boule
In spermatogenesis, a major transition occurs as the mitotically amplifying population of spermatogonia cease mitosis and develop into primary spermatocytes. These primary spermatocytes become committed to undergoing the meiotic divisions, and then differentiating into spermatozoa. This change in cell behavior is associated with a dramatic switch in the transcript profile: some genes are downregulated and many are upregulated or switched on for the first time. The 'meiotic arrest' genes of Drosophila are crucial for regulating transcription in primary spermatocytes.
The Drosophila always early (aly) gene is involved in this switch in spermatocyte transcriptional regulation. aly coordinately
regulates meiotic cell cycle progression and terminal
differentiation during male gametogenesis. aly is required
for transcription of key G2-M cell cycle control genes and
of spermatid differentiation genes, and for maintenance of
normal chromatin structure in primary spermatocytes. 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. 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 homolog Twine, and thus link differentiation to the cell
cycle. 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 (Jiang, 2002 and references therein).
Transcriptional activation in early spermatocytes involves hundreds of genes, many of which are required for meiosis and spermatid differentiation. A number of the meiotic-arrest genes have been identified as general regulators of transcription; however, the gene-specific transcription factors have remained elusive. To identify such factors, the protein that specifically binds to the promoter of spermatid-differentiation gene Sperm-specific dynein intermediate chain (Sdic) was purified and identified as Modulo, the Drosophila homologue of nucleolin. Analysis of gene-expression patterns in the male sterile modulo mutant indicates that Modulo supports high expression of the meiotic-arrest genes and is essential for transcription of spermatid-differentiation genes. Expression of Modulo itself is under the control of meiotic-arrest genes and requires the DAZ/DAZL homologue Boule that is involved in the control of G2/M transition. Thus, regulatory interactions among Modulo, Boule, and the meiotic-arrest genes integrate meiosis and spermatid differentiation in the male germ line (Mikhaylova, 2006).
Although the general regulators of transcription in testes have been extensively characterized, the gene-specific transcription factors have long been elusive. To characterize such factors, attempts were made to identify the protein that binds to the conserved positive regulatory element b2UE1/b2UE2/TSE (see Nurminsky, 1998, for TSE consensus) that is necessary for activity of the β(2)tubulin promoter in Drosophila testes and is present in the promoter of the testes-specific gene Sdic. Modulo is required for transcription of a number of spermatid-differentiation genes, including β(2)tubulin and Sdic. Expression of Modulo itself in testes is positively regulated by the meiotic-arrest genes at the posttranscriptional level and requires the DAZ/DAZLA homologue Boule, the protein that also controls the G2/M meiotic transition through posttranscriptional regulation of Cdc25/Twine (Mikhaylova, 2006).
The promoter of the testes-specific gene Sdic contains the TSE motif that shows similarities to the conserved elements b2UE1 and b2UE2 found in other testes-specific promoters. An abundant TSE-binding protein was detected in protein extracts from Drosophila testes but not from gonadectomized males by using EMSA. Formation of the DNA–protein complex was completely inhibited by a 100-fold molar excess of the unlabeled TSE probe. At the same time, the presence of a 104-fold molar excess of the heterologous double-stranded oligonucleotide competitor 1 in all EMSA reactions did not inhibit formation of the DNA–protein complex, indicating that binding of the protein is sequence-specific. Further addition of the different oligonucleotide competitor 2 in 100-fold excess to the probe did not interfere with the complex formation. To corroborate this finding, five more different heterologous oligonucleotides were tested using the same conditions, and none of them impeded complex formation. Thus, a protein that specifically binds to the conserved TSE promoter motif is up-regulated in testes and may be involved in transcriptional regulation of Sdic (Mikhaylova, 2006).
To identify the TSE-binding factor, a multistep procedure was developed for its biochemical purification from whole adult flies
Modulo is a broadly expressed protein that has been detected in ovaries, embryonic epidermis and mesoderm, larval imaginal discs, salivary glands, and brain and in cultured cells. Western blot analysis showed that the size of Modulo differs between testes and somatic tissues represented by the gonadectomized males. In testes, mobility of the protein is more consistent with the predicted 60.3-kDa size of the Modulo polypeptide; no signal was detected in testes of the mod07570 male sterile mutant, thus confirming the specificity of the assay. However, the apparent molecular mass of the Modulo variant expressed in somatic tissues is ~50 kDa, which corresponds with the size of the TSE-binding protein that was purified from the whole-fly extracts and identified as Modulo (Mikhaylova, 2006).
A analysis indicated that the 50-kDa protein is a truncated variant missing the N terminus of the full-size Modulo. All of the identified trypsin-generated peptides were located in the C-terminal portion of the molecule, whereas not a single peptide was identified near the N terminus. In addition, an unusual peptide flanked with the trypsin cleavage site at only one (the C-terminal) end was repeatedly identified with high confidence during the analysis. The N-terminal end of the peptide thus possibly represents the N terminus of the truncated 50-kDa Modulo variant, and its position is consistent with the size of the protein (predicted molecular mass, 46.2 kDa). In this case, the 50-kDa variant is missing the highly acidic N-terminal domain that is present in the full-size protein (Mikhaylova, 2006).
The 50-kDa somatic Modulo variant is capable of specific binding to the TSE motif. Thus, a specific variant of Modulo is expressed in testes, where it is capable of specific binding to the TSE-containing promoters. This full-size Modulo variant contains the N-terminal acidic domain, the structure of which is characteristic for the acidic activators that facilitate assembly of the core transcription machinery on the promoter and recruitment of chromatin-remodeling factors. Known acidic activators interact with the TFIID complex and facilitate interaction of TFIID with TFIIA and TFIIB. In testes, TFIID is represented by a specific variant encoded by the meiotic-arrest genes of the sa group. Modulo coimmunoprecipitates with the testes-specific TFIID subunit Sa and, thus, probably interacts with the testes-specific TFIID during transcriptional activation of testes-specific genes (Mikhaylova, 2006).
The suggested activity of Modulo as transcriptional activator in testes is not consistent with its role in somatic tissues, where it is involved in multiple vital activities that probably include chromatin-mediated transcriptional repression [based on the demonstrated Su(var) phenotype of the modulo mutants]. Structural differences between the Modulo variants may underlie this apparent discrepancy. The 50-kDa somatic Modulo variant is able to bind to DNA but is missing the N-terminal acidic domain and, thus, cannot establish the activating interactions observed in testes. Instead, the somatic Modulo variant may contribute to repression of testes-specific genes in somatic tissues (Mikhaylova, 2006).
The Modulo-binding element TSE is present in the promoter of the testes-specific gene Sdic that is up-regulated in primary spermatocytes. To regulate Sdic expression, Modulo has to be present at the same or earlier stage of spermatogenesis. Localization of the zone of up-regulation of Modulo in adult whole-mount testes using immunofluorescence showed that this zone, indeed, precedes and overlaps the zone of Sdic expression. To visualize Sdic expression, advantage was taken of the Sdic::GFP fusion transgene. Modulo is weakly expressed in early spermatogonia and stem cells located at the tip of the testis, but is up-regulated in late spermatogonia/early spermatocytes. Within the cells, Modulo is localized in both the nucleus and the cytoplasm. Specificity of the immunofluorescence staining was confirmed by the absence of appreciable signal in testes of the mod07570 and achi1 mutants that show severe down-regulation of Modulo; also, no staining was observed in wild-type testes when the primary antibody was omitted (Mikhaylova, 2006).
Thus, in spermatogenesis, up-regulation of Modulo precedes activation of its putative regulation target, Sdic. Attempts were made to see whether this pattern is also present for the general transcriptional regulators (the meiotic-arrest genes) and their regulation targets (the spermatid-differentiation genes). To dissect the temporal order of expression of these genes, transcript levels were quantitated in testes dissected from developing larvae using real-time RT-PCR. In Drosophila, spermatogenesis begins early in larval development, and the first wave of meiosis commences at the time of pupation. All of the five tested spermatid-differentiation genes, including Sdic, show drastic up-regulation in late third-instar larvae, i.e., during spermatocyte maturation before meiotic divisions. This finding is consistent with the observed pattern of up-regulation of the Sdic::GFP transgene in adult testes. However, up-regulation of the three studied meiotic-arrest genes precedes up-regulation of the spermatid-differentiation genes. Hence, Modulo is up-regulated in the male germ line in a manner similar to known general transcriptional regulators, before the major wave of transcriptional activation that includes spermatid-differentiation genes (Mikhaylova, 2006).
Although knockout modulo mutations result in lethality, a male sterile hypomorphic mutation mod07570 has been described; thus, mutation, caused by a transposon insertion, results in testes-specific modulo knockdown. To analyze the role of Modulo in transcriptional regulation, transcripts of a number of spermatogenesis-related genes were quantitated in testes of the mod07570 mutant and of the wild type, using real-time RT-PCR. The constitutive transcript of the ribosomal protein gene Rp49 was used as the cDNA template-loading reference. The ubiquitous transcripts RpL9 and Act5C and the broadly expressed spermatogenesis-related genes des and twe were not significantly affected by the mod07570 mutation (Mikhaylova, 2006).
Conversely, a number of genes with testes-biased expression were down-regulated to various extents in the mod07570 mutant testes. In particular, several meiotic-arrest genes (including aly, can, nht, and rye) were down-regulated 5- to 7-fold. Among the 13 other testes-biased genes examined, 7 showed moderate 2- to 5-fold down-regulation. However, four testes-biased genes showed >10-fold down-regulation, and two more genes were down-regulated 7- to 9-fold. Thus, there is a subset of testes-biased genes [including Sdic, Ssl, fzo, dhod, β, and dj] that are specifically affected by the Modulo deficiency (Mikhaylova, 2006).
Because the meiotic-arrest genes themselves are involved in transcriptional regulation in testes, some effects of the Modulo deficiency may be mediated by their down-regulation. Such effects should be similar to the effects caused by mutations in the meiotic-arrest genes themselves. This possibility was addressed by analyzing gene expression patterns in testes of the meiotic-arrest mutants achi1, sa1, and Taf12LKG00946 (rye), and by comparing them to the pattern of gene expression in the mod07570 mutant testes. Among the 13 testes-biased genes analyzed, four genes were affected differently by the meiotic arrest and the modulo mutations. The genes Mst98Ca, Pros28.1B, and CG10934 were very sensitive to mutations in meiotic-arrest genes but not in modulo and, conversely, the gene Ssl did not show striking sensitivity to the mutations in the meiotic-arrest genes sa and rye but was severely affected in the modulo mutant. Therefore, the effect of Modulo deficiency on transcription in testes cannot be reduced to down-regulation of the meiotic-arrest genes. It is possible that such down-regulation leads to the subpar performance of the meiotic-arrest genes that is still sufficient to carry the germ line of the mod07570 mutant through the meiotic divisions but results in moderate (e.g., 3- to 4-fold) down-regulation of testes-biased genes, such as Mst98Ca, Pros28.1B, and CG10934. However, a more severe effect of the Modulo deficiency on a subset of spermatid-differentiation genes probably reflects disruption of gene-specific transcriptional regulation and provides a molecular basis for the spermatid-differentiation failure observed in the mod07570 mutant (Mikhaylova, 2006).
The subset of genes strongly affected in the mod07570 mutant includes Sdic and β(2)Tubulin. These genes possess the TSE-like Modulo-binding motifs and, thus, probably represent the direct regulatory targets of Modulo. Interestingly, it was not possible to detect specific binding of Modulo to the promoters of fzo and dj that are also strongly affected by the modulo mutation. At the same time, the studied dj promoter fragment contained all sequences necessary for efficient testes-specific transcription. This finding implies that Modulo has indirect target genes such as dj and, probably, fzo that may be regulated by transcription factors that are, in turn, under the control of Modulo. A broad survey of 96 transcriptional regulators expressed in testes identified nine putative transcription factors that are down-regulated >10-fold in the mod07570 mutant testes. Thus, mutation in modulo can lead to disruption of the downstream cascade of transcriptional regulation that includes Modulo-dependent transcription factors and their regulation targets (Mikhaylova, 2006).
To determine whether Modulo is sufficient to induce ectopic transcription of spermatid-differentiation genes, recombinant full-size Modulo was expressed in the Schneider-2 cultured cells under the control of metallothionein promoter. Stable transfected clones were selected, and expression of the transgene was induced by various concentrations of Cu2+ in the culture media. Unexpectedly, it was observed that the Schneider-2 cells naturally express the full-size Modulo variant. Nevertheless, these cells do not show significant expression of the Modulo-dependent testes-specific genes Sdic, Ssl, and dhod, and increase of the Modulo dose by induced expression of the transgene did not affect the levels of these transcripts. Therefore, other, presumably testes-specific factors, (such as the testes-specific TFIID) have to cooperate with Modulo to induce expression of spermatid-differentiation genes, thus defining tissue specificity of the Modulo-mediated transcriptional regulation (Mikhaylova, 2006).
To analyze the regulation of Modulo expression in testes, a number of mutants that control different stages of spermatogenesis were analyzed. In testes of the bam mutant, both the Modulo protein and modulo transcript are severely down-regulated. Thus, high levels of Modulo expression in the testes require the onset of the meiosis/differentiation program. Furthermore, mutations in the meiotic-arrest genes achi/vis, sa, and rye result in severe down-regulation of Modulo protein in testes; however, modulo transcription is not affected. Therefore, Modulo expression in the testes is regulated by the meiotic-arrest genes at posttranscriptonal levels, similar to the regulation of the meiotic entry control protein Cdc25/Twine. Translation of Twine in the testes requires the RNA-binding protein Boule. To investigate whether a similar mechanism is involved in the regulation of Modulo, testes of the boule mutants were examined, and it was found that Modulo expression in testes is severely affected by the Boule deficiency (Mikhaylova, 2006).
Regulation of Modulo expression in testes by Boule provides a mechanistic link between meiosis and spermatid differentiation in the male germ line. The meiotic-arrest genes are required for expression of a number of spermatogenesis-related genes, including boule. Boule is required for expression of Modulo, which, in turn, is necessary to maintain expression of several meiotic-arrest genes. These events establish a positive regulatory loop that sustains high levels of expression of Boule, Modulo, and the meiotic-arrest genes after the onset of the meiosis/differentiation program in spermatocytes. Boule further regulates the G2/M transition in meiosis by positive translational regulation of Cdc25/Twine, and Modulo and the products of the meiotic-arrest genes are required for expression of a number of spermatid-differentiation genes. Thus, the pathways that lead to meiosis and to expression of the spermatid-differentiation genes in the male germ line are integrated in a single mechanism to ensure coordinated execution of meiotic divisions and spermatid differentiation (Mikhaylova, 2006).
During spermatogenesis, cells coordinate differentiation with the meiotic
cell cycle to generate functional gametes. The gene off-schedule (ofs) was identified as being essential for this
coordinated control. During the meiotic G2 phase, Drosophila
ofs mutant germ cells do not reach their proper size and fail to execute
meiosis or significant differentiation. The accumulation of four cell cycle
regulators -- Cyclin A, Boule, Twine and Roughex -- is altered in these mutants,
indicating that ofs reveals a novel branch of the pathway controlling
meiosis and differentiation. Ofs is homologous to eukaryotic translation
initiation factor eIF4G. The level of ofs expression in spermatocytes
is much higher than for the known eIF4G ortholog (known as eIF-4G or eIF4G),
suggesting that Ofs substitutes for this protein. Consistent with this, assays
for association with mRNA cap complexes, as well as RNA-interference and
phenotypic-rescue experiments, demonstrate that Ofs has eIF4G activity. Based
on these studies, it is speculated that spermatocytes monitor G2 growth
as one means to coordinate the initiation of meiotic division and
differentiation (Franklin-Dumont, 2007). A second studied, co-published with the Franklin-Dumont paper, has reported similar findings (Baker, 2007).
Initiation is the rate-limiting step in translation and is the most common target of translational control. The mRNA 5' cap is bound by eIF4F, a heterotrimeric protein complex that is the focal point for initiation. eIF4G is the backbone of this complex; it interacts not only with eIF4E, but also with eIF4A, an RNA helicase that facilitates ribosome binding and its passage along the 5' untranslated region (UTR) towards the initiation codon. eIF4G also associates with eIF3, a multisubunit factor that bridges the proteins bound to the mRNA's 5' end with the 40S ribosomal subunit. This ribosomal subunit comes 'pre-charged' as a ternary complex composed of eIF2, GTP and the initiator methionine-transfer RNA. With the aid of eIF4 initiation factor as well as ATP, this agglomeration of RNA and protein is thought to scan the mRNA in the 5' to 3' direction. When it encounters an AUG start codon in an optimal context, other factors as well as the 60S ribosomal subunit are recruited and polypeptide chain elongation begins (Richter, 2005).
The eIF4E-eIF4G interface is an important target for translational control. The core portion of eIF4G that interacts with eIF4E is small -- about 15 amino-acid residues (Mader, 1995). Strikingly, several other proteins contain similar peptide motifs, and it is this region that competes with eIF4G for binding to eIF4E; in this manner they control the rate of 40S ribosomal subunit association with mRNA, and hence translation initiation. A clear demonstration of why the competition between eIF4G and other proteins for interaction with eIF4E is so effective in preventing translation comes from X-ray crystallographic analysis. Peptides derived from the regions of eIF4G and an eIF4E inhibitory protein called 4E-BP (for 4E-binding proteins, also known as PHAS-I for phosphorylated heat and acid soluble protein stimulated by insulin; see Drosophila Thor) form nearly identical α-helical structures that lie along the same convex region of eIF4E, some distance from this protein's cap binding site (Marcotrigiano, 1997; Matsuo, 1997). Peptides with the general sequence YXXXXLphi, where phi is any hydrophobic amino acid, would probably form similar α-helical structures, implying that other proteins containing this peptide motif could control translation initiation (Richter, 2005).
The original three eIF4E inhibitory proteins, the 4E-BPs, prevent eIF4F complex formation by sequestering available eIF4E. This sequestration results in the inhibition of translation of certain mRNAs that normally require high levels of available eIF4E (Gingras, 1999). eIF4E-binding proteins interact with the eIF4E on only specific mRNAs, and do so either because they also interact with certain RNA elements directly, or do so through affiliations with RNA binding proteins (Richter, 2005).
In spermatogenesis, progenitor cells must execute the meiotic divisions in
coordination with acquiring the specialized morphology and functionality of
sperm. This conserved process is particularly amenable to analysis in
Drosophila. The fly testis is a blind-ended tube organized as an
assembly line for spermatogenesis. Germline stem cells at the blind end give
rise to gonialblasts, which divide mitotically four times with incomplete
cytokinesis to produce a cyst of 16 interconnected spermatogonia. These cells
exit the mitotic cycle and enter meiosis as spermatocytes, exhibiting an
extended G2 phase characterized by a significant increase in cell
mass and robust transcription. At the end of G2, the spermatocytes
undergo the meiotic divisions and begin the conversion from round spermatids
to specialized spermatozoa (Franklin-Dumont, 2007).
Ten 'spermatocyte arrest' genes are required for both meiosis and
differentiation and are sorted into two classes according to their molecular
targets and specific role in promoting transcription. The
always early (aly) class affects the transcription of meiotic genes such
boule, twine and cyclin B, as well as that of
differentiation genes such as fuzzy onions (fzo) and don
juan. Notably, these mutations do not effect transcription of
other spermatocyte genes, such as pelota, cyclin A and
roughex. The Aly class proteins are thought to alter chromatin
structure to permit the high levels of transcription necessary in
spermatocytes. The cannonball (can) class affects boule and
twine expression post-transcriptionally only and has no effect on
cyclin B. The post-transcriptional effects must be indirect, because
all can class loci encode testis-specific components of the general
transcriptional machinery. Together, the spermatocyte arrest genes reveal how a
diverse set of genes is selectively transcribed in spermatocytes (Franklin-Dumont, 2007).
The transcriptional regulatory pathway does not address the timing of
meiotic entry and differentiation, however. Although transcripts necessary for
these processes accumulate in early spermatocytes, the corresponding proteins
do not appear until much later.
Because there is little, if any, transcription after the G2-M
transition in flies, spermatocytes must delay meiotic division until
all the necessary transcripts have accumulated. A similar dilemma exists
during the mitotic cycle in yeast. For cells to maintain the same average size
over several divisions, control points act during the gap phases and allow
cell cycle progression only when the cell has reached a threshold size, with
G1 predominating in budding yeast and G2 in fission
yeast. Cell growth rates also feed back on mitotic cell cycle progression in
Drosophila cells. Less is known about how growth might affect the
specialized meiotic cell cycle (Franklin-Dumont, 2007).
Identification and characterization of off-schedule provides evidence that cell growth is linked to the
coordination of meiosis and differentiation. Spermatocytes in ofs
mutant males fail to execute the G2-M transition of meiosis or
substantive post-meiotic differentiation and have a significant cell size
defect. The Off-schedule protein resembles the eukaryotic
initiation factor 4G (eIF4G), which is a member of the eIF4F translation
initiation complex and bridges mature mRNAs and the ribosome
(Prevot, 2003). The eIF4G activity of Ofs is apparent in its ability to associate with mRNA caps and to functionally replace canonical eIF4G in
cell culture. Because translation is primarily regulated at initiation, eIF4G
is instrumental in determining the translational capacity of a cell and thus
its ability to accumulate mass. Thus, the ofs mutant phenotype
suggests that sufficient cell mass must accumulate before spermatocytes
execute meiosis and differentiation (Franklin-Dumont, 2007).
Alignment among eIF4G sequences suggests that Ofs would be part of the
eIF4F complex with eIF4A and eIF4E, and demonstration of its association
with 7-methyl GTP Sepharose strongly supports this. Although
binding of Ofs directly to eIF4A was not measured, alignment of human and fly eIF4G proteins shows conservation of three out of four sets of amino acids necessary to bind eIF4A (Imataka, 1997). Of 12 crucial residues, ten were identical in Ofs, one was a conservative (L>I) change, and the twelfth diverged in
Drosophila eIF4G as well. With regard to eIF4E
binding, the putative binding site in Ofs has an arginine substituted for the
usual hydrophobic residue. However, a similar substitution is tolerated in
Drosophila eIF4E binding protein 1
(Miron, 2001), and Baker (2007) presents evidence for interaction with Drosophila eIF4E1. Taken together, it is quite likely that Ofs participates in
cap-dependent translation initiation (Franklin-Dumont, 2007).
eIF4G (CG10811) and Ofs (CG10192) appear to be the only two eIF4G proteins
encoded in the fly genome. One other candidate, l(2)01424, is more related to
the proposed translational inhibitor, NAT1/p97 (Rpn1)/DAP5, than to eIF4G
proteins (Takahashi,
2005). Although the novel N-terminus of Ofs raised the possibility
that it would play a role distinct from eIF4G, the data suggest that Ofs can
act as the only eIF4G in cultured cells. Whether these two proteins always act
redundantly in vivo cannot be assessed without mutations in eIF4G.
Nevertheless, eIF4G, at its endogenous level, cannot substitute for Ofs in
spermatocytes. Perhaps this is simply due to a relatively lower level of eIF4G
compared with Ofs. Alternatively, Ofs might uniquely aid in the translation of
a special class of mRNAs, specific to spermatocyte development. Perhaps
sequences in its novel N-terminus assist in such a role. Although further
experiments are needed to distinguish between these possibilities, one reason
for a distinction between spermatocytes and other cells might be in their
respective mode of growth control. In cultured eIF4G-deficient mitotic cells,
the cell cycle effect observed was on G1, whereas the defect in
spermatocyte progression is in G2. Although the G1-S
transition is the major control point for growth sensing in mitotic cells of
the fly, G2 might make more sense as the control point
for meiosis, because it is during this phase of the cycle that spermatocytes
need to prepare not just for division, but for differentiation. Furthermore,
spermatocytes might commit to the meiotic cycle, versus returning to the
mitotic cycle, during G2, as is the case for the yeast
Saccharomyces cerevisiae. Perhaps expressing a unique eIF4G (Ofs) in
spermatocytes helps serve this role. Given the functional role for
ofs, it is proposed that ofs henceforth be known as eIF4G2 (Franklin-Dumont, 2007).
Because ofs (eIF4G2) encodes the predominant eIF4G in
spermatocytes, one might expect that mutant cells would exhibit decreased
translation of many mRNAs. Just as a striking delay was found in in Boule
accumulation, other proteins would be expected to be similarly affected. Such
a global deficit could account for the delayed development of these cells, and
would be predicted to influence cell size, because the translational capacity
of a cell predicts its ability to accumulate mass.
Indeed, one of the earliest phenotypes in eIF4G2 spermatocytes was
their small size. Yet, Aly accumulation appeared normal and
Rux protein appeared to accumulate to an excess degree in early
spermatocytes. These data demonstrate that some mRNAs are not affected by the
translational deficit, and raise an alternative scenario wherein spermatocytes
actively monitor their size. If they do not achieve proper growth, a
checkpoint is induced to prohibit meiosis and differentiation. Because meiosis
involves two cell divisions with little intervening interphase, size
monitoring would be especially important before these cells commit to
divide (Franklin-Dumont, 2007).
Circumstantial support for a growth checkpoint includes the accumulation of
the Cdk inhibitor Rux, which leads to aberrant behavior of Cyclin A. In this
model, the postulated checkpoint causes the striking delay in the accumulation
of Boule, which, in turn, explains the delay in Twine accumulation. Eventually, Boule does accumulate to reasonable levels, perhaps as cells leak through the checkpoint, just as eventually occurs in mitotic checkpoints. However, by then, Cyclin A has been degraded, and without it, the eventual accumulation of Twine cannot trigger meiosis, so the checkpoint has succeeded (Franklin-Dumont, 2007).
To establish that a checkpoint exists, one would need to identify the
sensor, which detects the problem, and effectors, which execute inhibitory
functions until the cell resolves the problem. No candidate is available for
the sensor that detects growth at this time, nor for effectors controlling
differentiation. However, it can be speculated that Rux is one effector regulating
the meiosis branch, where it could serve to inhibit Cyclin A-driven Cdc2
kinase activity (Avedisov,
2000). Rux is not the only effector regulating meiosis, however.
Previous work showed that directly increasing the level of Rux only blocked
entry into the second meiotic division
(Gönczy, 1994). Consequently, the accumulation of Rux that is observed in eIF4G2 mutants cannot fully explain the absence of the first meiotic division or the defect in differentiation. As would be typical for cell cycle regulation,
several effectors must be activated at once to completely block the
G2-M transition (Franklin-Dumont, 2007).
The existence of other effectors could explain why forcing early Twine
accumulation failed to restore meiotic entry to eIF4G2 mutants in a
rux background. Alternatively, there might be additional positive
factors necessary for G2-M transition that have not accumulated in
eIF4G2 spermatocytes. Consistent with this, prior work driving
expression of another Cdc25, string (stg), in early
spermatocytes directed a normal rather than a precocious G2-M
transition. Thus, advancing Cdc25 activity is insufficient to trigger a
precocious G2-M even in the absence of a growth defect. Perhaps
early spermatocytes have not had enough time to accumulate an essential
component, such as Cyclin B, for the meiotic divisions. It was found that
eIF4G2 mutant clones exhibit Cyclin B levels comparable to
neighboring heterozygous cells. However, there is a peak in
Cyclin B accumulation just prior to meiosis I, and Baker (2007) describes a deficit of this Cyclin B peak in eIF4G2 mutants. Thus, Cyclin B remains a candidate factor (Franklin-Dumont, 2007).
Whether a growth checkpoint exists or not, mass accumulation could be used
to time the G2-M transition by coupling rate-limiting cell cycle
proteins to the translational capacity of the cell. In the budding yeast,
S. cerevisiae, cyclin CLN3 (also known as YHC3) contains an upstream
open reading frame in the 5' UTR that slows its translation in
G1 under poor growth conditions.
Similarly, during G2 in the fission yeast, Schizosaccharomyces
pombe, accumulation of CDC25 is disproportionately affected by defects in
translation. Perhaps the translation of Boule, along with a few other
meiotic cell cycle regulators, is disproportionately affected when translation
is compromised in spermatocytes. Although this should be investigated, this
simpler model does not explain the aberrant accumulation of Rux and the
nuclear sequestration of Cyclin A that was observed (Franklin-Dumont, 2007 and references therein).
The defects in differentiation in eIF4G2 mutants are not secondary
to the meiotic block, because several cell cycle mutants fail to divide but
still undergo substantial post-meiotic differentiation. Several
spermatid differentiation genes, such as don juan and fuzzy
onions, are transcribed in primary spermatocytes under the control of
spermatocyte arrest genes. Translational control delays the accumulation of their
protein products. This delay is functionally relevant, because precocious
don juan accumulation leads to sterility. In
principle, then, the lack of significant differentiation in eIF4G2
mutants could simply be due to a more pronounced translational delay for key
differentiation genes. Alternatively, the block in differentiation might
reflect a direct effect of the proposed growth checkpoint. Consistent with
either model, the accumulation of the mitochondrial fusion protein Fuzzy
onions is delayed, although this was not timed precisely.
It is expected that other differentiation targets will also be abnormally delayed
in eIF4G2 mutants (Franklin-Dumont, 2007).
There are striking parallels to the role of eIF4G2 during spermatogenesis
in other organisms. For instance, there are also two major isoforms of eIF4G
in Caenorhabditis elegans, encoded by ifg-1. When the longest isoform was depleted from the germ line, oocytes arrested in meiosis I (B. D. Keiper, personal communication to Franklin-Dumont, 2007). The requirement for ifg-1 in spermatogenesis has not yet been examined. However, one of the five isoforms of eIF4E in the worm, IFE-1, is clearly essential for spermatogenesis. RNA interference against ife-1 results in delayed meiotic progression, and in defective sperm, in both hermaphrodites and males (Amiri, 2001). Furthermore, mouse testes carrying the Y chromosome deletion Spy (also known as Eif2s3y-Mouse Genome Informatics) have a meiotic arrest phenotype due to a lack of EIF2 (also known as EIF2S2-Mouse Genome Informatics) function (Mazeyrat, 2001). Taken together, these examples suggest that translational control, and therefore possibly growth control, is a common theme for meiotic cycle cells (Franklin-Dumont, 2007).
The Drosophila boule gene is expressed exclusively in the male germline. Boule localization was examined in flattened germ cell preparations (testis squashes) by immunocytochemistry. In wild-type testes, Boule is first detected at the beginning of the spermatocyte growth phase, following completion of the premeiotic S phase. Boule staining is diffuse at this stage. However, as the primary spermatocytes mature, Boule becomes highly concentrated in nuclei. Within these nuclei, Boule protein staining is not uniform but is instead consistently restricted to a crescent-shaped region. To define the nature of the subnuclear localization of Boule, the Boule staining pattern was compared to prominent nuclear structures. Comparison with chromatin localization, as assayed with the DNA-binding dye Hoechst, indicates that the Boule crescent is near, but not coincident with, one of the three readily detectable chromosome pairs. The associated chromosomes have been identified as the XY pair, based on the distinct morphology of this chromosome set relative to the autosomes. In most cells, Boule staining does not overlap with that of the XY pair, but is very frequently found nearby, usually at the periphery of the nucleus (Cheng, 1998).
Since the X and Y chromosomes contain the rRNA loci and are therefore closely associated with the nucleolus, phase-contrast microscopy was used to determine the location of Boule relative to this organelle. The spermatocyte nucleolus appears as a roughly spherical, phase-dense structure within the nucleus. The crescent-shaped region of Boule staining very often abuts and embraces the nucleolus, but is not coincident with it. The distinctive Boule crescent thus represents a perinucleolar pattern of protein localization. Boule nuclear localization persists until the end of the spermatocyte growth phase. At the time of nucleolar breakdown, prior to meiosis, Boule is in the cytoplasm rather than the nucleus. As a result, during meiosis Boule is excluded from the chromatin-containing region and is largely cytoplasmic. Boule remains cytoplasmic in postmeiotic spermatids, where it appears to be uniformly distributed. Boule localization in spermatocytes is thus biphasic, being first nuclear and then cytoplasmic. Boule localization was examined in twine mutant spermatocytes, which fail to activate the cell cycle oscillator and hence do not undergo the meiotic divisions. Boule undergoes a wild-type transition from the nucleus to the cytoplasm in the twine mutant. It is concluded that the translocation of Boule to the cytoplasm is not dependent on meiotic entry (Cheng, 1998).
Nuclear localization was dependent on RNA and on the presence of the Y chromosome. Boule nuclear localization is absent from primary spermatocytes in samples treated with RNase. However, the cytoplasmic localization of Boule in postmeiotic onion stage spermatids is unaffected by RNase treatment, indicating that nuclear but not cytoplasmic Boule localization is primarily dependent on RNA (Cheng, 1998).
Deletion of the Y chromosome ks-1 fertility locus eliminates Boule nuclear localization, although it does not perturb entry into meiosis. Based on these observations it is proposed that Boule acts in the cytoplasm to regulate the stability or translation of messenger RNA encoding an essential meiotic factor. The Y chromosome encodes six fertility loci, each spanning four or more megabases of DNA, that are heavily transcribed in the primary spermatocyte. Indeed, several of the resulting lampbrush chromosome structures, or Y loops, are visible by phase microscopy at the nuclear periphery of un-stained primary spermatocytes. It was hypothesized, therefore, that Boule might bind to one or more Y loop structures. To compare Y loop localization with that of Boule, testis squashes were immunostained with an antibody that recognizes Y loop associated antigens in Drosophila chromatin. This antiserum recognizes a subset of the Y loops, predominantly the antigen associated with the kl-5 and to a lesser extent the ks-1 fertility factors. Like Boule, the Y loop antigens occupy a crescent-like region that is often adjacent to the nucleolus. In flattened spermatocytes double stained with anti-Boule and anti-Y loop sera, Boule and the Y loop antigens have been found localized to neighboring regions that occasionally overlap. The similarity and close spatial association of the localization patterns suggests that Boule associates with Y loops (Cheng, 1998).
To delineate the region of the Y required for Boule nuclear localization, a deletion analysis was carried out. Four of the Y chromosome fertility factors lie on the long arm (kl-1, kl-2, kl-3, kl-5) and two on the short arm (ks-1 and ks-2), with numbering on each arm ordered from proximal to distal. Eight distinct sets of Y chromosome synthetic deficiencies were analyzed for alterations in Boule staining. Only the three deficiencies affecting ks-1 disrupt the nuclear localization of Boule. In all three such cases Boule is predominantly cytoplasmic in primary spermatocytes. Staining in later spermatids is not affected by any of the deficiencies. These results indicate that Boule nuclear localization depends on the ks-1 region of the Y. However, since the ks-1-dependent localization of the RNA binding protein RB97D is wild-type in a boule mutant, the Y loop structure derived from the ks-1 region is not substantially altered in the absence of Boule (Cheng, 1998).
Spermatocytes are formed in boule mutants, but fail to undergo meiotic divisions. Comparison of the localization of Cyclin A in boule mutant and in wild-type germ cells supports the conclusion that the meiotic prophase is normal in boule mutants. Although the meiotic prophase appears wild type in boule mutant germ cells. subsequent stages are aberrant. Cyclin A is exclusively cytoplasmic in the extended premeiotic G2, and enter the nucleus at the transition between G2 and M phases only to be degraded rapidly after nuclear translocation, yet Cyclin A persists in boule mutants (Eberhart, 1996).
Agulnik, A. I., et al. (1998). Evolution of the DAZ gene family suggests that Y-linked DAZ plays little, or a limited, role in spermatogenesis but underlines a recent African origin for human populations. Hum. Mol. Genet. 7(9): 1371-7.
Alphey, L., et al. (1992). twine, a cdc25 homolog that functions in the male and female germline of Drosophila. Cell 69: 977-988.
Baker, C. C. and Fuller, M. T. (2007). Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog. Development 134(15): 2863-9. Medline abstract: 17611220
Castrillon, D. H., et al. (1993). Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: Characterization of male-sterile mu-tants generated by single P element mutagenesis. Genetics 135: 489-505.
Chai, N. N., et al. (1997). A putative human male infertility gene DAZLA: genomic structure and methylation status. Mol. Hum. Reprod. 3(8): 705-8.
Cheng, M. H., Maines, J. Z. and Wasserman, S. A. (1998). Biphasic subcellular localization of the DAZL-related protein boule in Drosophila spermatogenesis. Dev. Biol. 204(2): 567-76.
Cooke, H. J., et al. (1996). A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. Mol. Genet. 5(4): 513-6.
Courtot, C., et al. (1992). The Drosophila cdc25 homolog twine is required for meiosis. Development 116: 405-416.
Eberhart, C. G., Maines, J. Z. and Wasserman, S. A. (1996). Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia. Nature 381(6585): 783-5.
Fox, M., Urano, J. and Reijo Pera, R. A. (2005). Identification and characterization of RNA sequences to which human PUMILIO-2 (PUM2) and deleted in Azoospermia-like (DAZL) bind. Genomics 85(1): 92-105. 15607425
Franklin-Dumont, T. M., Chatterjee, C., Wasserman, S. A. and Dinardo, S. (2007). A novel eIF4G homolog, Off-schedule, couples translational control to meiosis and differentiation in Drosophila spermatocytes. Development 134(15): 2851-61. Medline abstract: 17611222
Houston, D. W., et al. (1998). A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule. Development 125(2): 171-80.
Houston, D. W. and King, M. L. (2000). A critical role for xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus. Development 127(3): 447-56.
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. 12490562
Johnson, A. D., et al. (2001). Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad. Dev. Bio. 234: 402-415. 11397009
Karashima, T., Sugimoto, A. and Yamamoto, M. (2000). Caenorhabditis elegans homologue of the human azoospermia factor DAZ is required for oogenesis but not for spermatogenesis. Development 127(5): 1069-1079
Maegawa, S., Yamashita, M., Yasuda, K. and Inoue, K. (2002). Zebrafish DAZ-like protein controls translation via the sequence 'GUUC'. Genes Cells. 7(9): 971-84. 12296827
Maines, J. Z. and Wasserman, S. A. (1999). Post-transcriptional regulation of the meiotic Cdc25 protein Twine by the Dazl orthologue Boule. Nat. Cell Biol. 1(3): 171-4. >
Maruyama, R., et al. (2005). Caenorhabditis elegans DAZ-1 is expressed in proliferating germ cells and directs proper nuclear organization and cytoplasmic core formation during oogenesis. Dev. Biol. 277: 142-154. 15572146
Mikhaylova, L. M., Boutanaev, A. M. and Nurminsky, D. I. (2006). Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line. Proc. Natl. Acad. Sci. 103(32): 11975-80. Medline abstract: 16877538
Nurminsky, D. I., Nurminskaya, M. V., De Aguiar, D. and Hartl, D. L. (1998). Selective sweep of a newly evolved sperm-specific gene in Drosophila. Nature 396(6711): 572-5. Medline abstract: 9859991
Richter, J. D. and Sonenberg, N. (2005). Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433(7025): 477-80. Medline abstract: 15690031
Reijo, R., et al. (1995). Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat. Genet. 10(4): 383-93.
Reijo, R., et al. (1996). Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics 35(2): 346-52.
Reijo, R. A., et al. (2000). DAZ family proteins exist throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice. Biol. Reprod. 63(5): 1490-6. 11058556
Ruggiu, M., et al. (1997). The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389(6646): 73-7.
Santel, A., et al. (1997). The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif. Mech. Dev. 64(1-2): 19-30.
Saxena R., et al. (1996). The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned. Nat. Genet. 14(3): 292-9.
Seboun E., et al. (1997). Gene sequence, localization, and evolutionary conservation of DAZLA, a candidate male sterility gene. Genomics 41(2): 227-35.
Shan, Z., et al. (1996). A SPGY copy homologous to the mouse gene Dazla and the Drosophila gene boule is autosomal and expressed only in the human male gonad. Hum. Mol. Genet. 5(12): 2005-11.
Sigrist, S., Ried, G. and Lehner, C. F. (1995) Dmcdc2 kinase is required for both meiotic divisions during Drosophila spermatogenesis and is activated by the Twine/cdc25 phosphatase. Mech. Dev. 53: 247-260.
Tsui, S., Dai, T., Warren, S. T., Salido, E. C., Yen, P. H. (2000). Association of the mouse infertility factor DAZL1 with actively translating polyribosomes. Biol. Reprod. 62(6): 1655-60. 10819768
Urano, J., Fox, M. S. and Reijo Pera, R. A. (2005). Interaction of the conserved meiotic regulators, BOULE (BOL) and PUMILIO-2 (PUM2). Mol. Reprod. Dev. (3): 290-298. 15806553
Venables, J. P., Ruggiu, M. and Cooke, H. J. (2001). The RNA-binding specificity of the mouse Dazl protein. Nucleic Acids Res. 29(12): 2479-83. 11410654
White-Cooper, H., et al. (1998). Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila. Development 125: 125-134.
Xu, E. Y., et al. (2003). Human BOULE gene rescues meiotic defects in infertile flies. Hum. Mol. Genet. 12(2): 169-75. 12499397
Yen, P. H., Chai, N. N. and Salido, E. C. (1996). The human autosomal gene DAZLA: testis specificity and a candidate for male infertility. Hum. Mol. Genet. 5(12): 2013-7.
Yen, P. H., Chai, N. N. and Salido, E. C. (1997). The human DAZ genes, a putative male infertility factor on the Y chromosome, are highly polymorphic in the DAZ repeat regions. Mamm. Genome 8(10): 756-9.
date revised: 20 June 2005
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.