always early


Nuclear entry of Aly and Cookie monster is mutually dependant

In Drosophila spermatogenesis, meiotic cell cycle progression and cellular differentiation are linked by the function of the meiotic arrest genes. The meiotic arrest genes control differentiation by regulating the transcriptional activation of many differentiation-specific genes. The meiotic arrest genes have been subdivided into aly and can classes, based on the mechanism by which they control cell cycle progression. aly has previously been shown to encode a chromatin-associated protein. This study presents the identification, cloning and characterization of a novel Drosophila meiotic arrest gene, cookie monster (comr), that has a mutant phenotype indistinguishable from that of aly. A null mutant allele of comr is viable but male sterile. Mutant primary spermatocytes fail to initiate transcription of a large number of genes, and arrest before entry into the meiotic divisions. In adult males, expression of comr is testis specific, low levels of transcripts are detected at other stages of development. comr encodes a novel acidic protein, which is nuclear and primarily localized to regions of chromatin in primary spermatocytes. The nuclear localization of Aly and Comr proteins are mutually dependent. Finally, it has been shown that active RNA polymerase II is found in distinct domains in the nucleus that constitute a subset of the total Comr stained chromatin (Jiang, 2002).

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. 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, characterized 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 individualizing to form motile sperm (Jiang, 2002).

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 1996). 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 (White-Cooper, 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 (Jiang, 2002).

Three meiotic arrest genes have been cloned to date, aly, can and no hitter (nht). can and nht encode testis-specific homologs 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. aly encodes a homolog 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, 1998; Solari, 2000; Jiang, 2002 and references therein).

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 localization of LIN-9 in C. elegans has not been determined. Aly protein localizes to chromatin in maturing Drosophila primary spermatocytes. This localization suggests that the lin-9 homolog, Aly, may act in close concert with a NURD complex on chromatin (White-Cooper, 2000). The nuclear localization 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 localization signal (Jiang, 2002).

cookie monster is 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. Comr protein is localized 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. The nuclear localization of these two proteins is mutually dependent. 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 localization domain, but the level of Comr protein does not predict the level of active RNA polymerase II (Jiang, 2002).

A large collection of EMS-induced male sterile mutations were examined by phase-contrast microscopy of testes squashes to identify potential new meiotic arrest loci. Wild-type testes contain cysts of cells at many different stages of spermatogenesis: pre-meiotic, meiotic and post-meiotic. By contrast, testes from one of the Zuker lines (Z2-1340) contain mature pre-meiotic primary spermatocytes, but no cysts undergoing meiotic divisions or attempting post-meiotic spermatid differentiation. Z2-1340 testes were smaller than wild type because of the lack of later stages of spermatogenesis. The arrested cells degenerated towards the distal end of the mutant testes. Z2-1340 females are fertile. Since Z2-1340 maps to the second chromosome, and all of the previously characterized meiotic arrest genes were on the third chromosome, Z2-1340 must represent a previously uncharacterized meiotic arrest locus, which has now been called cookie monster because the cells look like a 'whole bunch of cookie monster eyes' (Jiang, 2002).

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 are transcribed in can-class mutants (can, mia and sa) but not in aly (White-Cooper, 1998). A set of diagnostic RNA in situ hybridization experiments reveal that comr resembles aly rather than the can-class mutant mia. comr mutant testes transcribed polo, but fail to transcribe both boule and Mst87F (Jiang, 2002).

The phenotype of comr also resembles 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 have apparently normal chromosome morphology whereas aly mutants have defects in chromatin structure. The major chromosome bivalents of wild-type primary spermatocytes form three discrete, clearly delineated domains within each nucleus. By contrast the chromosome bivalents of comrZ1340 mutant primary spermatocytes are fuzzy, with indistinct boundaries. Therefore, comr mutant spermatocytes 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 (Jiang, 2002).

The comr ORF, based on genome project predictions and RT-PCR encodes a novel 600 amino acid protein, with a predicted molecular weight of 68.4 kDa, and a predicted pI of 5.1. The Comr protein shows no significant homology to any protein in the protein or translated EST databases. The predicted protein contains an acidic domain in the C terminus of the protein (amino acids 518-570), and a predicted nuclear localization sequence (NLS) (amino acids 583-589). In addition, a region that may represent a very divergent PB1 domain (amino acids 348-431) has been identified. PB1 domains have been found in several signal transduction proteins, including kinase C iota (KPCI), and have been shown to mediate protein-protein interactions (Jiang, 2002).

The developmental expression profile of comr was determined by RT-PCR. The transcript is testis specific in adult males, being undetectable in gonadectomized males, consistent with the fully viable but male sterile phenotype of the mutant. Low levels of the transcript were also detected in whole females, embryos and larvae. RNA in situ hybridization revealed a uniform transcript distribution in embryos (Jiang, 2002).

In testes, the comr transcript is expressed at highest levels in early primary spermatocytes, as revealed by RNA in situ hybridization. The transcript level decreases as the spermatocytes grow, and becomes undetectable as the primary spermatocytes enter the meiotic divisions. This expression pattern of comr is essentially identical to the pattern reported for aly (White-Cooper, 2000). The earliest defect 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 (Jiang, 2002).

An antibody raised against a peptide from the C terminus of Comr protein recognized a 100 kDa protein in wild-type testes that was absent from comrZ1340 mutant testes. Although this protein is significantly larger than the predicted size for Comr (68 kDa) the low pI of the protein could affect its mobility in SDS-PAGE. Comr was expressed 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 shows that ectopically expressed tagged Comr protein migrates as a single band with an apparent molecular weight of 100 kDa. 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 (Jiang, 2002).

In wild-type testes the anti-Comr antibody recognizes the nuclei of young and maturing primary spermatocytes. 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 recognized the single spot within each nucleus, but the general nuclear staining was absent. Therefore Comr protein appears to localize 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 (Jiang, 2002).

To explore the relationship between Comr localization and chromatin in primary spermatocytes, indirect immunofluorescent staining was carried out of Comr double labelled with the DNA dye propidium iodide. Staining with the anti-Comr antibody is restricted to the nuclei of primary spermatocytes. Meiotic and post-meiotic cells do 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 shows two distinct types of staining pattern. A brightly stained 'stringy' region 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 is found throughout the nucleus, concentrated near the condensed regions of chromatin. Again, to determine which component(s) of this pattern were attributable to the crossreacting antigen, comrZ1340 mutant testes were stained. The stringy staining in a small region of the nucleus was still detected in comr mutant spermatocytes. However the general nuclear staining was absent from these cells, confirming the findings from the immunohistochemistry. It is concluded that Comr protein is expressed only in primary spermatocytes in the testis, that it is nuclear and that is associated with regions of chromatin (Jiang, 2002).

The identical mutant phenotypes of comr and aly suggest that they act in the same pathway; it was therefore of interest to examining whether comr regulates aly or vice versa. To determine how comr interacts with known meiotic arrest loci, the expressions of comr and aly were examained in various mutant backgrounds. comr transcript was detected by in situ hybridization and RT-PCT at a level similar to wild type in aly mutant testes. Transcription of comr was also detected in testes of can3. 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. 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 are similar to wild type in comrZ1340 mutant testes (Jiang, 2002).

The similar localization pattern of Comr and Aly proteins, combined with their identical mutant phenotypes, suggests that the proteins may interact directly or indirectly. To dissect this relationship, the subcellular localization pattern of one protein in a background mutant for the other was examined. Since 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 is localized to the nuclei of maturing primary spermatocytes. Nuclear staining of primary spermatocytes in cysts gives a spotty appearance to the apical region of the testis. By contrast, Aly protein shows a honeycomb distribution pattern in comrZ1340 mutant spermatocytes. The holes in this pattern correspond to the nuclei; therefore, Aly protein fails to translocate to the nucleus and instead remains cytoplasmic in comrZ1340 mutant spermatocytes. This indicates that the nuclear localization of Aly protein is dependent on comr function (Jiang, 2002).

The reciprocal experiment revealed that Comr protein localization depends on normal function of aly. Immunofluorescence was carried out 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. mia mutant spermatocytes showed staining throughout the nucleus, in addition to the bright spot. By immunohistochemistry, the anti-Comr antibody staining of aly5 and comrZ1340 mutant spermatocytes were indistinguishable, 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. 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 localization of Comr depends on aly, but does not depend on the normal function of the can-class meiotic arrest genes can and mia (Jiang, 2002).

comr mutant spermatocytes are defective for transcription of a number of cell cycle and spermatid differentiation genes. To investigate how Comr protein localization in the nucleus is associated with mRNA transcription, an antibody specific to active RNA polymerase II phospho-C-terminal domain (P-CTD) was used in triple labelling experiments. Staining with anti P-CTD reveals that the regions of the nucleus with most active transcription are adjacent to, but not overlapping with, regions of visible (i.e. condensed) DNA. The active transcription was found in domains within the nucleus, not randomly distributed. Lower levels of detectable P-CTD colocalize with the more condensed DNA. The highest levels of active transcription partially overlap with high levels of Comr protein, 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, although P-CTD staining was not found in all regions containing Comr protein. Thus, Comr protein is not exclusively localized on the pol II-transcriptionally active chromatin, but all the chromatin where RNA polymerase II is transcriptionally active has at least some associated Comr protein (Jiang, 2002). 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. It is possible that the acidic domain on Comr interacts with the basic histone proteins to alter chromatin structure (Jiang, 2002).

aly encodes a homolog of the C. elegans SynMuvB gene lin-9. 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. 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. 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 (Jiang, 2002).

The predicted Comr protein does 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 localization 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 (Jiang, 2002).

The meiotic arrest gene can encodes a testis-specific homolog of dTAF80, a subunit of the basal transcription factor TFIID (Hiller, 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. The human and yeast homologs of can are also found in the histone acetyl-transferase (HAT) complex PCAF or SAGA. Since 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. 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, and so is expressed throughout development; twine is required for meiosis in the female germline and therefore is also expressed in ovaries, and boule transcripts have been found in cDNA libraries derived from heads. 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. Target promoters could be regulated, first by chromatin remodelling promoted by Aly and Comr, then by transcription factor binding and recruitment of TFIID complexes (Jiang, 2002).

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 is 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 (Jiang, 2002).

Physical interaction between Always early, Cookie monster and TGIF homeodomain proteins Vismay and Achintya

Two tightly linked and nearly identical homeobox genes of the TGIF (TG-interacting factor) subclass called vismay and achintya (often referred to as TGIF) are essential for spermatogenesis in Drosophila. 'achintya' is a Sanskrit word meaning 'that which is beyond thought and contemplation', and relates to initial difficulties in interpreting the mutant analysis; 'vismay' is a Hindi word meaning 'surprise', which described the reaction when the genome sequence revealed the tandem duplication. In flies deficient for both genes, spermatogenesis is blocked prior to any spermatid differentiation and before the first meiotic division. This suggests that vismay and achintya function at the same step as two previously characterized meiotic arrest genes, always early and cookie monster. Consistent with this idea, both always early and cookie monster are still expressed in flies deficient in vismay and achintya. Conversely, Vismay and Achintya proteins are present in always early mutant testes. Co-immunoprecipitation experiments further suggest that Vismay and Achintya proteins exist in a complex with Always early and Cookie monster proteins. Because Vismay and Achintya are likely to be sequence-specific DNA binding factors, these results suggest that they help to specify the spermatogenesis program by recruiting or stabilizing Always early and Cookie monster to specific target genes that need to be transcriptionally regulated during testes development (Wang 2003; Ayyar, 2003).

The control pathway underlying spermatogenesis is, as yet, poorly defined but a few 'meiotic-arrest' mutants have been identified. All the meiotic arrest mutants have a similar phenotype -- mature primary spermatocytes arrest development, and fail to enter either the meiotic divisions or spermatid differentiation. The currently identified meiotic arrest genes have been subdivided into two classes. The aly-class genes [always early (aly) and cookie monster (comr)] appear to be higher in the control hierarchy and regulate transcription of some genes involved in entry into meiosis (boule, twine, Cyclin B) and also of many spermiogenesis genes (e.g. fuzzy onions, janus B, don juan, gonadal) required for the differentiation of functional sperm. In contrast, can-class meiotic arrest genes (including cannonball, meiosis 1 arrest (mia) and spermatocyte arrest) do not affect transcription of the meiosis cell-cycle genes but are required for spermiogenesis gene transcriptional activation (Ayyar, 2003 and references therein).

To place achi/vis within this scheme the expression of a set of meiosis-related genes and a selected set of spermiogenesis genes were examined in Df(2R)achi1 homozygous mutant testes by RT-PCR analysis, and in homozygous mutant males by in situ hybridization. Both the set of spermiogenesis genes tested (fuzzy onions, janus B, don juan, gonadal) and the meiosis-related cell-cycle genes (boule, twine, Cyclin B) showed strongly reduced expression in the mutant, placing achi/vis in the aly class of meiotic arrest genes. Transcription of other genes (RP49, polo and Cyclin A) was not affected in the mutants. To determine whether Drosophila achi/vis is required upstream in the pathway for transcription of other meiotic arrest genes, the expression of aly and comr was tested in achi/vis mutant testes. In situ hybridization on achiZ3922 visZ3922 mutant testes revealed aly and comr transcripts at levels similar to wild type, and RT-PCR analysis on Df(2R)achi1 demonstrated robust expression of aly and can transcripts. In the RT-PCR analysis the levels of aly and can actually appear somewhat higher than wild type. This result is not interpreted, however, as indicative of a regulatory interaction but rather as a reflection of the altered cellular composition of the mutant testes. Similarly, aly and comr are not required for the expression of achi/vis because normal levels of achi/vis transcripts were found, by RT-PCR, in aly and comr homozygous mutant testes (Ayyar, 2003).

Neither of the two previously described aly-class meiotic arrest genes contain a predicted DNA binding domain, yet they are both chromatin associated, and are clearly required for transcriptional activation. A simple model would be that the gene products, Aly, Comr and Achi/Vis all act together as components of a single mechanism required for gene activation in spermatogenesis. If this were true it would predict that the phenotype of aly and comr mutations might be indistinguishable from the achi/vis loss-of-function phenotype. To test this, the phenotypes were examined in detail. As noted above the Df(2R)achi1 phenotype includes an expansion of early primary spermatocytes, indicative of an early role for achi/vis in the primary spermatocyte stage. A similar cellular defect has not previously been described for aly but, aly mutants also display expansion of the early primary spermatocyte population presumably due to a defect in progression through the primary spermatocyte differentiation program. This phenotype is not common to all meiotic arrest mutants and progression through the primary spermatocyte stages in mia mutants appears similar to wild type. In both aly and achi/vis mutants the primary spermatocytes do exhibit some spermatocyte differentiation; they increase in size and chromosomal reorganization occurs, giving clear chromatin clumps, as visualized by either DAPI or anti-histone labelling. However, the chromatin fails to organize as tightly in the aly mutant as in the wild type and the cells arrest with peripheral chromatin clumps with a fuzzy appearance. The chromatin morphology of comr is identical to that of aly. In achi/vis mutant testes the chromatin appears to follow a wild-type program up to the generation of mature primary spermatocytes with peripheral chromatin clumps, however, the cells arrest with rounded chromatin clumps that are not apposed to the nuclear periphery and that resemble the chromatin configuration in meiotic stages. It is concluded that the achi/vis phenotype is similar but not identical to that of aly and comr (Ayyar, 2003).

The normal chromatin association of Aly and Comr proteins is essential for their function, and the localization of these two proteins is mutually dependent, i.e., in an aly mutant Comr protein remains cytoplasmic, and vice versa. In contrast, both Comr and Aly proteins localize to chromatin in testes mutant for the downstream, can-class, genes. To determine whether achi/vis plays a role in the production or localization of the other aly-class proteins, the levels and localisation of Aly and Comr proteins were examined in achiZ3922 visZ3922 mutant testes. Both Aly and Comr proteins were detected by Western blotting in achiZ3922 visZ3922 mutant testes. Immunofluorescent staining revealed that Aly and Comr proteins are nuclear in achiZ3922 visZ3922 testes. This places achi/vis downstream of, or parallel to, comr and aly (Ayyar, 2003).

Many of the phenotypes observed in the achi/vis deficiency are also observed in aly and comr mutants. In addition, immunolocalization studies suggest that Vis, Achi, Aly and Comr proteins are co-expressed in the nuclei of primary spermatocytes. These observations prompted a test to see if these proteins may be present as a complex in wild-type testes. This was by carrying out immunoprecipitation (IP) experiments with the anti-Achi antibody and determining if either Aly or Comr is co-immunoprecipitated. Interestingly, both Aly and Comr can be co-immunoprecipitated with Vis/Achi from wild type, but not from pingpong testes. These results suggest that Vis and Achi proteins are present in a complex with Aly and Comr during wild-type testes development (Wang, 2003).

Tombola, a tesmin/TSO1-family protein, regulates transcriptional activation in the Drosophila male germline and physically interacts with Always early

During male gametogenesis, a developmentally regulated and cell type-specific transcriptional programme is activated in primary spermatocytes to prepare for differentiation of sperm. The Drosophila aly-class meiotic-arrest loci (aly, comr, achi/vis and topi) are essential for activation of transcription of many differentiation-specific genes, and several genes important for meiotic cell cycle progression, thus linking meiotic divisions to cellular differentiation during spermatogenesis. Protein interaction studies suggest that the aly-class gene products form a chromatin-associated complex in primary spermatocytes. This study identified, cloned and characterised a new aly-class meiotic-arrest gene, tombola (tomb), which encodes a testis-specific CXC-domain protein that interacts with Aly. The tomb mutant phenotype is more like that of aly and comr mutants than that of achi/vis or topi mutants in terms of target gene profile and chromosome morphology. tomb encodes a chromatin-associated protein required for localisation of Aly and Comr, but not Topi, to chromatin. Reciprocally, aly and comr, but not topi or achi/vis, are required to maintain the normal localisation of Tomb. tomb and aly might be components of a complex paralogous to the Drosophila dREAM/Myb-MuvB and C. elegans DRM transcriptional regulatory complexes (Jiang, 2007).

The meiotic-arrest genes of Drosophila regulate a developmental transition and associated gene expression switch, during which many hundreds of genes whose products are required during sperm formation are upregulated. Most meiotic-arrest genes described to date have been identified through classical genetics. To find additional gene products that act with those already isolated, a reverse genetics approach was undertaken; tomb was identified while screening for proteins that could bind Aly in a yeast two-hybrid system (Jiang, 2007).

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, a second region of homology shared between tomb and the other animal tesmin/TSO1 CXC-domain-containing proteins was identifed. This C-terminal domain has conserved secondary structure, and might be responsible for the Tomb-Aly interaction (Jiang, 2007).

Direct interactions have been demonstrated between Comr and Topi, whereas Aly, Comr and Achi/Vis have been found in a complex in vivo. This study shows that Aly and Comr can interact with Tomb. In support of the interaction data, a second study (Beall, 2007) 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. It has been shown that the nuclear localisations of Aly and Comr are mutually dependent, i.e., Aly remains cytoplasmic in comr mutants and vice versa (Jiang, 2003). It has 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, 2003; Perezgazga, 2004). tomb can now be placed into the pathway of complex assembly and activity. It is proposed 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, 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 (Matsuura, 2002; Sutou, 2003), in a similar manner to fly aly and tomb (Jiang, 2007).

modulo (mod) encoding Drosophila nucleolin, has been implicated in transcriptional activation of spermiogenesis genes (Mikhaylova, 2006). 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. 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 (Jiang, 2007).

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 (Jiang, 2007).

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. Mip120 and Mip130 have been described as components of the dREAM/Myb-MuvB complex found in embryos and tissue culture cells. The dREAM complex contains, in addition to Mip120 and Mip130, Myb, Caf1p55, Dp, Mip40, E2F2 and Rbf or Rbf2 (Korenjak, 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. dREAM/Myb-MuvB regulates DNA replication at chorion gene amplification origins in Drosophila ovarian follicle cells (Beall, 2004; Beall, 2002; Cayirlioglu, 2001; Frolov, 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. 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 (Jiang, 2007).

DRM, a complex containing the C. elegans homologues of the dREAM subunits has recently been described. The genes encoding DRM components act together in the SynMuvB genetic pathway that regulates vulval development redundantly with the SynMuvA and SynMuvC pathways. 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, 2004). LIN9, LIN54 (human Mip120) and hMip40 are all also capable of binding directly to Rb (Jiang, 2007).

Drosophila E2f2- and Rbf2-null mutants are viable and male fertile, but E2F2 females have reduced fertility (Cayirlioglu, 2001; Frolov, 2001; Stevaux, 2005), whereas Myb-, Dp- and Rbf-null mutants are lethal and males mutant for weak Dp alleles are sterile 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 (Stevaux, 2005) in repression of some testis-specific genes (Jiang, 2007).

There is remarkable evolutionary conservation of the interaction between dREAM/Myb-MuvB gene products in somatic tissues in mammals, flies and worms. It is suggested that gene duplications in Drosophila of lin-54 (tomb/mip120), lin-9 (aly/mip130) and lin-52 (CG12442/lin52), 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 (Jiang, 2007).

Wake-up-call, a lin-52 paralogue, and Always early, a lin-9 homologue physically interact, but have opposing functions in regulating testis-specific gene expression

A conserved multi-subunit complex (MybMuvB, MMB), regulates transcriptional activity of many different target genes in Drosophila somatic cells. A paralogous complex, tMAC, controls expression of at least 1500 genes in the male germline, and is essential for sperm production. The roles of specific subunits of tMAC, MMB or orthologous complexes in regulating target gene expression are not understood. MMB and orthologous complexes have Lin-52 as a subunit, but Lin-52 did not co-purify with tMAC. This study identified wake-up-call (wuc), a lin-52 paralogue, via a physical interaction with the tMAC lin-9-related subunit Aly, and Wuc was found to co-localises with known tMAC subunits. It was shown that wuc, like aly, is required for spermatogenesis. However, despite phenotypic similarities, the role of wuc is very different from that of previously characterised tMAC mutants. Unlike aly, loss of wuc results in only relatively mild defects in testis-specific gene expression. Strikingly, wuc loss of function partially rescues expression of target genes in aly mutant testes. It is proposed that wuc represses testis-specific gene expression, that this repression is counteracted by aly, and that aly and a testis-specific TF(II)D complex work together to promote high transcriptional activity of spermiogenic genes specifically in primary spermatocytes (Doggett, 2011).

always early: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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