes (Fig. 7B). The holes in this pattern correspond to the nuclei; therefore, Aly protein failed to translocate to the nucleus and instead remained cytoplasmic in comrZ1340 mutant spermatocytes. This indicates that the nuclear localisation of Aly protein is dependent on comr function.
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The reciprocal experiment revealed that Comr protein localisation depended on normal function of aly. We carried out immunofluorescence with anti-Comr antibody on aly5 and mia mutant testes. The anti-Comr staining of aly5 and comrZ1340 mutant primary spermatocytes was indistinguishable. A single stained spot, corresponding to the 120 kDa antigen was present in the mutant cells (Fig. 7D). mia mutant spermatocytes showed staining throughout the nucleus, in addition to the bright spot (Fig. 7G). By immunohistochemistry, the anti-Comr antibody staining of aly5 and comrZ1340 mutant spermatocytes were indistinguishable (data not shown), with only one small region of the nucleus stained. mia and can3 testes probed with the anti Comr antibody had staining throughout the nuclei of primary spermatocytes, in addition to the single strongly stained spot (data not shown). In both immunostaining techniques, the staining intensity of the spot in the nucleus was less than in wild type, consistent with the reduction in level of the 120 kDa antigen seen in the western blotting. Thus, the nuclear localisation of Comr depended on aly, but did not depend on the normal function of the can-class meiotic arrest genes can and mia.
Relationship of Comr to transcription
comr mutant spermatocytes are defective for transcription of a
number of cell cycle and spermatid differentiation genes
(Fig. 2). To investigate how
Comr protein localisation in the nucleus is associated with mRNA
transcription, we used an antibody specific to active RNA polymerase II
phospho-C-terminal domain (P-CTD) in triple labelling experiments.
Fig. 8 shows the relationship
between Comr, DNA and active transcription in primary spermatocytes. Staining
with anti P-CTD revealed that the regions of the nucleus with most active
transcription were adjacent to, but not overlapping with, regions of visible
(i.e. condensed) DNA (arrow in Fig.
8D). The active transcription was found in domains within the
nucleus, not randomly distributed. Lower levels of detectable P-CTD
colocalised with the more condensed DNA (arrowhead in
Fig. 8D). The highest levels of
active transcription partially overlapped with high levels of Comr protein
(arrows in Fig. 8F), although
some regions of strong P-CTD staining were associated with weaker Comr
staining. All of the regions where transcriptional activity was detected had
at least some Comr protein present (arrow in
Fig. 8F), although P-CTD
staining was not found in all regions containing Comr protein. Thus, Comr
protein was not exclusively localised on the pol II-transcriptionally active
chromatin, but all the chromatin where RNA polymerase II is transcriptionally
active had at least some associated Comr protein.
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DISCUSSION |
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comr and aly are mutually dependent
Comr and Aly are mutually dependent: Aly protein requires comr for
its normal nuclear localisation, and Comr protein requires aly for
its normal nuclear localisation. Thus, the phenotypes of the two mutants are
identical, because ablation of one essentially knocks out the other by
preventing its normal localisation. It seems likely that Aly and Comr proteins
physically interact or are both essential components of a multi-subunit
complex. Anchoring of either protein in the nucleus would then be dependent on
the formation of this complex, loss of either would give the aberrant protein
localisation patterns seen. To date we have been unable to detect any direct
physical interaction between Aly and Comr proteins (J. J. and H. W.-C.,
unpublished), so we would favour a hypothesis where Aly and Comr are in a
complex with at least one other protein. Aly nuclear localisation is
regulated; this regulation need not be solely dependent on Comr. For example,
cytoplasmic Aly could be phosphorylated, and dephosphorylation could allow
nuclear entry and therefore interaction with Comr. This protein complex would
then be able to attach to chromatin.
aly, comr and can transcripts are all transcribed in a
characteristic pattern in wild-type testes
(Hiller et al., 2001;
White-Cooper et al., 2000
).
High levels of transcripts are found in early primary spermatocytes, and the
levels decrease as the spermatocytes mature. None of the transcripts are
detectable as the cells enter the meiotic divisions. This pattern is entirely
consistent with the role of these genes in activation of transcription of many
new genes in early primary spermatocytes, and the continued high
transcriptional activity throughout primary spermatocytes development.
It is interesting to note that both comr and aly
transcripts appear to be expressed at low levels in tissues other than the
testis. We were able to detect some comr transcript by RT-PCR in
females and in all stages of embryos examined (0-1, 3-5 and 12-24 hours).
aly mRNA was also detected at low levels in the older embryo samples
(J. J. and H. W.-C., unpublished). However null mutants of both comr
and aly are fully viable and female fertile, showing that the
expression of these genes outside the testis is not essential. It is possible
that comr and aly have a redundant function in these other
tissues. A second Drosophila homologue of aly has been
identified (White-Cooper et al.,
2000), but there is no obvious comr homologue in the
genome. It is attractive to postulate that the meiotic arrest genes are all
under the control of the same transcription factor, so that they are all
activated early in the primary spermatocyte programme of development.
How do aly and comr regulate transcription?
The predicted comr protein is novel. While its predicted size is
68 kDa, Comr protein (from testes or expressed in mammalian tissue culture
cells) migrates at about 100 kDa in SDS-PAGE. This aberrant mobility on
SDS-PAGE gels may be due to the acidity of the protein retarding its
migration. The low predicted pI of the protein may provide some clues as to
its biochemical function. The protein is rather acidic throughout its length,
as well as having a very acidic region near the C terminus. In this regard, it
bears some similarity to the acidic histone chaperone protein nucleoplasmin,
which is important for nucleosome assembly and remodelling during
transcription (Chen et al.,
1994; Earnshaw et al.,
1980
). It is possible that the acidic domain on Comr interacts
with the basic histone proteins to alter chromatin structure.
aly encodes a homologue of the C. elegans SynMuvB gene
lin-9 (Beitel et al.,
2000; White-Cooper et al.,
2000
). The SynMuvA and B genes act in two genetically redundant
pathways to repress vulval cell fate and promote hypodermal cell fate in the
vulval precursor cells (Chen and Han,
2001
; Fay and Han,
2000
; Ferguson and Horvitz,
1989
). The SynMuvB genes include subunits of the NURD histone
deacetylase/nucleosome remodelling complex, and probably regulate genes
involved in vulval formation by altering chromatin structure
(Lu and Horvitz, 1998
;
Solari and Ahringer, 2000
). By
analogy, aly may activate such a NURD complex in primary
spermatocytes. comr could act with aly as a regulator of the
complex. Alternatively comr and maybe also aly could
function as testis specific components of the NURD complex. In this model Comr
(and Aly) proteins would be directly involved in nucleosome remodelling, Comr
protein perhaps interacting with histones as postulated above.
The predicted Comr protein did not contain any sequence motifs with known DNA-binding activity. Nevertheless, Comr protein is found in cells in close association with the chromatin, suggesting that the chromatin localisation of Comr may be mediated by protein-protein, rather than protein-DNA, interactions. A candidate region for mediating such protein-protein interactions is the PB-1-like motif. This region of Comr is not similar enough to the PB-1 consensus to score a significant match, therefore it is unlikely that the domain is a true PB-1 domain, interacting with the PC motif. However this PB-1 like region of Comr could be responsible for mediating protein-protein interactions by binding to a motif similar to PC.
It has recently been shown that the meiotic arrest gene can
encodes a testis-specific homologue of dTAF80, a subunit of the basal
transcription factor TFIID
(Hiller et al., 2001).
TFIID consists of TATA-binding protein and associated factors,
binds to the promoter region, interacts with transcriptional activator
proteins and helps in recruitment of the RNA polymerase II holoenzyme complex
to the transcription initiation site
(Dynlacht et al., 1991
;
Goodrich et al., 1996
;
Roeder, 1996
). The human and
yeast homologues of can are also found in the histone
acetyl-transferase (HAT) complex PCAF or SAGA
(Grant et al., 1998
;
Ogryzko et al., 1998
). As
can is not required for all transcriptional activation in primary
spermatocytes, it may be that spermatocytes have two TFIID
complexes, each with a different set of target genes. aly and
comr could function by altering the chromatin structure at the site
of target promoters so that the testis specific, Can-containing,
TFIID complex can bind and activate transcription. This would make
all the can-dependent transcripts also dependent on comr and
aly, consistent with our observations. However this simple model
cannot explain why some genes, namely cyclinB, twine and
boule, require aly and comr but not the
can-class genes for their expression. These
aly-class-dependent genes are all required for normal meiotic cell
cycle progression in testes; however, they are not transcribed exclusively in
primary spermatocytes. cyclinB is required for mitosis, so is
expressed throughout development (Lehner
and O'Farrell, 1990
), twine is required for meiosis in
the female germline so is also expressed in ovaries
(Alphey et al., 1992
), and
boule transcripts have been found in cDNA libraries derived from
heads (H. W.-C., unpublished) (Rubin et
al., 2000
). Their transcription in primary spermatocytes may
depend on particular chromatin structure to facilitate binding of a
spermatocyte specific transcription factor, which would act in conjunction
with the conventional TFIID complex. This, or a related, postulated
specific transcription factor could also be required for transcription of
can-class dependent genes by interacting with the testis specific
TFIID complex. Fig.
9 shows a model for how target promoters could be regulated, first
by chromatin remodelling promoted by Aly and Comr, then by transcription
factor binding and recruitment of TFIID complexes.
|
How does the pathway interact with the transcriptional pattern
When assessed using immunostaining, both Comr and Aly proteins persist
until the G2-M transition of meiosis I, but become undetectable as the
chromosomes condense in prometaphase I. At this point, transcriptional
activity is shut down. The cause and effect relationship between transcription
shut down and Aly/Comr disappearance events not clear. Perhaps transcription
shuts down because Comr and Aly are degraded in response to the same cues that
signal chromosome condensation. Alternatively, the proteins could become
physically excluded from the DNA during chromosome condensation, and then
degraded.
Transcription from a particular promoter can be viewed as a cycle of
polymerase binding, initiation of transcription, promoter clearance and
termination. During this cycle, the phosphorylation state of the CTD of RNA
polymerase II changes. Unphosphorylated pol II is competent to enter the
pre-initiation complex, as promoter clearing occurs the pol II becomes
multiply phosphorylated on the CTD (Conaway
et al., 2000; Dahmus,
1996
). At termination of transcription, RNA polymerase II is
dephosphorylated and released, ready for another cycle. Hence, antibodies
specific to phosphorylated CTD only label the pool of RNA polymerase II that
is actively transcribing. The antibody used in this study (H5) specifically
recognises phosphorylation of Ser2 in the heptapeptide CTD repeat
(Patturajan et al., 1998
).
We have demonstrated that the most active regions of mRNA transcription are not associated with strong DNA staining. Lower levels of transcriptional activity were found to be associated with this DNA. This is expected, as we would predict that active transcription occurs on the most decondensed, and therefore weakly staining, regions of DNA. The staining clearly shows that there are discrete domains within the nucleus with high levels of transcriptional activity, rather than the active regions of chromatin being randomly distributed within the nucleoplasm. Comr protein colocalised with DNA staining, and therefore it is not surprising that we found Comr protein associated with all regions of transcriptional activity. There was no clear correlation between the intensity of Comr staining and the presence or absence of P-CTD immunoreactivity. Within each nucleus some regions of high mRNA transcription were associated with high Comr, other regions were associated with low Comr levels. Not all Comr-positive regions of chromatin were transcriptionally active. Given this distribution how does Comr control the transcription in primary spermatocytes of some genes but not others? Comr protein seems to be present on more regions of chromatin than those that are actively transcribing, so it is unlikely that binding of Comr to chromatin regions is solely responsible for marking them for transcriptional activation. The Comr-containing complex must interact with another factor to give the promoter specificity we observe. For many promoters, that other factor may be the Can-containing TFIID complex; the other factor for can-independent promoters remains unidentified. Because neither of the aly-class genes described to date encode direct DNA-binding proteins, it is likely that more genes of this class, which encode DNA-binding transcription factors, remain to be identified. These would be likely to contribute also to the promoter specificity of the meiotic arrest transcriptional activation pathway through sequence-specific DNA binding.
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ACKNOWLEDGMENTS |
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