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).

GENE STRUCTURE

cDNA clone length - 2955

Bases in 5' UTR - 46

Exons - 5

Bases in 3' UTR - 80

PROTEIN STRUCTURE

Amino Acids - 942

Structural Domains

The cannonball gene encodes a homolog of dTAF_II80; a protein identified biochemically from Drosophila embryo extracts as a component of the general transcription factor TFIID (Dynlach, 1993; Kokubo, 1993). The predicted can protein has four distinct regions of significant homology to Drosophila dTAF_II80 and its human (hTAF_II100) and yeast (Saccharomyces cerevisiae; yTAF_II90 and Schizosaccharomyces pombe; pTAF_II72) homologs. Interspersed between the conserved regions are variable segments where the proteins share little or no homology. Overall, the predicted can protein was more related to dTAF_II80 than to hTAF_II100, but dTAF_II80 and hTAF_II100 are more related to each other than either is to can. The predicted can protein has a carboxy-terminal extension not present in dTAF_II80 or its yeast and human homologs. Secondary structure predictions have indicated that the carboxy-terminal extension of the can protein is likely to fold into an extended alpha-helix. The predicted can protein has a high degree of sequence identity/similarity to dTAF_II80 and hTAF_II100 within each WD40 motif in addition to the GH-X_21-40-D-X₅-WD backbone of the WD40 motif. The conserved spacing of the repeats, and the presence of three other conserved domains outside of the WD40 repeat region indicate that the can predicted protein is much more related to dTAF_II80 and its homologs than to other WD40-containing proteins (Hiller, 2001).

date revised: 15 January 2005

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