InteractiveFly: GeneBrief

Cannonball: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Alternate forms of the PolII transcription initiation machinery have been proposed to play a role in selective activation of cell-type-specific gene expression programs during cellular differentiation. The cannonball (can) gene of Drosophila encodes a homolog of a TBP-associated factor (dTAF5) protein expressed only in spermatocytes, where it is required for normal transcription of genes required for spermatid differentiation (Hiller, 2001). Drosophila primary spermatocytes also express four additional tissue-specific TAFs: no hitter (nht - homolog of dTAF4), meiosis I arrest (mia - homolog of dTAF6), spermatocyte arrest (sa - homolog of dTAF8) and ryan express (rye - homolog of dTAF12). Mutations in nht, mia and sa have similar effects in primary spermatocytes on transcription of several target genes involved in spermatid differentiation, and cause the same phenotypes as mutations in can, blocking both meiotic cell cycle progression and spermatid differentiation. The nht, mia, sa and rye proteins contain histone fold domain dimerization motifs. The nht and rye proteins interact structurally when co-expressed in bacteria, similarly to their generally expressed homologs TAF4 and TAF12, which heterodimerize. Strikingly, the structural interaction is tissue specific: nht did not interact with dTAF12 and dTAF4 did not interact with rye in a bacterial co-expression assay. It is proposed that the products of the five Drosophila genes encoding testis TAF homologs collaborate in an alternative TAF-containing protein complex to regulate a testis-specific gene expression program in primary spermatocytes required for terminal differentiation of male germ cells (Hiller, 2004).

The initiation of transcription by RNA polymerase II (PolII) remains a critical control point for regulation of differential gene expression during development and the differentiation of specialized cell types. The general transcription factor TFIID is thought to play a central role in interpreting and integrating molecular signals regulating the core PolII transcription machinery for initiation (Hochheimer, 2003). Recruitment of TFIID to sequences near the transcriptional start site is thought to in turn recruit and/or stabilize PolII binding in the preinitiation complex (Lemon, 2000; Orphanides, 1996; Roeder, 1996). The multisubunit TFIID complex contains the TATA-binding protein (TBP) and 12-14 TBP-associated factors (TAFs) (Albright, 2000). Recently, alternative forms of TBP and tissue-specific TAFs have been described in a number of organisms (reviewed by Hochheimer, 2003; Veenstra, 2001), raising the possibility that cell-type- or stage-specific forms of what was previously thought of as the general transcription machinery may play an important role in selective activation of certain PolII promoters (Verrijzer, 2001). To date, however, only a few tissue-specific TAF_IIs have been investigated for in vivo function in cell-type-specific gene expression programs. For example, an alternate form of TAF4 (TAF4b -- formerly hTAF_II105) highly expressed in granulosa cells was found to associate with TBP and TAF1 in a large, TFIID-like protein complex in ovarian extracts (Freiman, 2001) and to be required for normal follicular development and for expression of a number of genes in developing ovarian follicles in mice (Hiller, 2004 and references therein).

A striking example of a tissue-specific TAF homolog required for execution of a developmentally regulated transcriptional program appears during differentiation of male germ cells in Drosophila (Hiller, 2001). The cannonball gene (can) of Drosophila, which encodes a homolog of dTAF5 expressed only in male germ cells, is required for normal transcription in primary spermatocytes of a set of spermatid differentiation genes required for normal spermatogenesis (Hiller, 2001; Lin, 1996; White-Cooper, 1998). Flies null mutant for can are viable and female fertile but male sterile. The requirement for can function is gene selective: only a specific set of genes normally expressed in wild-type primary spermatocytes are affected, while a number of other genes are transcribed normally in spermatocytes from can mutant males. While it is now clear that cell-type-specific TAF homologs such as can and mTAF4b can play important roles in tissue-specific gene expression, the mechanisms by which they function at specific promoters are not understood. To identify proteins that might collaborate with the dTAF5 homolog can to regulate expression of specific target genes in Drosophila spermatocytes, the expression and function of other TAF_II homologs in the Drosophila genome were investigated (Hiller, 2004).

Drosophila primary spermatocytes are shown to express several additional tissue-specific TAF homologs that act to control expression of spermatid differentiation genes: nht (homolog of dTAF4), mia (homolog of dTAF6), sa (homolog of dTAF8), and rye (homolog of dTAF12). Mutations in nht, sa and mia cause the same phenotypes as can, blocking meiotic cell cycle progression and spermatid differentiation, and have similar effects on transcription in primary spermatocytes of several target genes involved in spermatid differentiation. It is proposed that these five Drosophila testis-specific TAF homologs collaborate in an alternative TAF-containing protein complex expressed in primary spermatocytes to regulate expression of a set of genes involved in spermatid differentiation (Hiller, 2004).

Therefore can, which encodes a homolog of the WD40 repeat containing dTAF5 (Hiller, 2001), and at least four histone fold domain containing TAFs, have tissue-specific homologs: the nht, mia, sa and rye genes encode homologs of dTAF4, dTAF6, dTAF8 and dTAF12, respectively. Null mutations in nht, can, sa and mia all cause the same mutant phenotype and have similar effects on transcription in primary spermatocytes of several target genes involved in spermatid differentiation. In addition, the protein encoded by the rye testis TAF homolog interacts structurally and specifically with nht, suggesting that the five testis TAF homologs function together or in a pathway to regulate a gene selective transcription program for terminal differentiation in the male germline. Loss-of-function mutations in the Drosophila testis TAFs block spermatogenesis at the G₂/M transition of meiosis I, after spermatocyte growth, and the mutant testes have large numbers of differentiating and mature spermatocytes (Lin, 1996; Hiller, 2004). Because transcription of spermatid differentiation genes occurs during the spermatocyte stage in Drosophila, the dramatic reduction in transcripts for target genes involved in spermatid differentiation in the mutants was not due to lack of the cell type in which the target genes are normally transcribed. Importantly, the requirement for the testis-specific TAF homologs for normal levels of transcript accumulation of targets is gene selective; transcripts from many genes normally expressed in primary spermatocytes are expressed at normal levels in the testis TAF mutants (White-Cooper, 1998; Hiller, 2004).

If the five testis TAFs identified to date act in a multi-subunit complex resembling TFIID, then the identity of the other subunits expected in a TFIID-like complex is a puzzle. The five TAF homologs identified may substitute for their more generally expressed homologs in a chimeric TFIID complex, where the other components are the generally expressed TAFs. Alternatively, the Drosophila testis TAFs may participate in a TFIID-like complex where the other components are highly diverged from their more generally expressed forms. It is noted that the nht, can, mia, sa, and rye predicted proteins are more diverged from their generally expressed homologs than the generally expressed Drosophila homologs are diverged from their human counterparts. Another possibility is that the testis TAF homologs act in a different type of protein complex, for example a HAT complex or with the Polycomb complex of chromatin modifying factors, to regulate the cell-type-specific expression of terminal differentiation genes required for spermatogenesis (Hiller, 2004).

The testis TAF4 homolog nht lacks the long, glutamine-rich N terminus characteristic of both the generally expressed Drosophila homolog and TAF4 from mammals. This glutamine-rich N-terminal domain can interact structurally with certain transcriptional activator proteins in vitro and has been proposed to help TFIID mediate activated transcription, perhaps by tethering TFIID to transcriptional activators bound at enhancers (reviewed by Hochheimer, 2003). If so, then the testis-specific homolog nht may render a possible testis-specific TFIID-like complex less sensitive to transcriptional activators that might normally interact through the glutamine rich N-terminal domain of dTAF4 (Hiller, 2004).

Northern blot analysis suggested low levels of alternate transcript forms of mia expressed in females and embryos. Alternate transcripts have also been described for TAF6 in human cells (Bell, 2001). However, analysis of null mutants, including an allele with an early stop codon in the mia open reading frame, suggested that wild-type function of mia is required for spermatogenesis but not for female fertility or embryonic development. It is possible that the mia protein is expressed only in spermatocytes, or that the generally expressed homolog dTAF6 can substitute for mia function in other tissues. Perhaps mia protein may be required to allow nht:rye to bind into a testis-specific TFIID or HAT-like complex, so that mia function is essential only where it is necessary to incorporate nht:rye. The histone fold domain containing dTAF6 forms a heterodimer partner with dTAF9 (Xie, 1996). However, searches of the Drosophila genome have not yet revealed an obvious candidate for a second homolog of dTAF9 that might serve as a binding partner with mia (Hiller, 2004).

The generally expressed TAF8 homolog Prodos binds specifically to dTAF10B in vitro and in yeast two-hybrid assays. The Drosophila genome encodes two homologs of TAF10. However, unlike the testis-specific TAF homologs described in this study, both dTAF10 and dTAF10B are expressed during embryogenesis, with some degree of tissue specificity (Georgieva, 2000). Preliminary tests in the bacterial co-expression assay did not reveal interaction between the testis-specific dTAF8 homolog sa and either dTAF10 or dTAF10B. Searches of the Drosophila genome have not yet revealed an obvious candidate for an additional dTAF10 homolog that might serve as an histone fold domsin heterodimer partner with sa (Hiller, 2004).

Several tissue-specific TAFs and other GTF component homologs have been found to be expressed in the testis in differentiating male germ cells in mammals as well as in Drosophila. Altering the composition of the general transcription machinery may be particularly important for gene expression in the male germline. Differentiation of male gametes in both Drosophila and mammals depends on a robust germline-specific transcriptional program. Expression of many genes required for spermatid differentiation takes place in spermatocytes in Drosophila and in spermatocytes and/or early round spermatids in mammals. Many genes that are transcribed in somatic cells at other stages of development are expressed in male germ cells from testis-specific promoters. In addition, a number of generally expressed genes in Drosophila have homologs that are only or mainly expressed in the testis. In several cases the cis-acting regulatory sequences that drive expression of the testis-specific transcripts have been shown to be contained within short regions positioned near the start of transcription. Humans express a testis-enriched subunit of TFIIA and a testis-specific TAF1 homolog (Ozer, 2000; Upadhyaya, 1999; Wang, 2002) while a testis-specific homolog of TAF7 has been identified in mouse (Pointud, 2003). In addition, wild-type function of the TATA-binding protein homolog TRF2 is required in mouse to produce mature sperm (Martianov, 2001; Zhang, 2001). Both studies of the testis TAFs of Drosophila and studies of knockout mutant mice lacking mTRF2 function indicate that these testis-specific homologs of GTF components are required for normal transcription and terminal differentiation in male germ cells. It is possible that chromatin may be in a different condensation state in spermatocytes and early round haploid spermatids than in many somatic cells and so may require specialized forms of the general transcription machinery to recognize or access testis-specific promoters within an altered chromatin landscape (Hiller, 2004).

One striking finding was the tissue specificity of the structural interactions between dTAF4:dTAF12 compared with the nht:rye proteins. Examination of the virtual structure of the histone fold domains of nht and rye threaded onto the crystal structure of the hTAF4:hTAF12 HFD heterodimer did not reveal any obvious single reason for the specificity of the dimer partners observed in biochemical assays, suggesting that the specificity of the binding partner interaction may be the result of an additive effect of a number of residue interactions across the histone fold domain. One notable difference between the predicted structures of dTAF4:dTAF12 compared with nht:rye, based on threading onto the hTAF4:hTAF12 HFD crystal structure, appeared in the region where loop 1 of TAF4 interacts with loop 2 of TAF12. Loop 1 of hTAF4 and loop 2 of hTAF12 both have a short region of beta sheet, which interacts in parallel to anchor the end of the alpha2 helix interaction in the hTAF4:hTAF12 crystal structure (Werten, 2002). The dTAF4:dTAF12 sequences fit well across this region, yielding predicted interacting beta sheets based on the threading algorithm used. However, the nht:rye predicted structures did not fit this region well. Both nht loop 1 and rye loop 2 lacked a predicted short beta sheet in the virtual heterodimer formed by threading onto the hTAF4:hTAF12 crystal structure. When the virtual structures of dTAF4 and rye predicted by threading and were placed in the heterodimer positions, an acidic clash between Asp732 of the dTAF4 HFD and Asp117 of rye was created in the region where loop1 of TAF4 would contact loop 2 of rye. A second notable difference in the predicted heterodimers of dTAF4:dTAF12 and nht:rye was that the nht His-75:rye Ser-104 interaction contained a hydrogen bond. This interaction would be expected to be much stronger than the weak Ala-Val interaction at the corresponding position in TAF4:TAF12. This interaction would also be abrogated in an nht:TAF12 or TAF4:rye heterodimer. However, the alpha-1 helix of TAF4, corresponding to the region where nht His-75 is located, was not required for TAF4-TAF12 dimerization in yeast TAF4 mutant rescue experiments (Thuault, 2002). The dTAF4:dTAF12 structure predicted by threading on to the hTAF4:hTAF12 crystal structure is likely to be relatively reliable. However, the amino acid sequences of the predicted HFD motifs of nht and rye were considerably diverged from the corresponding regions of both the human and the generally expressed Drosophila TAF4 and TAF12 proteins. As a result, the virtual structure of the nht:rye histone fold domain heterodimer, calculated from threading, is much less reliable and may underestimate the divergence of the protein structures from the heterodimer between the generally expressed homologs. Since the nht:rye heterodimer can be stably expressed in bacteria the best way to compare the structures and probe the molecular basis of the specificity between the generally expressed and the testis-specific predicted heterodimers may be through solving the crystal structure of the testis-specific nht:rye complex (Hiller, 2004).

REGULATION

Functional characterization of testis specific TAFs

Five genes encoding predicted second homologs of biochemically identified Drosophila embryo TAFs were found encoded in the Drosophila genome by tBlastn searches. In addition to cannonball (can), which encodes a homolog of dTAF5 (Hiller, 2001) the Drosophila genome contains predicted second homologs of several other TAFs: no hitter (nht) (CG15259) a homolog of TAF4, spermatocyte arrest (sa) a homolog of dTAF8 (CG11308), meiosis I arrest (mia) (CG10390) (see also Aoyagi, 2000) a homolog of TAF6 and ryan express (rye) (CG15632) a homolog of TAF12. In addition, the Drosophila genome encodes two previously described TAF10 homologs, dTAF10 and dTAF10b, both expressed during embryogenensis (Hernandez-Hernandez, 2001; Georgieva, 2000; Hiller, 2004 and references therein).

The full-length protein encoded by nht was homologous to the C-terminal portion of dTAF4 and its human homolog hTAF4, but lacked the characteristic N terminal glutamine rich domain. A 924 bp cDNA representing the nht transcript isolated from a testis cDNA library and confirmed by RT-PCR from testis mRNA closely matched the 1 kb size predicted by Northern analysis. Sequencing of this near full length nht cDNA revealed an open reading frame encoding a predicted 245 amino acid protein. Comparison with the genomic DNA sequence revealed no introns in the region covered by the cDNA. The initial methionine was preceded by in frame stop codons in the cDNA, suggesting that it represented the start of the protein. The yeast homolog Mpt1p (yTAF4) also does not contain the extended N-terminal glutamine rich domain (Hiller, 2004).

The C-terminal domain of TAF4 contains a histone fold domain (HFD), a dimerization domain present in several different TAF subunits. Through this characteristic alpha-helix – loop – alpha-helix – loop – alpha-helix structure (alpha1-L1-alpha2-L2-alpha3) several TAFs form specific heterodimer pairs, much like the histones H2A:H2B and H3:H4. Although amino acid sequence conservation was relatively low, the predicted Nht protein contained a domain (aa64-aa114 of nht) that could be aligned with the alpha1-L1-alpha2 region of the histone fold motif of TAF4 and histone H2A. The predicted alignment for the Drosophila TAF4 homologs was checked by threading the amino acid sequences on to the crystal structure obtained for the hTAF4:hTAF12 heterodimer. The dTAF4 sequence fits very well, with an RMSD of 0.14A for the 715-758aa region. The nht sequence also fit into the structure, although the nht sequence was more diverged than dTAF4 from hTAF4 and the RMSD was considerably higher (0.74A, even after optimizing by comparing RMSDs for three different possible local alignments) (Hiller, 2004).

The homology between the predicted nht protein and TAF4 proteins from yeast to humans was particularly striking in the well conserved pattern of alternating hydrophobic and charged or polar residues in the region of alpha helix 2, which forms the core of the interaction domain between TAF4 and its heterodimer partner TAF12. The region of hTAF4 that might contribute to the predicted alpha 3 helix was not included in the crystal structure and is the subject of some debate (Thuault, 2002; Werten, 2002). However, the predicted nht protein did share with the TAF4 homologs from human to yeast a characteristic conserved pattern of hydrophobic, charged and polar residues in the CCTD region near the C terminus of the proteins. In addition, multiple sequence alignments revealed two other extended regions between the HFD and the CCTD that had patterns of charged, polar and hydrophobic residues conserved from nht, to its Drosophila, mosquito, human and C. elegans homologs (Hiller, 2004).

The predicted protein encoded by rye is homologous to TAF12, the histone fold motif-containing binding partner of TAF4. A 550 bp rye cDNA isolated from a testis cDNA library closely matched the 600 bp transcript size estimated from Northern blots. Sequencing of this near full length rye cDNA revealed an open reading frame encoding a 138aa predicted protein with homology to dTAF12. An in frame stop codon located six base pairs upstream of the predicted initial methionine in the cDNA suggested that this corresponded to the start of the protein (Hiller, 2004).

The predicted Rye protein contains a histone fold domain region (amino acid residues 68-130 of rye) that aligns well with the histone fold domain of TAF12 and histone H2B. The sequence of the generally expressed dTAF12 threaded onto the crystal structure of hTAF12 in the hTAF4:hTAF12 dimer well, with an RMSD of 0.09Å for the region from aa91-aa164. Although rye is more diverged, the rye HFD sequence also fit onto the hTAF4:hTAF12 dimer crystal structure (RMSD=0.47). Again, rye and the TAF12 proteins from C. elegans to humans showed a striking pattern of alternating hydrophobic and charged or polar residues, echoing the similar pattern in histone H2B in the region of the predicted HFD, especially in the second helix (Hiller, 2004).

The mia gene encodes a protein homologous to dTAF6. A 2400 bp cDNA isolated from a testis cDNA library corresponded in size to the mia transcript expressed in testis estimated from Northern blots. The testis mia cDNA also contains almost 600 bp of 5' untranslated sequence containing multiple stop codons in all three reading frames upstream of the predicted initial methionine (Hiller, 2004).

The mia N-terminal region resembles corresponding regions of the generally expressed dTAF6 and TAF6 from other organisms, although conservation at the level of amino acid sequence is relatively low. However, multiple sequence alignments reveal that amino acid residues 35-94 of the predicted Mia protein align with the histone fold domain of TAF6 and histone H4 with respect to the pattern of hydrophobic, charged and polar residues. Conservation of this pattern is strongest in the region extending from the middle of the alpha 2 helix through loop 2 and the alpha 3 helix of the HFD, based on the dTAF6:dTAF9 crystal structure (Xie, 1996). In the nucleosome, the regions of alpha 2, loop 2 and alpha 3 of histone H4 are involved both in dimerization, involving residues internal to the H3:H4 pair, and in forming non-symmetrical tetrameric bonds between H4 and H2B via exposed residues (Luger, 1997). The sequence of Mia threaded onto the crystal structure of dTAF6 in the dTAF6:dTAF9 heterodimer with an RMSD of 0.79 for aa33-aa94. In addition to the N-terminal predicted HFD, the Mia protein contains an extended central region (aa176-437 of Mia) with a pattern of hydrophobic, charged, polar and proline residues conserved in TAF6 proteins from yeast to humans. Embedded in this 'TAF6 domain' are several regions of significant amino acid sequence conservation between the predicted Mia protein, dTAF6 and TAF6 from other organisms (Hiller, 2004).

The sa gene encodes a protein homologous to dTAF8, also known as Prodos. Sequencing of an 825 bp sa cDNA isolated by RT-PCR from testis mRNA revealed three protein coding exons separated by two introns (FBgn0037080). The cDNA has an in frame stop codon 84 base pairs upstream of the predicted initial methionine (Hiller, 2004).

The N-terminal region of sa resembles the corresponding regions of the generally expressed dTAF8 (Prodos) and TAF8 from other organisms. Although conservation at the level of amino acid sequence is relatively low, domain searches (PFAM) and multiple sequence alignments suggest that amino acid residues 4-70 of the predicted Sa protein contained a predicted histone fold domain, similar to TAF8 homologs from a variety of species with respect to the pattern of hydrophobic, charged and polar residues. Conservation of this pattern is strongest in the predicted alpha 2 helix region. Although no crystal structure is yet available for TAF8 homologs, secondary structure predictions also suggested an alpha-helix – loop – alpha-helix – loop – alpha-helix pattern in this region of the predicted sa protein, consistent with a histone fold domain structure. In support of the prediction of an N-terminal histone fold domain in dTAF8, deletion of amino acids 1-39 from the generally expressed Drosophila TAF8 homolog Prodos disrupts binding (Hernandez-Hernandez, 2001) between dTAF8 (Prodos) and its binding partner dTAF10B (Hiller, 2004).

The predicted Sa protein also contains an extended central region (aa107-186) with significant amino acid sequence conservation and a pattern of hydrophobic, charged, polar and proline residues conserved in TAF8 homologs from yeast to humans. This region is embedded in a larger domain (aa78-186) listed in the pFam database as pFamB-10670, an unannotated domain characteristic of this family of TAFs (Hiller, 2004).

Because TAF4 and TAF12 physically interact as binding partners through their histone fold domains (Gangloff, 2000; Werten, 2002; Yokomori, 1993), whether the Drosophila testis-specific TAF4 and TAF12 homologs nht and rye also interact structurally was tested using a bacterial co-expression and GST pulldown assay. In bacteria carrying a GST-nht fusion construct encoding full length Nht and an empty vector in place of a Rye-FLAG fusion construct, the GST-nht fusion protein was detected in total bacterial extracts (T) under inducing conditions, but was not soluble in the absence of Rye-FLAG fusion protein. A portion of Rye containing the histone fold region fused to a FLAG epitope was not stable and failed to accumulate when expressed in bacteria in the absence of Nht. However, when both the Nht and Rye fusion proteins were co-expressed in the same bacteria, both the GST-Nht fusion protein and the FLAG-tagged Rye HFD-containing fragment accumulated in the total bacterial extract (T) and were soluble. When the GST-Nht fusion protein was isolated from extracts from bacteria expressing both the Nht and Rye fusion proteins by binding to glutathione-Sepharose, the FLAG-Rye fusion bound and co-eluted with GST-Nht (Hiller, 2004).

The physical interaction between the Nht and Rye fusion proteins appeared to be specific for the testis-specific partners. The C-terminal region of the generally expressed Drosophila TAF4 homolog dTAF4, analogous to the full length Nht protein, interacts with dTAF12: a FLAG epitope-tagged fusion protein containing the histone fold region of dTAF12 is stable and co-purifies with the GST-dTAF4 C-terminal domain fusion protein on glutathione-Sepharose. However, under the same conditions, the C terminal (HFD containing) domain of the generally expressed dTAF4 fused to GST did not stabilize FLAG-tagged Rye when the two were co-expressed in bacteria. In similar assays, the GST-Nht fusion protein was not solubilized when co-expressed with the FLAG-dTAF12 HFD containing fragment, and so was not available to bind to and elute from the glutathione-Sepharose (Hiller, 2004).

DEVELOPMENTAL BIOLOGY

Adult

Expression of the can TAF_II homolog is stage and tissue specific. A 3.2-kb can transcript was detected in poly(A)⁺ mRNA from adult males but not in mRNA from adult females, embryos, or adult males lacking a germ line. In contrast, the can homolog dTAF_II80 is widely expressed. The can transcript is present but reduced in size to 3.0 kb in males homozygous for the internal deletion allele can¹, confirming that the mRNA corresponds to the can gene product. The male-specific and germ-line-dependent expression of can is consistent with the can phenotype; male can mutants are male sterile but viable and female fertile (Lin, 1996; Hiller, 2001).

Within the testis, expression of can mRNA is restricted to primary spermatocytes. In wild-type Drosophila testis, male germ-line stem cells and mitotically dividing spermatogonia are localized to the testis apical tip. The germ cells make the transition to the primary spermatocyte stage just below this region. can mRNA is detected in male germ cells by in situ hybridization beginning at the onset of the primary spermatocyte period. The can signal is highest in early spermatocytes but persists at lower levels in more mature primary spermatocytes until approximately the time of the meiotic divisions. can message is not detected in mitotically dividing gonial cells or stem cells at the apical tip of the testis, or in postmeiotic germ cells and elongating spermatids. The can message is not detected in testes from flies homozygous for the can¹² insertion allele. The cell type in which can mRNA is expressed in the testis is consistent with the defects observed in flies containing can loss of function mutations. All target genes identified to date that require wild-type can function for normal levels of transcription are expressed in male germ cells beginning early in the primary spermatocyte growth and gene expression period (White-Cooper, 1998; Hiller, 2001).

It is possible that primary spermatocytes have one complex containing can protein and a second alternative complex containing dTAF_II80. In situ hybridization to wild-type testis revealed dTAF_II80 message in both primary spermatocytes and spermatogonia (Hiller, 2001).

The newly identified TAF homologs nht, sa, mia and rye are expressed either exclusively or mainly in the testis. The 1 kb nht and the 0.6 kb rye transcript were detected by Northern blot analysis in adult males but not in adult females, adult males lacking a germline, or embryos. Likewise, RT-PCR indicated expression of sa mRNA in adult males and in testes, but not in adult males lacking germline, females or embryos. For mia, Northern blots revealed a predominant 3 kb transcript in adult males but not in agametic males, females or embryos. In addition, one or two smaller transcripts expressed at much lower levels were detected in females and embryos in Northern blots probed for mia. Expression of mia in embryos was confirmed by RT-PCR and mia ESTs were identified from embryo- as well as testis-derived libraries in the BDGP EST sequence database (Hiller, 2004).

Within the testis, transcripts for nht, sa, rye and mia were detected in primary spermatocytes upon in situ hybridization of an antisense RNA probe to whole-mount testes. nht, sa, rye and mia transcripts appeared first in male germ cells at the transition from the spermatogonial stage to the primary spermatocyte growth phase. Like can mRNA, the level of nht, sa, rye and mia mRNAs detected was characteristically highest in early spermatocytes, with the signal decreasing in more mature spermatocytes. nht, sa, rye and mia transcripts were not detected by in situ hybridization in the apical tip region of the testes containing stem cells and spermatogonia (Hiller, 2004).

To investigate whether the testis TAFs are transcribed independently at the onset of the primary spermatocyte program or whether some of the testis TAFs might regulate mRNA expression of the others, mRNA expression of nht, can, mia, sa and rye was examined in spermatocytes from males mutant for nht, can, mia or sa by in situ hybridization to whole-mount testes. In all cases examined, mRNA for the testis TAFs accumulated in the various mutant spermatocytes. At times, the testis TAF transcripts appeared sharply at the boundary between spermatogonia and spermatocytes, as in wild type. However, in some cases staining for the transcript appeared gradually in spermatocytes further from the testis apical tip. No clear pattern was observed among these variations, which may be due in part to the probe or the degree to which spermatocytes are less crowded up into the testis apical third in the absence of differentiating spermatids in the mutants. Notably, for the alleles examined, can mRNA accumulated in can mutant spermatocytes. The same was true for nht, sa, and mia mRNA in nht, sa, or mia mutant spermatocytes, respectively, indicating that transcription of any particular testis TAF did not depend on wild-type function of the respective protein itself. Transcripts for can, mia, sa, nht and rye also accumulated in spermatocytes mutant for aly, which has meiotic arrest and spermatid differentiation mutant phenotypes similar to the can class testis TAFs, although transcripts appeared to accumulate gradually rather than turn on abruptly in early spermatocytes. Accumulation of aly transcripts or protein is independent of wild-type function of the testis TAFs can, mia and sa (White-Cooper, 2000; Hiller, 2004).

EFFECTS OF MUTATION

To explore the molecular mechanisms that regulate the tissue- and stage-specific transcrption program for spermatid differentiation, the can gene was positionally cloned. The can¹ mutation was mapped between flanking marked P-element inserts by recombination and further localized with respect to restriction fragment polymorphism (RFLP) markers. Southern blot analysis of transposon-induced can alleles revealed that the can¹ allele was associated with an ~200-bp deletion. The can¹² mutation had RFLPs consistent with an ~7.5-kb insertion, and the can⁸ and can⁹ mutations had rearrangements consistent with ~4.5-kb insertions. A 5.7-kb genomic DNA fragment spanning the region of the can¹, can⁸, can⁹, and can¹² rearrangements rescued both the spermatogenic arrest phenotype and the male sterility associated with can mutants when introduced into flies by P-element-mediated germ-line transformation. A transcript was identified encoded in the 5.7-kb rescue fragment by a combination of cDNA library screening, RT-PCR, and 5' and 3' RACE (Hiller, 2001).

Sequence analysis revealed that the transcript from the 5.7-kb genomic DNA fragment encoded a predicted 943-amino-acid protein with five WD40 repeat motifs clustered near the carboxyl terminus. Sequence analysis of genomic DNA derived from homozygous mutant flies identified mutations within the ORF for several can alleles, confirming the identity of the predicted protein as can. The can¹ strain had a 198-bp deletion removing one and a half of the conserved WD40 repeats. The can² and can⁶ alleles each had a point mutation that changed a conserved tryptophan to a stop codon in the fifth or third WD40 repeat, respectively. The can³ mutation changed a conserved serine to proline in the second WD40 repeat. The can⁴ allele had two missense mutations: the first changed a signature-conserved aspartic acid to asparagine in the first WD40 repeat, and the second caused a similar conserved aspartic acid to asparagine substitution in the fifth WD40 repeat. The can⁷ allele had a missense mutation changing a conserved leucine to glutamine in the first WD40 repeat. Finally, the can¹³ line contained a single base pair deletion resulting in a frame shift and subsequent premature stop codon. The strong loss of function phenotype associated with the single amino acid substitutions in the can³, can⁴, and can⁷ missense alleles indicates that the WD40 repeat motifs are stringently required for can function. can³, can⁴, and can⁷ all cause the same male sterile phenotype as the null alleles (Hiller, 2001).

Wild-type function of can is required for normal accumulation of transcripts encoded by a suite of spermatid differentiation genes (White-Cooper, 1998). Early characterizations of TAF_II function using in vitro transcription assays indicate that TBP supports basal transcription, whereas TBP with TAF_IIs supports activated transcription in the presence of transcriptional activators. The level of expression of can target genes in can mutant testes is reminiscent of this quantitative effect. Null mutations in can significantly reduce but do not completely eliminate transcription of target gene messages relative to wild type. Nevertheless, in each case, a low level of transcript is detected in the mutants. Since each of these target genes is expressed for the first time during development in primary spermatocytes, the residual message is not likely to be due to transcripts expressed at earlier stages (Hiller, 2001).

The effects of can mutations on transcript levels in primary spermatocytes are gene specific. The twine cell cycle regulatory phosphatase is normally transcribed in primary spermatocytes, starting at the onset of the primary spermatocyte growth and gene expression stage. Expression of twine mRNA is not can dependent in primary spermatocytes, since similar levels of the twine message were detected in can mutant and wild-type male reproductive tracts (Hiller, 2001).

To explore whether wild-type can influences the levels of target gene expression by regulating transcription or message stability, the effect of can mutations on expression of a promoter lacZ fusion construct was assayed in vivo. Sequences from ~53 to +50 of the ß2t promoter fused to lacZ and introduced into flies allowed lacZ reporter mRNA expression in primary spermatocytes. Expression of the lacZ mRNA in primary spermatocytes is greatly reduced in can homozygotes compared to their can/+ siblings, indicating that can is likely to regulate expression of this reporter construct at the level of transcription (Hiller, 2001).

The nuclear arrest phenotype of meiotic arrest mutants

Wild-type function of four Drosophila genes, spermatocyte arrest, cannonball, always early and meiosis I arrest, is required both for cell-cycle progression through the G2/M transition of meiosis I in males and for onset of spermatid differentiation. In males mutant for any one of these meiotic arrest genes, mature primary spermatocytes with partially condensed chromosomes accumulate and postmeiotic cells are lacking. The arrest in cell-cycle progression occurs prior to degradation of Cyclin A protein. The block in spermatogenesis in these mutants is not simply a secondary consequence of meiotic cell-cycle arrest, since spermatid differentiation proceeds in males mutant for the cell cycle activating phosphatase twine. Instead, the arrest of both meiosis and spermiogenesis suggests a control point that may serve to coordinate the male meiotic cell cycle with the spermatid differentiation program. The phenotype of the Drosophila meiotic arrest mutants is strikingly similar to the histopathological features of meiosis I maturation arrest infertility in human males, suggesting that the control point may be conserved from flies to man (Lin, 1996).

To pinpoint the mutant arrest point, a time line of the cytological events of the wild-type G2/M transition of meiosis I was established, based on a series of still images of unfixed cells. At the end of the post-S growth phase, wild-type mature primary spermatocytes go through a G2/M cell-cycle transition, during which a number of morphological events mark onset of the first meiotic division. Mature primary spermatocytes in unfixed squashed preparations have large, blocky nuclei and prominent nucleoli. The bivalents are already paired and appear as three discrete structures next to the nuclear membrane in living cells viewed by fluorescence microscopy. At the initiation of the G2/M transition of meiosis I, the nuclei became round, the nucleoli gradually became pale and smaller. Additionally, an organized, aster-like array of cytoplasmic components became visible at one side of each nucleus and the bivalents begin to condense. The two autosomal bivalents often undergo condensation at different rates, resulting in one oblong and one rounded bivalent in the same nucleus. The sex bivalent, which is associated with the nucleolus, stains less brightly and appears punctate at this stage when visualized by Hoechst staining. At the next landmark stage, the nucleoli have broken down completely, a characteristic refractile body and array of small particles appear in the nucleus, and two distinct astral arrays of cytoplasmic components marking the meiotic spindle poles can be seen separating to opposite sides of the nucleus. The bivalents continue condensation and often begin to move inward away from the nuclear membrane, and the sex bivalent again becomes brightly stained at this stage. By prometaphase, the two asters are at opposite poles and bivalents are fully condensed and have moved inward from the nuclear membrane The nucleus then becomes oval shaped and the astral membranes and mitochondria assume the characteristic distribution around its perimeter. The bivalents often briefly align at a metaphase plate before the onset of anaphase (Lin, 1996).

The wild-type stages from the onset of G2/M transition to metaphase are relatively rare, suggesting that these phases of cell cycle proceed rapidly. In careful examination of 49 testes, an average of 0.9 cysts for 16 cells were observed at the G2/M transition of meiosis I, as compared to an estimated total of 150 cysts per wild-type testis. Among the cysts of spermatocytes at the transition phase that were examined by Hoechst stain of live squashes: 38% were at stage B, 27% were at stage C, 21% were in prometaphase and 14% were at metaphase. Wild-type spermatocytes within the same cyst often spanned more than one stage in the G2/M transition due to a wave of developmental asynchrony across the cyst. This developmental asynchrony helped in the ordering of the stages. For example, within a single cyst, the nucleoli might be completely broken down (stage C) in some cells while other cells in the same cyst still retain pale nucleoli and less condensed bivalents (stage B) (Lin, 1996).

aly, can, mia or sa spermatocytes arrest in the G2/M transition of meiosis I with partial chromosome condensation. In aly, can, mia or sa males, the progression of spermatogenesis appears to be blocked during the G2/M transition of meiosis I, so that what is a transient and rare stage in wild type becomes the predominant cell type in the mutant testis. The effect of each of the mutations is similar. In testes from males mutant for aly, can, mia or sa, the early stages of spermatogenesis appeared normal. However, large numbers of spermatocytes at the early stage of the G2/M transition accumulate (average of 206 cells per testis, n=40 testes). Spermatocytes in the mutants arrested prior to full chromosome condensation, spindle pole organization and nucleolar breakdown. Typically, the arrested cells had rounded nuclei and partially condensed chromosomes as in wild-type stage B. The partially condensed chromosomes in the mutants were typically round, but usually remained next to the nuclear membrane and never became fully condensed. The nucleolus remained dark and prominent in the mutant cells, resembling the nucleolus of wild-type primary spermatocytes at stage A. Especially in can, arrested spermatocyte nuclei often contain an array of small particles resembling those in wild-type stage C. The sex bivalent is usually pale in Hoechst, with several bright punctate dots as in stage B of wild type. Immunofluorescence staining with antibodies against tubulin shows that accumulating spermatocytes in the mutants do not have organized spindle asters. In contrast, in wild-type spermatocytes with chromosomes at the same degree of condensation, the nucleoli have already initiated breakdown and cytoplasmic astral arrays of microtubules are visible. Thus, in the mutants, the cytoplasm and the nucleolus of the accumulated spermatocytes appear to arrest at the mature primary spermatocyte stage, while the chromosomes appear to initiate condensation. The mutant spermatocytes fail to progress to subsequent stages of meiosis. Instead, they accumulate and then eventually degenerate at the base of the testis (Lin, 1996).

The appearance of the chromosomes in the arrested aly spermatocytes was subtly different from the chromosomes in the arrested can, mia and sa spermatocytes, which resembled wildtype stage B chromosomes. For several different aly allelic combinations examined, the chromosomes appeared less well condensed and their morphology less discrete than in can, mia, sa or wild type, suggesting that aly mutations might affect events of chromosome condensation as well as progression of spermatogenesis (Lin, 1996).

To test whether the arrested cells that accumulate in aly testes are still functional intermediates in spermatogenesis, advantage was taken of the temperature sensitivity of the aly¹ allele. Testes from aly¹/aly¹ or aly¹/Df males raised at 27°C showed the phenotype described above. In aly¹ males raised at 18°C, however, spermatocytes completed meiosis and underwent abnormal spermatid differentiation. When males raised at 27°C were shifted to 18°C, the G2/M spermatocytes that had accumulated at the non-permissive temperature did not synchronously proceed through meiosis. No cells undergoing meiotic divisions or spermatid differentiation were observed in aly¹/aly¹ or aly¹/Df mutant testes 48 hours after the temperature shift. At 75 and 96 hours after the shift, at most one cyst of early spermatid cells per testis was observed. Most of these early spermatid cells that progressed beyond the arrest point appeared abnormal. The delayed timing and small number suggest that the spermatids were most likely derived from spermatocytes that had not yet reached the arrest point at the time of the temperature shift. Later spermatid stages with elongated flagella were never observed in mutant testes even up to 168 hours (7 days) after the temperature shift. Thus, the cells that accumulate in aly¹ testes at non-permissive temperature do not appear to be normal, functional intermediates in spermatogenesis. This could be directly due to the aly lesion. Alternatively, the arrested cells may become incompetent to proceed through meiosis due to turnover of message stores during the arrested state. In wild type, transcription is largely shut down by the time spermatocytes enter the first meiotic division (Lin, 1996).

The subcellular localization of Cyclin A protein changes during the meiotic cell cycle. In wild-type testes, cyclin A protein is located largely in the cytoplasm in spermatocytes from early stages until the onset of the G2/M transition, prior to chromosome condensation (Lin, 1996).

Much of the Cyclin A protein moves into the nucleus early in the G2/M transition as the chromosomes undergo condensation and remain there through prometaphase. Cyclin A protein is abruptly degraded just prior to metaphase I and is not detected in subsequent stages of meiosis. In wild type, fewer than one cyst per testis (7 spermatocyte cysts in 19 testes) had cyclin A in the nucleus. The rapid disappearance of Cyclin A protein between prometaphase and metaphase provides a biochemical indicator of cell-cycle progression (Lin, 1996).

The behavior of cyclin A protein in the mutant testes supports the conclusion that spermatocytes arrest cell-cycle progression at the G2/M transition of meiosis I. In aly, can, mia or sa mutants, Cyclin A protein is mainly cytoplasmic in growing primary spermatocytes, as in wild type. However, Cyclin A protein persists in the arrested mutant spermatocytes until the cells themselves degenerate. The persistence of Cyclin A protein in the meiotic arrest mutants is consistent with cell-cycle arrest prior to metaphase I and the activation of the machinery for cell-cycle-dependent destruction of Cyclin A protein (Lin, 1996).

Although aly, can, mia and sa mutants block meiotic cell-cycle progression, the phenotypes of meiotic arrest mutants are strikingly different from the phenotype of mutants in the meiosis specific cdc25 homologue twine. Male germ cells in twine mutants skipped certain aspects of meiosis, but nevertheless proceeded through the spermatid differentiation program. In twine primary spermatocytes entering the first meiotic division, bivalents were observed with partially condensed chromosomes similar to those found in the wild-type spermatocytes at stage B of the G2/M transition. However, condensed chromosomes were not fully detected in twine males. Chromosome segregation and cytokinesis fails to occur and meiotic spindles do not form in the twine spermatocytes. In contrast to aly, can, mia and sa mutants, spermatocytes in twine males do not arrest in meiosis (average of 2.3 meiotic cysts per testis, n=20 testes). Nucleolar breakdown occurs and the cells proceed to the round spermatid stage and mitochondrial aggregates formed in twine males. Eventually, based on Hoechst staining, the bivalents decondense and assume a thin crescent shape closely apposed to the nuclear membrane (Lin, 1996).

In twine males, Cyclin A protein becomes nuclear at a similar stage as in wild type. However, degradation of Cyclin A protein is delayed in twine males compared to wild type. Germ cells at the round spermatid stage commonly had nuclear Cyclin A protein in twine males. In 14 homozygous twine testes examined, Cyclin A protein was nuclear in three spermatocyte and 70 spermatid cysts. In contrast, Cyclin A protein was never observed in spermatids in wild type. Cyclin A protein eventually is degraded in twine males, although the disappearance appears gradual rather than abrupt as in wild type. Occasionally cysts of 16 twine spermatids were observed with very low levels of residual nuclear Cyclin A protein. Also twine spermatid cysts were occasionally observed where some cells had nuclear Cyclin A protein while others did not (Lin, 1996).

Comparison of the phenotypes suggests that the meiotic arrest mutants arrest spermatogenesis at a more global point than twine. Consistent with this hypothesis, the phenotype of twine; sa¹ and twine; can³ double mutants is similar to the sa¹/Df and can³/can⁴ phenotype. twine; sa¹ and twine; can³ spermatocytes arrest at the G2/M transition stage, with partially condensed chromosomes and intact nucleoli. Cells do not progress beyond this arrest point (average of 346 cells per testis, n=15 testes) and the testes were devoid of postmeiotic spermatid stages (Lin, 1996).

Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila

The aly, can, mia and sa genes of Drosophila are essential in males both for the G2-meiosis I transition and for onset of spermatid differentiation. Function of all four genes is required for transcription in primary spermatocytes of a suite of spermatid differentiation genes. aly is also required for transcription of the cell cycle control genes cyclin B and twine in primary spermatocytes. In contrast can, mia and sa are required for accumulation of Twine protein but not twine transcript. It is proposed that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. It is also proposed that control of translation or protein stability regulates entry into the first meiotic division. It is suggested that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of twine protein, thus coordinating meiotic division with onset of spermatid differentiation (White-Cooper, 1998).

aly, can, mia and sa are required for accumulation of Twine protein but not twine transcript in late primary spermatocytes. These three meiotic arrest genes are required for the expression of fuzzy onions, whose product is required for mitochondrial fusion in early spermatids. Similarly, severe reductions in message level are observed for Male-specific RNA 87F (Mst87F), a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. gonadal, which is expressed as two differentially terminated variants in the testis, shows dramatic reduction of both variants in can, mia and sa mutant testis. It is proposed that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. It is also proposed that control of translation or protein stability regulates entry into the first meiotic division. It is suggested that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of Twine protein, thus coordinating meiotic division with onset of spermatid differentiation (White-Cooper, 1998).

aly, can, mia and sa are required for the transcription in primary spermatocytes of several genes involved in postmeiotic spermatid differentiation. The fuzzy onions (fzo) gene product is required for mitochondrial fusion in early haploid spermatids. fzo transcription initiates in early primary spermatocytes and the mRNA is present throughout the growing stages in wild type. fzo mRNA is greatly reduced in aly, can, mia and sa testes, despite the presence of primary spermatocytes in the mutant tissue. Message levels in mutant testes ranged from undetectable to low levels under conditions in which the in situ hybridization signal in wild type was strong, indicating that transcription may be reduced to a low basal level, but not entirely turned off. Similarly severe reductions in message level were observed for Mst87F, a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. Several other genes also showed dramatic reductions in transcript levels in meiotic arrest mutant testes when assayed by in situ hybridization. Reduced transcript levels in aly, can, mia and sa spermatocytes are not due to a general defect in transcription since a number of genes were transcribed at normal levels in mutant spermatocytes (White-Cooper, 1998).

Comparison of the effects of aly, can, mia and sa mutations on transcript levels suggests that genes normally transcribed in primary spermatocytes can be grouped into three classes. The transcription of the first (general) class of genes is independent of aly, can, mia and sa function. The second (meiotic) class of genes requires the normal function of aly, but not can, mia or sa. Expression of the third (spermiogenic) class of genes requires the wild-type activity of all four of the meiotic arrest genes (White-Cooper, 1998).

Although mutations in aly, can, mia and sa appear to cause arrest at the same point in the G2-M transition of meiosis I (Lin, 1996), the genes apparently control cell cycle progression by different biochemical mechanisms. aly, but not can, mia or sa, is required for the transcription of cyclin B and twine. The wild-type function of can, mia and sa instead appears to be required either to allow translation of twine message or to stabilize twine protein in mature primary spermatocytes. In either case aly, can, mia or sa mutations presumably cause cell cycle arrest at the same point in the G2-M transition, due to lack of active Cdc2/Cyclin B kinase complex. Cdc2 protein resolves into two distinct isoforms in Western blots. The slower migrating form, which is enriched compared to the faster migrating form in twine mutant testes, has been identified as a hyperphosphorylated, inactive form. The slower migrating form of Cdc2 also appears to be enriched compared to the faster migrating form in aly, can and sa. Production of Twine protein, but not Cyclin B, is dependent on can, mia and sa. Thus, although both Cyclin B and Twine protein accumulation are regulated posttranscriptionally in wild-type testes, the genetic control of their expression is different (White-Cooper, 1998).

It is proposed that can, mia, and sa act together or in a pathway to activate a tissue and stage-specific transcription program in primary spermatocytes, and that failure to initiate this program results in a global block in spermatid differentiation due to the lack of an array of gene products. The wild-type functions of can, mia and sa appear to be required for transcription in primary spermatocytes of a set of genes encoding products involved in post-meiotic spermatid differentiation. Transcription of these genes is initiated early in the primary spermatocyte stage, several days before the arrest point of the meiotic arrest mutants. Therefore the lack of transcription of this set of genes is likely to be a cause of the arrest rather than merely a downstream consequence (White-Cooper, 1998).

Of the eight genes identified so far that depend on can, mia and sa for transcription, some information about the function or time of action of the gene products is available for six. The product of the fzo gene is required for mitochondrial fusion, a post-meiotic event. Although fzo is transcribed in primary spermatocytes, the protein is not detected by immunofluorescence staining of testes until late in meiosis II. Expression of Mst87F, of four related genes at 84D and two related genes at 98C is regulated translationally. Although mRNAs are transcribed in primary spermatocytes, the proteins do not accumulate until days after the meiotic divisions. All of these genes encode proteins that are components of a structure in the sperm tail. Similarly the translation of janB and dj mRNAs is delayed until several days after the completion of meiosis. While the function of LanB is unknown, Dj is thought to serve a dual function; it is found in the sperm tail, but sequence comparisons suggest a possible role as a chromatin component (White-Cooper, 1998).

It is proposed that aly acts upstream of can, mia and sa, possibly to control expression or activation of components of the transcription machinery that drives expression of the spermatid differentiation genes. Wild-type function of aly is required for accumulation of at least three different mRNAs in primary spermatocytes that are not dependent on can, mia and sa, suggesting that aly is able to act independently of can, mia and sa. However aly mutations cause the same phenotype, and fail to express the same set of spermatid differentiation genes, as can, mia and sa mutations. This strongly suggests that aly might affect spermatid differentiation through an effect on expression or activity of either can, mia or sa. The block in meiotic cell cycle progression in can, mia and sa mutant testes could be due to a cross-regulatory mechanism that serves to coordinate meiosis and the spermatid differentiation program. It is proposed that a gene or genes transcribed in primary spermatocytes under the control of can, mia and sa encode(s) product(s) required either directly or indirectly to relieve the translational repression of twine message or to stabilise the Twine protein. Such a cross-regulatory mechanism between the pathways leading to spermatid differentiation and meiosis could serve in wild type to ensure that spermatocytes do not enter meiotic division until the proposed transcription program for post-meiotic spermatid differentiation genes has been successfully initiated. A late cross-regulatory mechanism may also explain why mutations that block spermatid differentiation but not meiotic cell cycle progression have not yet been isolated (White-Cooper, 1998).

The signal that activates the G2/M transition in male meiosis could be accumulation of the product of the proposed crossregulatory gene to a threshold sufficient to allow expression of twine protein. Alternatively, timing of the G2/M transition for meiosis I could be set via a less direct mechanism, involving the proposed cross regulatory gene, but not set directly by its level. For example accumulation of Twine protein may require an extrinsic signal received or transduced by a gene or genes controlled by the can, mia and sa transcription program. The degenerative spermatocyte (des) gene, encoding a novel protein that may be membrane associated, is a possible candidate for a component of such a signalling pathway (White-Cooper, 1998).

Mutations in des, like aly, can, mia and sa, cause a block in both meiotic cell cycle progression and the onset of spermatid differentiation. des mutations are also semi-lethal, suggesting a role for this gene outside the testis. Pole cell transplantation experiments also implicate extracellular signals in the regulation of meiotic progression and spermatid differentiation. Male (XY) germ cells transplanted into a female (XX) host initiate spermatogenesis in the host ovary. However the transplanted cells arrest as primary spermatocytes and fail to undergo the meiotic divisions or initiate spermatid differentiation. Part of the program of spermatid differentiation regulated by can, mia and sa could act to destabilize or turn off transcription of certain messages expressed in primary spermatocytes but not needed or deleterious after meiosis. In wild-type testes, cyclin A mRNA is present in primary spermatocytes but not detectable in post-meiotic cells. Loss of cyclin A mRNA could be an important mechanism to prevent DNA replication during meiosis II or in haploid spermatids. In wild-type testes Cyclin A protein is degraded at metaphase I and is not resynthesised for the second meiotic division. In males mutant for aly, can, mia or sa, cyclin A message and Cyclin A protein (Lin, 1996) persist in the arrested primary spermatocytes, suggesting that the wild-type function of the meiotic arrest genes and/or the transcription program they control is required directly or indirectly for disappearance of cyclin A message midway through spermatogenesis. A similar effect on message stability was seen for all of the other pre-meiotic genes tested (White-Cooper, 1998).

Yeast meiosis bears striking similarities to Drosophila spermatogenesis. In both cases S phase is followed by an extended G2 phase, characterized by high levels of transcription of genes required for meiosis and subsequent differentiation into spores or sperm. Many yeast mutants, including certain alleles of cdc2 in S. pombe, are analogous to twine, in that the mutant cells fail to complete one or both of the meiotic divisions, but still differentiate into spores. However meiosis and differentiation are coordinated, since mutations in some genes, mei4 in S. pombe or NDT80 in S. cerevisiae, like the meiotic arrest mutants of Drosophila, block both the meiotic division cycle and subsequent differentiation. The failure to accumulate both cell cycle and spermiogenesis mRNAs in aly mutants suggests that there may be parallels in the genetic control of animal spermatogenesis and yeast sporulation (White-Cooper, 1998 amd references therein).

The testis TAF homologs mediate a common gene expression program in spermatocytes

To assess the role of the TAF homologs in vivo, null mutant alleles were identified. Two nht mutants were identified by screening a large collection of male sterile lines for mutants with a meiotic arrest phenotype similar to can. The mutation causing the nht meiotic arrest phenotype was localized to polytene region 35C by recombination mapping and deficiency complementation. Since this region contained CG15259, the dTAF4 homolog, genomic DNA amplified from CG15259 was sequenced by PCR. Both nht alleles carried base changes from the background chromosome that caused premature stop codons in the predicted CG15259 open reading frame. A transgene with a 10.5 kb fragment of genomic DNA containing CG15259 fully rescued the spermatocyte arrest phenotype and male sterility of nht^z-5347/Df. Together these results establish that nht encodes the TAF4 homolog (Hiller, 2004).

A second allele of mia was identified in the same phenotypic screen. mia had previously been mapped to either polytene interval 78C9-81F or 83A-C2 (Lin, 1996). Since 83A-C2 contains the dTAF6 homolog CG10390, genomic DNA corresponding CG10390 were sequenced from the two mia alleles. mia¹ and mia^z-3348 had base changes resulting in stop condons, at aa297 and aa55, respectively. A 6.1 kb fragment of genomic DNA containing CG10390 rescued the male sterility and spermatogenesis phenotype of mia mutants (Hiller, 2004).

The sa locus was previously mapped to polytene chromosome interval 78C9-D2 (Lin, 1996). Additional local deletions made by mobilization of the EP(3)0339 P-element insert further localized sa proximal to EP(3)0339. The sa gene was found to lie in a large intron of CG6014, transcribed from the opposite strand. Sequencing of genomic DNA amplified from the two sa alleles by PCR reveled a 414 bp deletion and 158 bp insertion in the predicted protein coding region in sa¹, which had been recovered from the wild as VO45 by D. Lindsley in the Rome screen (Sandler, 1968) and a C to T transition resulting in a premature stop codon in the EMS-induced sa² compared to its background chromosome. A 3.2 kb genomic DNA fragment containing CG11308 fully rescued the spermatocyte arrest and male sterility phenotypes of sa¹/sa² males when introduced into flies by P-element-mediated germline transformation, establishing that sa corresponds to CG11308 (Hiller, 2004).

Mutations in nht, mia or sa caused the same meiotic arrest phenotype as loss of function of the testis-specific dTAF5 homolog can. Testes from nht males contained cells at the earliest stages of spermatogenesis up through primary spermatocytes, but germ cells failed to initiate meiotic cell division and the testes filled with mature primary spermatocytes. No stages of spermatid differentiation were detected in nht testes and mature spermatocytes eventually degenerated at the testis base. Loss of function of mia or sa caused a similar meiotic arrest phenotype, with mature spermatocytes arrested at the G2/M transition of meiosis I and lack of spermatid differentiation (Lin, 1996). Strikingly, although mia mRNA expression was not specific to the adult male germline, mia function appeared to be required mainly for spermatogenesis: mia¹/mia^z-3348 flies were viable and female fertile (Hiller, 2004).

Like can, loss of function of nht, mia or sa caused defects in expression of spermatid differentiation genes in primary spermatocytes. Transcripts from the can target genes fuzzy onions (fzo) and don juan (dj) were expressed at much lower levels in spermatocytes in nht mutant testes compared to wild type, based on in situ hybridization using RNA probes. Similar results were obtained for other target genes that also require can for normal levels of expression in primary spermatocytes, including janB and mst35B. As observed for can, although the level of target gene transcripts was significantly reduced in nht mutant testes, low levels of target gene messages were still detected by Northern blot analysis of poly(A)⁺ testis RNA. Also as observed for can, wild-type function of nht, mia or sa was not required for expression of all spermatocyte transcripts: mRNAs for the meiotic cell cycle regulators cyclin B and twine (a cdc25 homolog) were expressed normally in nht mutant testes, based on both in situ hybridization and Northern blots. Loss of function of mia or sa had previously been shown to have similar effects on the expression of the same genes as can (White-Cooper, 2000). With respect both to levels of target transcripts and effects on expression of twine and cyclin B transcripts in primary spermatocytes, nht was clearly like can, mia and sa rather than like the aly class meiotic arrest genes aly, comr, topi and achi/vis (Hiller, 2004).

Tissue-specific TAFs counteract Polycomb to turn on terminal differentiation

Polycomb transcriptional silencing machinery is implicated in the maintenance of precursor fates, but how this repression is reversed to allow cell differentiation is unknown. Testis-specific TAF (TBP-associated factor) homologs required for terminal differentiation of male germ cells may activate target gene expression in part by counteracting repression by Polycomb. Chromatin immunoprecipitation revealed that testis TAFs bind to target promoters, reduce Polycomb binding, and promote local accumulation of H3K4me3, a mark of Trithorax action. Testis TAFs also promoted relocalization of Polycomb Repression Complex 1 components to the nucleolus in spermatocytes, implicating subnuclear architecture in the regulation of terminal differentiation (Chen, 2005).

Male germ cells differentiate from adult stem cell precursors, first proliferating as spermatogonia, then converting to spermatocytes, which initiate a dramatic, cell type–specific transcription program. In Drosophila, five testis-specific TAF homologs (tTAFs) encoded by the can, sa, mia, nht, and rye genes are required for meiotic cell cycle progression and normal levels of expression in spermatocytes of target genes involved in postmeiotic spermatid differentiation. Requirement for the tTAFs is gene selective: Many genes are transcribed normally in tTAF mutant spermatocytes. Tissue-specific TAFs have also been implicated in gametogenesis and differentiation of specific cell types in mammals. In addition to action with TBP (TATA box–binding protein) in TFIID, certain TAFs associate with HAT (histone acetyltransferase) or Polycomb group (PcG) transcriptional regulatory complexes. To elucidate how tissue-specific TAFs can regulate gene-selective transcription programs during development, the mechanism of action of the Drosophila tTAFs was investigated in vivo (Chen, 2005).

The tTAF proteins were concentrated in a particular subcompartment of the nucleolus in primary spermatocytes. Expression of a functional green fluorescence protein (GFP)–tagged genomic sa rescuing transgene revealed that expression of Sa-GFP turns on specifically in male germ cells soon after initiation of spermatocyte differentiation and persists throughout the remainder of the primary spermatocyte stage, disappearing as cells entered the first meiotic division. Some Sa-GFP was detected associated with condensing chromatin. However, most Sa-GFP localized to the nucleolus, in a pattern complementary with Fibrillarin, which marks a fibrillar nucleolar subcompartment. Staining with antibodies against endogenous Sa, Can, Nht, or Mia proteins showed similar temporal expression and nucleolar localization in primary spermatocytes, consistent with collaborative function of the tTAFs. In contrast, the generally expressed sa homolog TAF8 and its binding partner TAF10b are excluded from the nucleolus (Chen, 2005).

Several components of the Polycomb Repression Complex 1 (PRC1) transcriptional regulator appear in the nucleolus in spermatocytes, coincident with tTAF expression and dependent on tTAF function. Polycomb (Pc) protein expresses from a Pc-GFP genomic transgene localized on chromatin, but in addition becomes concentrated in the nucleolus in primary spermatocytes. Both Pc-GFP and staining of endogenous protein with antibody against Pc (anti-Pc) revealed localization to the same nucleolar subcompartment as the one containing tTAFs. Recruitment of Pc to the nucleolus exactly coincides with onset of expression of the tTAFs after early G₂ phase in spermatocytes. Relocalization of Pc depends on wild-type tTAF activity: Pc localizes to chromatin but is not concentrated in the nucleolus in tTAF mutant spermatocytes. Two other components of the PRC1 core complex, Polyhomeotic (Ph) and Drosophila Ring protein (dRing), also become concentrated in the nucleolus in primary spermatocytes dependent on tTAF function. Failure of PRC1 components to localize to the nucleolus in tTAF mutants is not caused by nucleolar loss because Fibrillarin staining appears normal in the mutants. H3K27me3 laid down by action of the PRC2 complex acts as a docking site for the Pc chromodomain to recruit PRC1 and block transcription initiation. H3K27me3 localizes on chromatin in spermatocytes, along with Pc. However, no H3K27me3 was detected in the nucleolus in spermatocytes, suggesting that PRC1 components may be recruited to the nucleolus by a different mechanism independent of chromatin (Chen, 2005).

The tTAFs are required for activation of robust transcription of several spermatid differentiation genes, whereas the PcG proteins are known to mediate transcriptional repression. Chromatin immunoprecipitation (ChIP) suggested that the tTAFs might allow robust transcription of spermatid differentiation genes in part by counteracting repression by Pc, perhaps causing dissociation of PRC1 from cis-acting control sequences at target genes (Chen, 2005).

ChIP from wild-type testes using anti-Sa revealed enrichment of tTAF binding at three different known target genes (fzo, Mst87F, and dj), compared with binding at intergenic regions 10 to 20 kb away or at a tTAF-independent gene expressed in the same cell type (cyclin A or sa itself), suggesting that the tTAFs are in occupancy at target genes. Real-time polymerase chain reaction (PCR) analysis revealed ~10-fold enrichment of Sa at a target (mst87F) compared with a non-target gene (sa) (Chen, 2005).

ChIP analysis also revealed that Pc protein binds to tTAF-dependent target genes in tTAF mutant testes, and that wild-type function of the tTAFs reduce Pc binding. ChIP with anti-Pc from can mutant testes preferentially precipitates the three tTAF target promoters, compared with intergenic regions or promoters from two different nontarget controls. Quantification by real-time PCR showed more than 50-fold enrichment of Pc at the target gene mst87F compared with the tTAF-independent control sa. In contrast, relative occupancy of Pc at the tTAF targets was not significantly different from that at the non-targets in wild-type testes (Chen, 2005).

The tTAFs may act near the promoter of target genes (fzo) to allow expression by directly or indirectly reducing nearby binding of PRC1. ChIP using primer pairs across the promoter region of fzo revealed that the tTAF enrich most strongly for sequences just upstream of the transcription start site. In contrast, Pc-containing protein complexes (in tTAF mutant testes) enrich for a broader distribution, including sequences near and downstream of the transcription start site, consistent with localization of Pc at Ultrabithorax (Ubx) locus in wing discs and on the hsp26 promoter in vivo (Chen, 2005).

Binding of the tTAFs at target promoters may allow expression through recruitment or activation of the Trithorax group (TrxG) transcriptional activation complex, which often acts in opposition to repression by PcG proteins. Trx, like its mammalian homolog MLL, creates an H3K4me3 epigenetic mark. ChIP from wild-type testes revealed H3K4me3 at or near the promoter regions of the three tTAF targets tested, as well as at nontargets. Analysis using primer pairs across the tTAF target fzo region revealed that H3K4me3 associated most strongly with sequences spanning the promoter. In contrast, ChIP with anti-H3K4me3 from can mutant testes did not enrich for the tTAF target promoters. Quantitative PCR revealed 36-fold enrichment of the promoter region of the tTAF-dependent mst87F gene by ChIP for H3K4me3 in wild-type compared with can mutant testes (Chen, 2005).

Consistent with the presence of H3K4me3 at target promoters in wild-type testes, trx function appears to be required for continued expression of two different kinds of tTAF-dependent targets. Boule triggers the G₂/M transition in meiosis I by allowing translation of twine and requires tTAFs for protein accumulation, setting up a cross-regulatory mechanism so that meiotic cell cycle progression awaits expression of terminal differentiation genes. When temperature-sensitive trx¹ flies grown at permissive temperature were shifted to nonpermissive temperature as adults, the Boule protein level in mutant testes substantially decreased over time at nonpermissive temperature compared with the level in wild-type flies shifted in parallel or trx¹ flies held at permissive temperature. Likewise, analysis of mRNA levels by semiquantitative PCR revealed a ~40% decrease in transcript level for the tTAF target gene fzo, but not for the tTAF-independent gene cyclin A, in testes from trx¹ mutant flies shifted to non-permissive temperature compared with the level in testes from similarly treated wild-type flies (Chen, 2005).

In summary, occupancy of tTAFs and Pc at target promoters appears to be mutually exclusive in wild-type and tTAF mutant spermatocytes, suggesting that the tTAFs may turn on target gene expression by counteracting repression by Polycomb, either directly or indirectly reducing Pc binding and allowing local action of Trx. Loss of function of Pc in marked clones of homozygous mutant cells does not restore terminal differentiation in a tTAF mutant background, suggesting that in addition to counteracting repression by Pc, tTAFs may also be required at the promoter region independent of Pc, possibly to recruit Trx or other cofactors for transcription activation. Transcriptional derepression by sequestration of PcG proteins has been observed during HIV-1 infection, when the viral Nef protein recruits the PRC2 component Eed to the plasma membrane. Likewise, the tTAFs may sequester Pc to the nucleolus. The tTAFs Nht, Can, and Mia are homologs of the generally expressed TAF4, TAF5, and TAF6, which are found as stoichiometric components of the PRC1 complex purified from fly embryos, raising the possibility that the tTAFs might associate with a population of Pc-, Ph-, and dRing-containing complexes in the nucleolus. If so, interactions in the nucleolus are likely to differ from interactions at the promoters of target genes, because the ChIP results indicate immunoprecipitation of tTAFs without Pc (Chen, 2005).

The PcG and TrxG proteins act to maintain cell fates set during embryogenesis throughout development. Emerging evidence indicates that PcG and TrxG complexes also play critical roles in decisions between proliferating precursor cell fates and terminal differentiation, for example, in the blood cell lineages. In particular, the mammalian PcG protein Bmi-1 promotes proliferation and blocks differentiation of normal and leukemic stem cells, and is required for establishment or maintenance of adult hematopoietic stem cells in mouse. Transcriptional silencing by PcG action may allow self-renewal and continued proliferation of precursor cells by blocking expression of terminal differentiation genes. This repression must be reversed to allow production of terminally differentiated cells, whereas failure may allow overproliferation of precursors and eventually cancer. Although central for both normal development and understanding the genesis of cancer, little is known about the mechanisms that reverse such epigenetic silencing to allow expression of the terminal differentiation program. These findings in the male germ line provide an example of how cell type– and stage–specific transcriptional regulatory machinery, turned on as part of the developmental program, might allow onset of terminal differentiation by counteracting repression by the PcG and highlight the importance of subnuclear localization in regulation of transcriptional regulation (Chen, 2005).

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date revised: 15 November 2005

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