BIOLOGICAL OVERVIEW

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. aly encodes a homolog of the C. elegans gene lin-9, a negative regulator of vulval development that acts in the same SynMuvB genetic pathway as the LIN-35 Rb-like protein. The aly gene family is conserved from plants to humans. Aly protein is both cytoplasmic and nuclear in early primary spermatocytes, then resolves to a chromatin-associated pattern. It remains cytoplasmic in a loss-of-function missense allele, suggesting that nuclear localization is critical for Aly function, and that other factors may alter Aly activity by controlling its subcellular localization. MAPK activation occurs normally in aly mutant testes. Therefore aly, and by inference lin-9, act in parallel to, or downstream of, activation of MAPK by the RTK-Ras signalling pathway (White-Cooper, 2000).

aly appears to play a crucial role at the head of the pathway controlling transcription of both cell cycle and differentiation genes. aly is required for expression of both cyclin B and twine in primary spermatocytes. In addition to regulating the expression of cell cycle genes, aly also regulates spermatid differentiation by controlling transcription in primary spermatocytes of a suite of spermatid differentiation genes (White-Cooper, 1998). The behavior of Aly protein, its homology to the C. elegans SynMuvB gene lin-9, and the abnormal chromatin structure in aly mutant spermatocytes suggest that aly may control transcription of cell cycle and terminal differentiation genes by regulating a chromatin remodelling complex (White-Cooper, 2000).

Numerous genes require the activity of the meiotic arrest genes for their transcription. aly is different from the other meiotic arrest genes cannonball (can), meiosis I arrest (mia) and spermatocyte arrest (sa), in that it is required for the transcriptional activation of more target genes, and for normal chromosome structure. Based on this, it is proposed that aly acts upstream of the can-class genes in primary spermatocytes. cookie monster (comr) is a novel meiotic arrest gene. comr mutant spermatocytes fail to transcribe twine and Male-specific RNA 87F (mst87F) as well as many other target genes, and the cells arrest with abnormal chromatin morphology. The nuclear localization of Aly and Comr proteins are mutually dependent. The demonstration that the comr mutant phenotype is indistinguishable from that of aly supports the segregation of the meiotic arrest genes into aly and can classes. The chromosome morphology defect seen in both aly and comr mutant lines supports the idea that the pathway in which they act has a role in the maintenance of normal chromatin structure (Jiang, 2003)

The aly gene and its homologs act at the intersection of tumor suppressor, cell cycle control and terminal differentiation pathways. The Aly protein of Drosophila regulates both male meiotic cell cycle progression and the terminal differentiation program of spermiogenesis by activating the transcription of genes required for both processes. Germ cells in aly mutant testes fail to progress beyond the mature primary spermatocyte stage, owing to lack of both key cell cycle transcripts required to enter the meiotic divisions, and of transcripts for proteins involved in the morphological changes of spermatid differentiation. aly is expressed in primary spermatocytes, the cells that show defects in aly mutants, suggesting a cell autonomous function. Aly protein is localized to the nucleus of maturing primary spermatocytes, where it appears to be associated with chromatin. Mutations in aly cause defects in the appearance of primary spermatocyte chromosomes, consistent with a role for aly in chromatin structure (Lin, 1996; White-Cooper, 2000).

The C. elegans homolog of aly, lin-9, acts in a pathway with the Rb tumor suppressor protein LIN-35 to antagonize RTK-Ras-MAPK signalling during vulval development, influencing the choice of terminal differentiation pathway. Vulval formation in C. elegans is controlled via an inductive signalling pathway in which the anchor cell of the gonad signals to the overlying ventral ectodermal cells of the vulval equivalence group, P3.p-P8.p. This signal activates an RTK-Ras-MAPK signal transduction pathway in P6.p, causing this cell to adopt a primary vulval cell fate, and to induce its neighbours P5.p and P7.p to adopt a secondary vulval fate. The remaining cells in the equivalence group (P3.p, P4.p and P8.p) do not adopt a vulval cell fate, instead they become hypodermal. The inductive pathway is antagonized by the SynMuv genes, which fall into two groups that represent two genetically redundant pathways. In animals doubly homozygous mutant for any one of the five SynMuvA genes and any one of the 12 SynMuvB genes, including lin-9, all the cells in the vulval equivalence group adopt an induced cell fate. The SynMuvB pathway has been proposed to repress expression of vulval genes via a complex of LIN-35 (an Rb homolog), LIN-53 (an Rb associated protein) and histone deacetylase, with the RTK-Ras-MAPK signal relieving this repression to activate vulval gene transcription (Lu, 1998). Ras pathway signalling has been shown to result directly in inactivation of Rb after mitogen stimulation in proliferating mammalian tissue culture cells via the interaction of Rb with Raf1 (White-Cooper, 2000 and references therein).

Activation of MAP kinase in the testis is not dependent upon the activity of the lin-9 homolog aly. If aly antagonizes a MAPK signalling pathway by preventing the phosphorylation and activation of ERK it would be expected that the mutant testes would have excess di-phosphorylated, active, ERK when compared with wild type. No differences were detected in the level of total or active ERK between wild-type and mutant testes, indicating that aly acts at the level of downstream effectors, or in a parallel pathway. This is consistent with the model proposed by Lu (1998) that the role of the SynMuv B genes is to maintain a repressor complex at the promoters of vulval differentiation genes. The activation of MAP kinase in P6.p in response to the anchor cell signal would then lead to relief of this repression and transcription of target genes (White-Cooper, 2000).

Genetic mosaic analysis of mutations in the SynMuvB pathway have suggested that some members act in the hypodermis (lin-15B, lin-37), while others function in the vulval precursor cells (lin-35, lin-36, lin-53). The SynMuvB pathway was therefore proposed to comprise an intercellular signalling pathway from the hypodermis to the vulval precursor cells (reviewed in Kornfeld, 1997). The lineage requirement for lin-9 function in C. elegans has not been tested. The cell autonomous activity of aly suggests that lin-9 will also have a cell autonomous role. In the nucleus, Aly protein could interact with homologs of other cell autonomous, nuclear, components of the SynMuvB pathway. Drosophila homologs of lin-35 (RbF), lin-53 (p55 subunit of chromatin assembly factor) and hda (histone deacetylase) have been described, although no Drosophila homolog of lin-36 has yet been identified (White-Cooper, 2000).

How might Aly control transcription? Aly protein contains neither a predicted DNA binding domain nor any domain that matches known transcriptional activators, yet it is required for the transcriptional activation of many target genes in primary spermatocytes. The Aly protein could act as a transcriptional co-activator; a physical interaction between Aly and one or more transcription factors could be responsible for the observed localization of Aly protein to chromatin. Drosophila E2F2, which is transcribed in the testis in primary spermatocytes in a pattern very similar to that of aly, is a candidate aly regulated or associated transcription factor since one role of Rb is to bind to and regulate the transcription factor E2F. Drosophila E2F1 promotes S-phase in embryos and induces PCNA expression in tissue culture cells; under the same conditions Drosophila E2F2 inhibits PCNA expression in tissue culture cells (White-Cooper, 2000).

Mutations in several SynMuvB genes dramatically reduce expression of transgenes in repetitive extrachromosomal arrays in C. elegans, without affecting expression of the endogenous genes or transgenes in non-repetitive arrays. Additionally several SynMuv pathway genes encode components of the NURD nucleosomal remodelling and histone deacetylase complex (Solari, 2000). These results suggest that one function of the SynMuvB genes is to activate transcription of genes contained within specialized chromatin architectures. Although de-acetylation of histones is often thought of in the context of transcriptional repression, the yeast histone deacetylase RPD3, a homolog of the NURD complex histone deacetylase HDAC1, was originally identified as a factor that is required to achieve maximal levels of both transcriptional repression and activation. The Aly protein of Drosophila may recruit or regulate a NURD-like complex on the bivalents in primary spermatocytes. Action of this complex could have a dual effect, reducing expression of genes not part of the terminal differentiation program, while allowing transcription of spermatogenic genes in a specialized chromatin domain. The proposed aly modulated specific chromatin domain could then be a target for a downstream transcription factor. The observation that wild-type function of aly is required for the normal appearance of chromatin in primary spermatocytes (Lin, 1996) is consistent with this proposed role for aly in chromatin structure (White-Cooper, 2000).

Translocation of Aly protein from the cytoplasm to the nucleus may represent an important control point. The protein encoded by the aly^z3-1393 allele fails to enter the nucleus, despite the presence of two consensus predicted nuclear localization signals. Failure of aly^z3-1393 mutant protein to enter the nucleus could be explained if translocation to the nucleus is inhibited by phosphorylation of Aly protein, in a manner similar to that observed for the cell cycle-regulated nuclear localization of the yeast SWI5 transcription factor. Like aly SWI5 contains a bipartite NLS. Phosphorylation of three serine residues close to this NLS prevents the nuclear accumulation of the SWI5 protein. S161 of Aly protein, two residues from the second basic domain of the bipartite NLS, is a good match to the consensus for cAMP-dependent protein kinase. This serine is conserved in all the aly homologs identified, where it lies two residues away from a classical NLS. The defective aly^z3-1393 protein has an acidic residue close to the NLS, which may allow it to adopt a conformation mimicking that of the phosphorylated form (White-Cooper, 2000).

The C. elegans SynMuvA and SynMuvB pathways are genetically redundant in vulval development, but not in all tissues. Similarly, defects have been detected only in the male germline in aly mutant flies (Lin, 1996), aly may function at other stages of development if a genetically redundant pathway is active. This remains a possibility since a very low level of aly message is detected in adult females by RT-PCR. Alternatively the second Drosophila lin-9 homolog, 86E4.4, could carry out the lin-9-like function at earlier stages of Drosophila development. The conservation of aly in many phyla suggests a SynMuvB like pathway may be a conserved feature in many different organisms, ranging from plants to vertebrates. It will be interesting to determine whether the aly protein family functions in mammals to coordinate meiotic divisions with gamete production. Transcription of boule lies downstream of aly function (White-Cooper, 1998). If the mechanisms are conserved, it might be expected that transcription of the boule homologs Daz and Dazl in mammalian spermatogenesis are dependant on aly homologs. The recent discovery in plants of both Rb proteins and other components of the Rb pathway supports the hypothesis that a SynMuvB pathway may have a conserved role in allowing multicellular organisms to evolve complex structures consisting of many different cell types, whose normal development depends on the coupling of cell cycle controls with cellular differentiation (White-Cooper, 2000).

The mechanism by which SynMuv genes control the choice of differentiation pathway is still very poorly understood. The SynMuv genes, including Rb and aly, may not always be involved in negative regulation of RTK-RASMAP kinase signalling. Rather, SynMuv pathway genes and aly could play a more general role in regulating differentiation, both repressing transcription of certain genes and being required for activation of others in a specialized chromatin context. In the case of the C. elegans vulval precursor cells, Rb and the SynMuv genes could counteract EGFR pathway signalling by affecting chromatin in the region of EGFR-RAS-MAP kinase target genes. In Drosophila primary spermatocytes, aly and its partners could also affect expression of meiotic cell cycle and terminal differentiation genes via effects on chromatin (White-Cooper, 2000).

GENE STRUCTURE

aly was cloned by a combination of fine structure recombination mapping, deletion analysis and mapping of RFLPs associated with insertion alleles. aly encodes a 1.85 kb germline-dependent transcript in males. A probe encompassing 18 kb of genomic sequence from -24 kb to -42 kb of a chromosome walk detected a single 1.85 kb transcript in Northern blots of poly-A+-selected RNA from wild-type males but not from germline-less males. A 1.5 kb cDNA obtained by screening a testis library with the same probe also recognized the 1.85 kb transcript in poly-A+ RNA from wild-type males. This transcript was detected in RNA from males homozygous for aly¹, a temperature-sensitive allele, but not in males homozygous for the hybrid-dysgenesis induced alleles aly⁴, aly⁵ or aly⁶. The 1.85 kb transcript is likely to be a product of the aly locus rather than a downstream transcriptional target of the meiotic arrest gene pathway since it was detected in Northern blots of poly-A+ RNA from males homozygous for can¹, mia and sa1. A much less abundant transcript at 1.5 kb was also detected in the more heavily loaded lanes using the cDNA probe (White-Cooper, 2000).

Comparison of the sequences of the 1.5 kb cDNA, a 5' RACE product derived from testis RNA and genomic clones from the Aly region indicated that the 1.85 kb transcription unit has two small introns. The first intron is in the 5' UTR, the second 92 codons into the predicted protein. Conceptual translation revealed an ORF encoding a predicted protein of 534 amino acids, 62 kDa (White-Cooper, 2000).

cDNA clone length - 2267

Bases in 5' UTR - 199

Exons - 3

Bases in 3' UTR - 463

PROTEIN STRUCTURE

Amino Acids - 534

Structural Domains

aly is a member of a conserved gene family that includes the C. elegans negative regulator of vulval induction, lin-9 BLAST searches of sequence databases identified aly as one of two Drosophila homologs of the C. elegans gene lin-9 (Beitel). The other homolog (86E4.4) had been identified by the European Drosophila genome project. Sequences with significant homology to this family of proteins were also identified from the Arabidopsis thaliana genomic sequence project (two closely related genes), and from Zea mays (maize), Oryza sativa (rice), Schistosoma mansoni, zebrafish, mouse and human EST projects. Multiple sequence (Clustal W) alignments have revealed two distinct domains of homology. On average, pairwise comparisons (Clustal W) within Region 1 show 32% amino acid identity, 53% similarity. The second homology domain (Region 2) was less well conserved, especially in the Arabidopsis sequence, where a putative divergent Region 2 was identified. On average, pairwise comparisons within Region 2 show 22% aa identity, 42% similarity, excluding those between the very well conserved vertebrate proteins, and the divergent Region 2 from Arabidopsis. The spacing between Regions 1 and 2 varies somewhat between the homologs. No significant similarities between the proteins in pairwise comparisons were detected outside the two conserved domains, nor were any similarities detected to any other proteins in the sequence databases. The second Drosophila homolog has a long C-terminal region that includes a leucine zipper motif. The Arabidopsis homolog appears to have an extended N-terminal region. However, since the Arabidopsis protein is based on GRAIL and GenScan predictions on genomic sequence, rather than on cDNA, it is not clear if these predicted exons are actually present in the mature transcript. All the database entries for the vertebrate sequences were derived from single sequencing runs on cDNAs, so the sequences of the entire transcription units are not available. However, given that both homology regions are present in the zebrafish predicted protein, first homology region are expected to be found in the mouse and human homologs when full-length cDNA sequences become available (White-Cooper, 2000).

A strikingly conserved feature of all the homologs where sequence of the first conserved region was available is the presence of a nuclear localization signal (NLS) predicted by PSORT within this region. In aly, this fits the consensus for a bipartite NLS: two basic residues, a ten residue spacer, and another basic region consisting of at least three out of five basic residues. The predicted NLS of the other homologs fits the classic consensus of four residues, at least three basic, the other any of K, R, P or H. The missense allele aly3-1393 changes Val150 to glutamic acid within region 1 of aly. This residue is conserved as an aliphatic amino acid (I, L or V). V150 of aly falls within the ten amino acid spacer region of the bipartite NLS (White-Cooper, 2000).

date revised: 20 March 2003

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