InteractiveFly: GeneBrief

achintya and vismay: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Cell type-specific transcriptional programs that drive differentiation of specialized cell types are key players in development and tissue regeneration. One of the most dramatic changes in the transcription program in Drosophila occurs with the transition from proliferating spermatogonia to differentiating spermatocytes, with >3000 genes either newly expressed or expressed from new alternative promoters in spermatocytes. This study shows that opening of these promoters from their closed state in precursor cells requires function of the spermatocyte-specific tMAC complex, localized at the promoters. The spermatocyte-specific promoters lack the previously identified canonical core promoter elements except for the Inr. Instead, these promoters are enriched for the binding site for the TALE-class homeodomain transcription factors Achi/Vis and for a motif originally identified under tMAC ChIP-seq peaks. The tMAC motif resembles part of the previously identified 14-bp beta2UE1 element critical for spermatocyte-specific expression. Analysis of downstream sequences relative to transcription start site usage suggested that ACA and CNAAATT motifs at specific positions can help promote efficient transcription initiation. These results reveal how promoter-proximal sequence elements that recruit and are acted upon by cell type-specific chromatin binding complexes help establish a robust, cell type-specific transcription program for terminal differentiation (Lu, 2020).

Transcriptional regulation plays a central role in producing different cell types from the same genomic content. Throughout embryonic development, cells make and respond to cell fate decisions by turning on new transcription programs required to generate progressively more specialized cell types. Similar events drive differentiation of specialized cells from proliferating precursors in the adult stem cell lineages that maintain and repair many tissues throughout the life span. Understanding how cell type-specific transcription is achieved forms the very basis of understanding differentiation and development in multicellular organisms (Lu, 2020).

Tissue and stage-specific transcription programs are established by intricate interplay among promoter-proximal and distal DNA elements, and protein complexes that interact with them. Much recent work has focused on the role of stage or tissue-specific transcriptional activators and repressors acting upon distal enhancer elements to control the time and place of expression of developmental genes. However, evidence has emerged that variant forms of core promoter motifs and their recognition factors can play roles in cell type-specific transcription programs in certain tissues (Lu, 2020).

Several canonical core promoter motifs and promoter types have been identified and extensively studied. In Drosophila, TATA-box and/or downstream promoter element (DPE)-containing promoters tend to initiate transcription from a narrow region. The TATA box is bound by the TATA-binding protein (TBP), while the DPE is bound by certain TBP-associated factors (TAFs) in the general transcription factor TFIID to help precisely position RNA polymerase II for transcript initiation. On the other hand, promoters containing the DNA replication-related element (DRE) and/or other Ohler motifs (Ohler, 2002) tend to initiate transcription from a broad region and are thought to be associated with housekeeping genes. Recent work in Drosophila has shown that thousands of enhancers exhibit a distinct preference for one or the other of these promoter types (Zabidi, 2015; Rennie, 2018), suggesting that sequences near the transcription start site can play key roles in geneselective transcriptional regulation. However, the extent to which these canonical versus other core promoter motifs contribute to cell type-specific gene regulatory programs in differentiating cells and the molecular mechanisms by which they do so are not understood (Lu, 2020).

Male germ cell differentiation in Drosophila provides an excellent opportunity to study cell type-specific transcriptional regulation, as more than a thousand genes turn on for the first time in development when male germ cells become spermatocytes. In Drosophila, one germline stem cell normally produces a new stem cell and a gonialblast, which founds a clone of proliferating spermatogonia through four rounds of mitosis. The resulting 16 interconnected germ cells undergo a last round of DNA synthesis; then, as spermatocytes, they enter meiotic prophase. During this ~3-day period the spermatocytes express the vast majority of genes needed for later stages of male germ cell development. A recently developed heat-shock-Bam time course system (Kim, 2017) provided a way to obtain large quantities of germ cells at similar stages and greatly empowered study of the temporal events and molecular mechanisms that underlie the dramatic, cell type-specific change in transcriptional regulation that accompanies the transition from spermatogonia to spermatocyte (Lu, 2020).

Genetic and biochemical studies have identified two sets of cell type-specific proteins that regulate the spermatocyte transcription program, the tMAC complex (Beall, 2007) required for turning on most of the spermatocyte-specific gene expression program (Perezgasga, 2004; Doggett, 2011) and the testis-specific TAFs (tTAFs) required for full levels of expression of many genes in that program (Doggett, 2011; Lu, 2015). tMAC interacts physically with Achi/Vis, two highly similar TGIF-related TALE-class homeodomain proteins encoded by tandemly duplicated genes that are required for transcription in spermatocytes of tMAC-dependent genes (Ayyar, 2003; Wang, 2003). In flies null mutant for tMAC components, Achi/Vis, or tTAFs, germ cells arrest as mature spermatocytes. tMAC is a spermatocyte-specific version of the widely expressed and evolutionarily conserved MuvB core complex, which binds different protein partners, including members of the E2F, DP, Rb, and Myb protein families, to repress or activate key cell cycle and developmental genes (Sadasivam, 2013; Fischer, 2017). The tMAC complex expressed in Drosophila spermatocytes contains two proteins shared with the MuvB core: p55 Caf1 (RBBP4 in humans) and Mip40 (LIN37). tMAC also contains testis-specific paralogs of three of the other MuvB core components: Aly (paralog of Mip130 [LIN9]), Tomb (paralog of Mip120 [LIN54]), and Wuc (paralog of Lin52 [LIN52]). In addition, tMAC includes the testis-specific proteins Topi and Comr. Among the known tMAC subunits, Tomb, Topi, and Comr have predicted DNA-binding domains. Despite the importance of tMAC for turning on expression of most of the genes newly expressed in spermatocytes, the mechanism by which tMAC carries out this function is not known (Lu, 2020).

To investigate how the cell type-specific gene expression program for spermatocyte differentiation turns on, this study used RNA-seq to map transcript levels, CAGE to quantitatively map transcription start site (TSS) usage, and ATAC-seq to map chromatin accessibility as proliferating spermatogonia transition to differentiating spermatocytes. Combining these data, the promoters that turn on when germ cells become spermatocytes were shown to lack most of the canonical core promoter motifs. Instead, these promoters are enriched for the tMAC-ChIP motif and putative Achi/Vis-binding motif, and require tMAC function to become accessible and initiate active transcription as germ cells transition to the spermatocyte state. The findings from genome-wide analyses and selected reporter constructs are consistent with published results that regulatory elements sufficient for expression in spermatocytes lie close to the promoter regions in several genes expressed specifically in spermatocytes (Lu, 2020).

One of the striking findings from this study is that many genes expressed both in bam^-/- mutant testes, in which spermatogonia continue to proliferate, and in 72 hr post-heat shock testes, in which many germ cells have progressed to the spermatocyte state, use an alternate promoter that turns on only in the spermatocyte-containing sample. It is possible that the conditions for productive transcription initiation are so different in spermatocytes compared with spermatogonia that many genes evolved alternative promoters to allow transcription in both cell types. Use of promoters with distinct proximal motifs bound by cell type-specific promoter-interacting factors such as are describe in this study may contribute to down-regulation of the old program as well as to turning on a new cell type-specific differentiation program. Indeed, the DRE motif, enriched at broad promoters down-regulated when spermatogonia transition to spermatocytes, is bound by the DRE-binding factor protein DREF, which recruits the TBP paralog TRF2 to facilitate transcript initiation. Because DREF protein is expressed in spermatogonia but down-regulated as spermatocytes mature, promoters that depend on the DRE motif and DREF-TRF2 may no longer express efficiently in late spermatocyte stages (Lu, 2020).

Function of the testis-specific tMAC complex is required for the vast majority of both the off-to-on gene and alternative new spermatocyte-specific promoters to become open and accessible as spermatogonia transition to the spermatocyte state. Where a new alternative spermatocyte-specific promoter is located within the stretch of DNA that wraps around the -1 nucleosome of the old spermatogonial promoter, displacement of this -1 nucleosome is likely a prerequisite for the expression of the new promoter. Together with the requirement for tMAC function for opening and expression of the new alternative promoter, this suggests that tMAC may play a role in remodeling nucleosomes. It is not known whether tMAC opens local chromatin by binding DNA that is transiently detached from nucleosomes due to loosening or breathing of nucleosomal arrays or other chromosomal events, and then holding it in an open position, or whether tMAC can bind to DNA wrapped around nucleosomes, similar to a pioneer transcription factor. The tMAC subunit Comr has a winged helix domain, and certain winged helix domain proteins are able to bind DNA on one side and allow simultaneous histone binding. Although none of the core components of tMAC have as yet been shown to have nucleosome remodeling activity, genetic analysis in C. elegans suggests that the generally expressed MMB/dREAM components (SynMuvB genes in worms) can interact with and may recruit components of the nucleosome remodeling and histone deacetylase (NuRD) complex (Lu, 2020).

It is possible that many of the alternative new promoters arose as a by-product of an ability of tMAC to promote chromatin opening and/or transcription initiation at many sites in the genome. In fact, mechanisms have evolved to keep this propensity under restraint: Recent work has showen that action of the multiple zinc finger protein Kmg expressed in spermatocytes is required to limit activity of tMAC to its normal target genes. With loss of Kmg function, tMAC binds to many additional sites in the genome that are not previously annotated promoters, with some of these being activated for transcription initiation (Kim, 2007; Lu, 2020 and references therein).

These motif and TSS analyses suggest that the off-to-on genes that express from a narrow region of efficient transcription initiation differ from the canonical, previously described narrow promoters. Among the down-regulated genes, most of the promoters with a narrow 'region of efficient transcription initiation' also had narrow total span of cap analysis gene expression (CAGE) signals, likely a result of TFIID precisely positioning Pol II for transcription initiation. In contrast, most of the off-to-on genes with narrow regions of efficient transcript initiation had a wide total span of CAGE signals. In other words, although these off-to-on promoters had many usable and permissive TSS positions, they were dominantly expressed from just a few TSSs within a narrow region, likely facilitated by the upstream and downstream promoter motifs and Inr at optimal positions (Lu, 2020).

Traditionally, there has been much focus on the importance of distal enhancer elements rather than promoters in specifying cell type-specific transcription programs. This view, however, may be somewhat biased by the intense analysis of developmental regulatory genes like even-skipped, the cell cycle regulatory phosphatase string/cdc25, or the close-range developmental signaling molecule BMP5, which are expressed and function in several disparate places in the body. Because the regionally expressed transcriptional activators and repressors that combine to establish positional identity differ in different regions of the body, it stands to reason that a gene that is expressed in different specific places, such as Pitx1 in jaw, pituitary, and pelvis, would need to use different enhancer elements to specify activation in different regions, with the regulatory input from the different enhancers perhaps feeding in to a common generic promoter. The situation and constraints may be quite different for terminal differentiation genes that are only expressed in a single tissue. In this case, as is shown in this study for differentiation of male germ cells, it may be more possible for the key regulatory sequences that specify cell type-specific transcriptional activation to be built into the core promoter and promoter-proximal regulatory sequences (Lu, 2020).

Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis

Two tightly linked and identical homeobox genes of the TGIF (TG-interacting factor) subclass called vismay and achintya (often referred to as TGIF) are essential for spermatogenesis in Drosophila. 'achintya' is a Sanskrit word meaning 'that which is beyond thought and contemplation', and relates to initial difficulties in interpreting the mutant analysis; 'vismay' is a Hindi word meaning 'surprise', which described the reaction when the genome sequence revealed the tandem duplication (Ayyar, 2001). In flies deficient for both genes, spermatogenesis is blocked prior to any spermatid differentiation and before the first meiotic division. This suggests that vismay and achintya function at the same step as two previously characterized meiotic arrest genes, always early and cookie monster. Consistent with this idea, both always early and cookie monster are still expressed in flies deficient in vismay and achintya. Conversely, Vismay and Achintya proteins are present in always early mutant testes. Co-immunoprecipitation experiments further suggest that Vismay and Achintya proteins exist in a complex with Always early and Cookie monster proteins. Because Vismay and Achintya are likely to be sequence-specific DNA binding factors, these results suggest that they help to specify the spermatogenesis program by recruiting or stabilizing Always early and Cookie monster to specific target genes that need to be transcriptionally regulated during testes development (Wang 2003; Ayyar, 2003).

TGIF is a transcription factor of the TALE homeodomain class (Burglin, 1996) that has been implicated in a number of distinct pathways. TALE-superclass homeodomains are characterized by the presence of an additional three amino acids (Three Amino-acid Loop Extension) between helices 1 and 2. They comprise an ancient family, with representation from yeast to humans, and they act as transcription factors, often in collaboration with other homeodomain proteins. Members of the PBC and Meis classes of TALE proteins function as cofactors of Hox homeodomain proteins. TGIF was first identified as a competitor of the retinoic acid receptor for binding to retinoic acid response elements (Bertolino, 1995). Subsequently TGIF was demonstrated to interact with Smads, and a role has been proposed for it as a negative regulator of TGFß signalling based on in vitro and cell culture experiments (Wotton, 1999a). The findings that TGIF binds transcriptional repression proteins such as HDAC, mSin3A and CtBP and that TGIF can displace the CBP/p300 co-activator from Smad complexes suggest that it acts to build a repression complex on Smad target gene promoters (Wotton, 1999b; Wotton, 2001a; Wotton, 2001b). TGIF acting as a Smad co-repressor has been proposed to impose a response ceiling on transcription from TGFß response genes. TGIF has also been suggested to act as a competitive inhibitor of the TALE-class homeodomain protein Meis2 (Yang, 2000) in neuronal cell lines (Ayyar, 2003 and references therein).

Consistent with an in vivo role in TGFß/BMP signalling, TGIF has been identified as one of a small group of genes implicated in the human developmental disorder holoprosencephaly (HPE). This failure of forebrain formation is a relatively common developmental disorder affecting 1 in 250 conceptuses and 1 in 16000 live births. Loss-of-function mutations in TGFß family members in the mouse and zebrafish exhibit holoprosencephaly phenotypes. Four regions in the human genome (HPE 1-4) have been correlated with HPE and HPE 4 has been mapped to a 6 Mb region on chromosome 18 at p11.3, which includes the TGIF locus. Collections of HPE families have revealed TGIF alleles with mutations that affect protein function, which provide a plausible case for the relevance of TGIF to HPE but surprisingly these mutations do not appear to be more prevalent in the HPE group. Another potentially relevant gene, twisted gastrulation has also been recently found to be located at 18p11.3 (Ayyar, 2003 and references therein).

During spermatogenesis in Drosophila, stem cells located at the tip of the testes divide asymmetrically to give a stem cell daughter and a primary spermatogonial cell. The primary spermatogonial cells divide four times mitotically to give a cluster of 16 spermatogonia encased by two somatic cyst cells. After the fourth mitotic division, the cells undergo DNA replication and, now called primary spermatocytes, they enter a long (approximately 3.5 days) G₂ phase which is a period of extensive transcription preceding the first meiotic metaphase. During this period the cells enlarge 25-fold. Upon entry into meiosis, transcription is shut down. The spermiogenesis genes, required to build functional sperm, tend to be transcribed during the long G₂ phase and then held under translational control for later protein production after meiosis (Ayyar, 2003).

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

To place achi/vis within this scheme the expression of a set of meiosis-related genes and a selected set of spermiogenesis genes were examined in Df(2R)achi¹ homozygous mutant testes by RT-PCR analysis, and in homozygous mutant males by in situ hybridization. Both the set of spermiogenesis genes tested (fuzzy onions, janus B, don juan, gonadal) and the meiosis-related cell-cycle genes (boule, twine, Cyclin B) showed strongly reduced expression in the mutant, placing achi/vis in the aly class of meiotic arrest genes. Transcription of other genes (RP49, polo and Cyclin A) was not affected in the mutants. To determine whether Drosophila achi/vis is required upstream in the pathway for transcription of other meiotic arrest genes, the expression of aly and comr was tested in achi/vis mutant testes. In situ hybridization on achi^Z3922 vis^Z3922 mutant testes revealed aly and comr transcripts at levels similar to wild type, and RT-PCR analysis on Df(2R)achi¹ demonstrated robust expression of aly and can transcripts. In the RT-PCR analysis the levels of aly and can actually appear somewhat higher than wild type. This result is not interpreted, however, as indicative of a regulatory interaction but rather as a reflection of the altered cellular composition of the mutant testes. Similarly, aly and comr are not required for the expression of achi/vis because normal levels of achi/vis transcripts were found, by RT-PCR, in aly and comr homozygous mutant testes (Ayyar, 2003).

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

The normal chromatin association of Aly and Comr proteins is essential for their function, and the localization of these two proteins is mutually dependent, i.e., in an aly mutant Comr protein remains cytoplasmic, and vice versa. In contrast, both Comr and Aly proteins localize to chromatin in testes mutant for the downstream, can-class, genes. To determine whether achi/vis plays a role in the production or localization of the other aly-class proteins, the levels and localisation of Aly and Comr proteins were examined in achi^Z3922 vis^Z3922 mutant testes. Both Aly and Comr proteins were detected by Western blotting in achi^Z3922 vis^Z3922 mutant testes. Immunofluorescent staining revealed that Aly and Comr proteins are nuclear in achi^Z3922 vis^Z3922 testes. This places achi/vis downstream of, or parallel to, comr and aly (Ayyar, 2003).

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

It is concluded that Drosophila TGIF is required for a specific transcriptional program in Drosophila spermatogenesis. The extended G₂ phase that primary spermatoctyes undergo prior to entry into meiotic division is an important period in spermatogenesis. It lasts for about 3.5 days and during this time the cells increase in volume 25-fold, execute the transcription program required to produce all the transcripts necessary for sperm differentiation and undergo a striking sequence of chromatin reorganization. The switch in transcriptional activity that occurs in primary spermatocytes is one of the most dramatic of any differentiation pathway, with a great many genes being expressed exclusively in this cell type. At the end of this stage virtually all transcription is switched off; many of the transcripts produced during this period are held under translational inhibition to be released after meiosis in a coordinated program of protein production that mediates sperm differentiation. Most male sterile mutations affect these later stages of sperm manufacture and typically result in a block in spermiogenesis at the very late stage of sperm individualization. A relatively small set of mutations have been characterized that block spermiogenesis during the extended primary spermatocyte G₂ phase. In these mutants, spermatocytes fail to enter meiotic division and fail to initiate spermatid differentiation (Lin, 1996). These meiotic arrest genes are required to initiate the primary spermatocyte-specific transcriptional activation program. Previous genetic and biochemical analyses have suggested that the aly-class genes act before the can-class genes, at the top of a regulatory hierarchy (White-Cooper, 1998). Therefore the aly-class meiotic arrest genes provide an entry point into the mechanisms that initiate and orchestrate the transcriptional program of spermiogenesis, that control spermatocyte differentiation and that regulate the entry into the meiotic divisions (Ayyar, 2003).

The relatively small number of these meiotic arrest genes currently identified presents the beguiling prospect that the transcriptional program of spermiogenesis may be controlled by an ancient simple mechanism. Interestingly, two of the can-class meiotic arrest genes (cannonball and no hitter) encode testis-specific components of the general transcription factor TFIID, suggesting that the genes activated during the primary spermatocyte stage may share a distinct core promoter type (Aoyagi, 2000; Aoyagi, 2001; Hiller, 2001). The homology of aly to a C. elegans gene implicated in a pathway leading to chromatin remodelling factors suggests that aly may have a role in chromatin reorganization to allow access for specific transcription factors and the testis specific TFIID to target promoters (Beitel, 2000; White-Cooper, 2000). The characterization of Drosophila TGIF represents the first description of a sequence-specific transcription factor implicated in this pathway. Combined mutations in achi and vis or deletion of both genes lead to a recessive male sterile meiotic arrest phenotype. A markedly decreased expression was found for both the spermiogenesis genes and also for CycB and twine, required for entry into meiosis, placing achi/vis into the aly-class of meiotic arrest mutants. Drosophila TGIF does not appear to be required for the transcriptional activation of other meiotic arrest genes since aly, comr and cannonball are all expressed in achi/vis mutants. Similarly, function of other meiotic arrest genes is not required for transcription of achi/vis (Ayyar, 2003).

Although the gross transcriptional consequences of loss of either achi/vis or aly appear similar, the mutant phenotypes are distinct. Both show effects on early primary spermatocytes with an expansion of this cell type presumably due to a slowing of the progress of differentiation through this stage. All the aly-class mutants (aly, comr and achi/vis) exhibit defects in chromatin organization but whereas the aly and comr mutant spermatocytes arrest with 'fuzzy' chromatin condensation (Lin, 1996), in achi/vis mutants the chromatin rounds up in condensed 'blobs' which are reminiscent of meiotic pro-metaphase. This difference in the phenotype is consistent with the finding that Drosophila TGIF is not required for the normal localization of Aly and Comr proteins, and suggests that TGIF is also not required for the chromatin remodelling mediated by Aly and Comr. This raises the question of whether the aly-class genes all act together as components of a simple transcription activation switch or whether they may be a somewhat heterogeneous collection with more diverse roles within the spermatogenesis transcriptional program (Ayyar, 2003).

DEVELOPMENTAL BIOLOGY

Sequencing ESTs made possible the generation of intron-exon maps for achi/vis and revealed alternative spliced products from the two transcription units. The major difference between splicing products is the presence or absence of exon6, which is 387 bp in length and encodes an additional 129 aa. The expression profiles of these transcripts were investigated by RT-PCR. The achi/vis transcripts are present from embryogenesis through to adulthood. Interestingly, adult males predominantly expressed the larger (class 2) splice variant and females predominantly the smaller (class 1). Using RNA derived from gonadectomised males compared to RNA derived from testes it was determined that the larger splice isoform was testis specific, whereas the shorter was present in both the carcass and the testis. Sequencing of subclones of this testis-specific large isoform confirm that both achi and vis class 2 transcripts are expressed in the testis (Ayyar, 2003).

In keeping with the meiotic arrest phenotype, RNA in situ analysis of achi/vis expression shows strong expression in the primary spermatocyte stage, which decays as the cells progress through meiosis. There is no detectable expression in the tip of the testis where the stem cells and spermatogonial cells reside (Ayyar, 2003).

To analyze the vis and achi expression patterns a polyclonal antibody was generated against AchiS. Using this antibody, nuclear expression is detected in nearly all cells and stages of development, including cells in the testes. The specificity of this antibody was confirmed by observing only background staining in the pingpong deficiency (deficient for both vis and achi). Moreover, a wild-type testes staining pattern is generated by either the P{vis} or P{achi} transgenes demonstrating that this antibody recognizes both Vis and Achi proteins (Wang, 2003).

Because of the male sterile phenotype of homozygous pingpong flies the Vis/Achi expression pattern was examined in more detail in wild-type testes and its pattern was compared to that of Aly, a chromatin-associated protein that is required for male meiosis in Drosophila (White-Cooper, 2000). At the most apical tip of the testes are somatic 'hub' cells, germline stem cells, and mitotically dividing spermatogonia. Further from the tip is a zone of larger primary spermatocytes organized into 16-cell cysts. These cells, which grow in volume ~25-fold over a period of ~3.5 days, remain in meiotic prophase, a specialized stage of the meiotic cell cycle that precedes the meiotic divisions. Like Aly (White-Cooper, 2000), Vis/Achi is most strongly observed in the nuclei of primary spermatocytes. In addition, weaker nuclear Vis/Achi staining is also observed in the mitotically dividing cells at the apical tip of the testes. This is not a background staining because it is not observed in the pingpong deficiency, which still have these cells. In addition, weak Aly staining is also observed at this stage (Wang, 2003).

The various stages of meiosis can be identified by the state of the chromosomes, as revealed by a fluorescent DNA dye such as propidium iodide. At the large primary spermatocyte stage, when both Aly and Vis/Achi are nuclear, the three major chromosome pairs can be seen as three separate but diffuse signals, indicating that the chromosomes are partially condensed. At metaphase of meiosis I the chromosomes are fully condensed and appear as nuclear dots. At this stage, Aly appears to associate with the spindle and Vis/Achi is predominantly in the cytoplasm. A similar pattern is seen for both proteins at metaphase of meiosis II. Following the second meiotic division spermatid differentiation begins with the onion stage, in which each cell has a characteristically round and specialized mitochondrion adjacent to its haploid nucleus. At this stage, the anti-Aly antibody labels a single dot per nucleus that co-localizes with the DNA. In contrast, Vis/Achi is observed throughout these cells except that it is excluded from the large and specialized mitochondrion next to each nucleus. Finally, at an even later stage of spermatid differentiation, Aly is barely detected above background and Vis/Achi partially co-localizes with the DNA. Vis/Achi is not detected in the somatic cyst cells that surround the primary spermatocytes (Wang, 2003).

Effects of Mutation or Deletion

To examine the in vivo role of Drosophila TGIF deletions were generated by imprecise excision of a P-element that mapped within the 5'-UTR of the achi transcription unit. Several small deletions were obtained together with a larger deletion, Df(2R)achi¹, that removes the whole of the achi transcription unit and part of the vis unit. In addition, using P-element induced male recombination, several large deletions were produced that removed achi and vis together with some neighboring genes. The largest of these deletions Df(2R)achi⁴ is homozygous lethal. In contrast the smaller deletions Df(2R)achi² and Df(2R)achi³ have the same phenotype as Df(2R)achi¹: they are homozygous viable, the males are completely sterile and females crossed to wild-type males exhibit a delay in egg laying. All three deletions failed to complement Df(2R)BSC3 with respect to the male-sterile phenotype (Ayyar, 2003).

Examination of the testes from homozygous mutants revealed complete absence of mature sperm. Developmental stages up to late primary spermatocyte were present but no meiotic stages could be seen. A mutant with the same phenotype, Z3922 was identified in a screen of EMS mutants. The mutation in Z3922 was mapped by meiotic recombination to 63.3±1.8 mu. The Z3922 chromosome failed to complement Df(2R)BSC3, placing the mutation in the 48F-49A chromosomal region. Fine scale deficiency and recombination mapping relative to P-element insertions in this region located the Z3922 locus to within 7 genes immediately distal to the P-element insertion P{w⁺}l(2)k17040. PCR sequencing of these candidate genes from Z3922 revealed mutations in both achi and vis; there is a premature stop in exon 5 of achi together with a 56 bp deletion just 5' of the vis homeodomain. The Z3922 chromosome (achi^Z3922 vis^Z3922) fails to complement the Df(2R)achi¹ allele (Ayyar, 2003).

The effects of the achi/vis mutations on spermatogenesis were examined in more detail. Labelling the DNA with DAPI provides an easy way to visualize the stages of spermatogenesis. In wild-type whole mounts, cells at the apical tip of the testis label strongly, but during the primary spermatocyte stage the intensity decreases correlating with chromosomal reorganization and increasing cell size. An expanded zone of high DAPI labelling was observed in Df(2R)achi¹ mutant testes. This experiment was repeated using confocal microscopy with anti-histone labelling to study the chromatin morphology. The expanded zone of cells with higher DAPI labelling corresponds to an expanded population of small primary spermatocytes with diffuse chromatin surrounding a prominent nucleolus (Ayyar, 2003).

Squashed preparations of cells from the testes were used to study the defects in greater detail using phase contrast microscopy and DAPI labelling. Spermatocytes from stages S1 to S6 were identified in Df(2R)achi¹ mutant testes. However, a marked defect was observed in the chromatin of the mature primary spermatocytes (stages S5 and S6). While these cells were large and had prominent nucleoli characteristic of this stage, the chromatin was condensed into tight central blobs normally observed only in later meiotic cells. Hence, in these mutants there appears to be an uncoupling between the nuclear/nucleolar differentiation and the chromatin condensation events of meiosis. Similar results were observed comparing wild-type and mutant histone-labelled preparations (Ayyar, 2003).

TGIF has been extensively characterized as a transcriptional repressor in vertebrates, yet in Drosophila the predominant effect seen in achi/vis mutants is failure to activate a testis-specific developmentally regulated transcriptional program. In Drosophila, achi/vis must be acting either directly as a transcriptional activator, or indirectly, as a repressor of a repressor. To investigate the transcriptional effects of lack of achi/vis in more detail, RNA expression profiling was undertaken, looking particularly for genes whose expression was increased in the mutants. This system is well suited to expression analysis since a viable infertile phenotype allows the easy generation of mutant tissue for comparison with wild type. Preliminary microarray analysis, comparing RNA from Df(2R)achi¹ mutant testes to wild-type, has demonstrated, as expected, a large number of transcripts (including the spermiogenesis genes) showing decreased expression relative to wild-type. However, many transcripts were also observed with increased levels in the mutant. The strongly increased transcripts of extra sex combs (esc), in the Df(2R)achi¹ mutant testis, was particularly intriguing and esc was further investigated as a possible candidate for repression by achi/vis. The microarray result was supported by RT-PCR comparison of esc transcript in wild type and Df(2R)achi¹. Esc is a component of the transcription silencing machinery and hence provided a possible link between Drosophila TGIF and gene repression. If TGIF normally represses esc transcription in primary spermatocytes this might allow the activation of the spermatogenesis transcription program. According to this model TGIF mutants would overexpress esc and this would prevent the program activation (Ayyar, 2003).

The interpretation of altered levels of transcripts in the mutant RNA population is complicated by the gross disruption of the RNA and cellular content of the mutant testes due to the failure of spermatogenesis at the primary spermatocyte stage. The expression of esc was therefore investigated in the testes at a cellular level using an antibody against Esc protein. This analysis did not support the model proposing repression of esc by TGIF. In wild-type testis, Esc is expressed in the early mitotic cells and was also robustly expressed in early primary spermatocytes; clearly esc expression is not normally switched off in primary spermatocytes by achi/vis. Esc immunolocalization also revealed the unexpected observation that as the primary spermatocytes mature, the Esc labelling, at first rather evenly distributed in the nucleus (but excluded from the nucleolus) progressively accumulates in nuclear spots. These nuclear spots are highly reminiscent of the labelling of a variety of functional silencing complexes in various cell types. Levels of Esc protein diminish as wild-type primary spermatocytes mature (Ayyar, 2003).

In the achi/vis mutant testes the overall expression levels of Esc protein are similar to wild type. How then can the increase in esc transcript level be explained? Esc protein level appears highest in early primary spermatocytes and this cell population is markedly expanded in achi/vis mutants. It is thought that expansion of cells with the highest level of esc is the likely cause of the observed increase in esc transcript abundance (Ayyar, 2003).

Despite no change in the apparent level of Esc protein in mutant cells, the Esc localization was strikingly altered in Df(2R)achi¹ mutant testes. Although in achi/vis mutants the primary spermatocytes apparently differentiate to the final primary spermatocyte stage, the concomitant accumulation of Esc in nuclear spots fails to occur. It appears achi/vis function is required for assembly of the Esc complexes (Ayyar, 2003).

vis and achi are located on the right arm of chromosome 2 in the Drosophila genome, in cytological position 49A. Starting with a P element [EP(2)2107] inserted close to the 5' end of achi, P element-mediated male recombination was used to generate a ~40 kilobase deletion that removes both vis and achi. Because this deficiency deletes two nearly identical genes and results in male sterility, it was named Df(2R)pingpong and is referred to simply as pingpong. In addition to deleting vis and achi, pingpong also removes four additional genes. However, homozygous flies carrying this deficiency are viable, suggesting that none of these genes are required for embryonic or larval development. Moreover, pingpong homozygous females are fertile and can give rise to viable pingpong progeny, eliminating the possibility that any of these genes have an essential maternal function. The only highly penetrant phenotype observed in pingpong homozygous flies is male sterility, which can be rescued by transgenes carrying genomic regions for either vis (P{vis}) or achi (P{achi}. Thus, it is concluded that vis and achi encode redundant functions required for male fertility (Wang, 2003).

The role of vis and achi in male fertility was quantitated by comparing the fertility of pingpong males with pingpong; P{vis} and pingpong; P{achi} males. Individual males were crossed to wild-type females and the number of progeny after 15 days was counted. In this assay wild-type (yw) males yielded an average of 132 progeny/male. pingpong males were completely infertile. In contrast, pingpong; P{achi} males were nearly as fertile as wild type and pingpong; P{vis} males were partially rescued. Although both P{vis} and P{achi} appear to generate a wild-type Vis/Achi expression pattern in the male germline these results suggest that achi is better able to rescue the mutant phenotype than vis (Wang, 2003).

The male sterile phenotype, together with its strong nuclear expression in primary spermatocytes, suggested that vis or achi may be required for meiosis. Therefore pingpong and pingpong; P{achi} testes were examined by phase contrast microscopy, staining the chromosomes with propidium iodide. In contrast to wild type, pingpong mutant testes are much smaller and are blocked before the first meiotic division. Specifically, no evidence of any elongated or onion stage spermatids is observed in the pingpong mutant. Instead, the testes are filled with cells that appear to remain at the primary spermatocyte stage. 16-cell cysts are still present, suggesting that the four mitotic divisions proceed normally in this mutant. However, the cells are smaller and not as round as in wild type. Far from the apical tip of the testes cells appear to degenerate. In addition, some of the chromosomes fail to fully condense in the absence of Vis and Achi. Typically, three spots of DNA are observed per cell, but the appearance of these spots ranges from diffuse (partially decondensed) to fully condensed. This phenotype indicates that the normally synchronized events leading to chromosome condensation fail in the pingpong mutant. Furthermore, these results suggest that although the pingpong mutant initiates meiosis in the male, meiosis is blocked before the first meiotic division, probably prior to the G₂ to M transition. In addition to being blocked in meiosis, pingpong testes do not show any signs of spermatid differentiation, such as the distinctive onion stage cysts or spermatid elongation (Wang, 2003).

Bromodeoxyuridine (BrdU) labeling was used to determine if the 16-cell cysts in pingpong testes undergo DNA synthesis before arresting. BrdU-labeled 16-cell cysts were observed in both wild-type and pingpong testes. Thus, the block in meiosis occurs after DNA synthesis but before the first meiotic division (Wang, 2003).

The genetic analysis of spermatogenesis in Drosophila has identified several genes that are required for the normal progression through meiosis in males. Two of these genes, aly and cookie monster (comr) can be distinguished from the others because they are required for the expression of twine, which encodes a cdc25-like phosphatase, and boule, an ortholog of the human gene Deleted in Azoospermia. In contrast, the gene cannonball (can) is also required for meiosis in males, but is not required for the expression of twine or boule mRNAs. To gain additional insight into the pingpong mutant phenotype mutant testes were stained for several markers known to be expressed in testes, including twine and boule. Like aly and comr, but unlike can mutant testes, the pingpong mutant does not express twine or boule mRNAs. Also like aly and comr mutants, pingpong testes do not express mst87F, a gene that is required for spermatid differentiation. These results suggest that vis and achi function at a similar step as aly and comr (Wang, 2003).

To determine if vis and achi act upstream of or in parallel with aly and comr, pingpong mutant testes were stained with antibodies for the aly and comr gene products, which are also highly expressed in primary spermatocytes (Jiang, 2003; White-Cooper, 2000). Strikingly, both Aly and Comr are observed in the nuclei of most cells in pingpong mutant testes. Thus, vis and achi are not required for the expression or nuclear localization of these genes. Conversely, Vis/Achi proteins are still observed in aly mutant testes (as is Comr). Consistent with these results, Vis and Achi are also expressed and are nuclear in testes mutant for spermatocytes arrest (sa) and can, genes that appear to act downstream of aly and comr. Thus, neither sa, can, or aly are required for the expression or nuclear localization of Vis or Achi. Taken together, these data suggest that Vis and Achi function in spermatogenesis in parallel with Aly and Comr (Wang, 2003).

The lack of twine expression in pingpong mutant testes is consistent with it causing a meiotic arrest phenotype. In addition to twine, meiosis is also controlled by the availability of Cyclins A and B, which are required to activate Cyclin dependent kinase 1 (Cdk1). Both Cyclins A and B are normally expressed during male spermatogenesis, at high levels in the mitotically dividing cells at the apical tip of the testes, at lower levels during the primary spermatocyte stage, and at higher levels in 16-cell cysts just prior to the G₂ to M transition. The cyclins are rapidly degraded at the end of metaphase. Cyclin levels were examined in pingpong mutant testes. Although there is some variation between individual testes, in general, intermediate levels of both Cyclin A and Cyclin B are observed throughout pingpong testes. Cyclin A and B levels persist, although at lower levels, up to the point when the cells appear to degenerate. In addition, although there is a transient nuclear localization of both Cyclins prior to cell division in the wild type, both Cyclins are observed predominantly in the cytoplasm in the pingpong mutant. Therefore, unlike in the wild type, Cyclin levels are not modulated in the pingpong mutant, consistent with a block prior to the G₂/M transition. In addition, the levels of Polo, a protein kinase required for cytokinesis during meiosis, indicate that in pingpong testes the block occurs prior to the first meiotic division (Wang, 2003).

The characterization of vis and achi cDNAs suggests the existence of two isoforms that differ because of the presence or absence of an alternatively spliced exon. To analyze the protein products derived from vis and achi the anti-Achi antibody was used in immunoblot experiments. Two bands with apparent molecular masses of ~60 kDa and ~80 kDa are detected in whole lysates of wild-type testes. In contrast, only a ~60 kDa band is detected in wild-type ovaries. Neither band is detected in the testes or ovaries from the pingpong mutant, suggesting that both are derived from vis and achi. Furthermore, based on the sizes of proteins observed in pingpong; P{vis} or pingpong; P{achi} flies, it is deduced that VisL and AchiL both migrate at ~80 kDa and both VisS and AchiS migrate at ~60 kDa. This assignment was confirmed by expressing the AchiL or AchiS cDNAs under the control of the alpha1-tubulin promoter in Df(2R)pingpong flies. Furthermore, in wild-type flies the slower migrating forms (AchiL and VisL) are testes specific because only the ~60 kDa species is observed in male or female somatic tissues (Wang, 2003).

Tests were performed to see if either AchiL or AchiS2 is sufficient to rescue the pingpong male infertility phenotype by ubiquitously expressing these isoforms under the control of the tub promoter. Flies containing either tub-AchiL or tub-AchiS in an otherwise wild-type background appear normal. In addition, although the long isoforms are testes specific, both Df(2R)pingpong; tub-AchiL and Df(2R)pingpong; tub-AchiS2 males have normal appearing testes, as demonstrated by both phase contrast microscopy and immunostaining with several markers. However, in both cases these males are only weakly fertile Df(2R)pingpong; tub-AchiL males give rise to an average of 10 progeny/male. One explanation for this result is that the tub promoter fails to provide accurate levels or timing of Achi expression to fully rescue pingpong sterility. However, males in which both long and short forms of Achi are co-expressed exhibit better fertility, suggesting that expression of both isoforms is required for complete rescue (Wang, 2003).

Cloning of TGIFs

Yp11.2/Xq21.3 is a human-specific homology block that constitutes the largest shared region among the sex chromosomes, spanning some 3.5 Mb. Only two transcribed sequences have been mapped to this segment: the protocadherin genes PCDHX/Y, and the X-linked poly(A)-binding protein PABPC5 gene, whose Y-homolog has been lost during human evolution. This paper reports the genomic structure, expression, and evolutionary conservation of a third (X-Y homologous) transcribed sequence, TGIFLX/Y (TGIF-like X/Y), mapping to this region. TGIFLX/Y has a 2666-bp mRNA encoded by two exons separated by a 96-bp intron. TGIFLX/Y are homeodomain-containing genes related to the TALE superclass gene family. Comparative DNA analysis indicates that TGIFLX originated from retrotransposition of TGIF2, located on 20q11.2-12, onto the X Chromosome. RT-PCR analysis reveals that both X- and Y-linked genes are specifically expressed in adult testis. Cloning and sequencing of TGIFLX homologs in hominoids and Old World monkeys provides evidence for an open reading frame in the eight species studied. Interestingly, a single base pair deletion in the human TGIFLY (as compared with TGIFLX) creates a different reading frame where the C-terminal residues shared by TGIFLX and other TGIF proteins are missing. The conservation, similarity to protein-encoding transcription factors, and specific expression in testis points to a transcriptional role for TGIFLX/Y in this tissue (Blanco-Arias, 2002).

Homeodomain transcription factors play important roles in directing cellular proliferation and differentiation. TGIF is a TALE-superclass homeodomain protein that serves as a multifunctional repressor of TGFbeta-induced transcription. TGIF2 is a novel TALE-superclass homeodomain protein that shows distinct homology with TGIF, especially in its DNA-binding domain. TGIF2 is expressed ubiquitously in human tissues, with the highest levels being found in heart, kidney, and testis. The TGIF2 product contains a putative nuclear localization signal; translocation of the protein to the nucleus was confirmed by transfection of epitope-tagged cDNA. TGIF2 lies on chromosome 20q11.2-12. Since amplification of 20q is often observed among ovarian cancers, the status of DNA copy-number and expression of TGIF2 was determined in 14 ovarian-cancer cell lines. This gene is over-expressed in all lines that showed amplification by FISH analysis. The results suggest that TGIF2 may play an important role in the development and/or progression of some ovarian tumors through a mechanism of gene amplification (Imoto, 2000).

Expression of TGIFs

The TGIF homeobox gene encodes a homeoprotein that represses the 9-cis retinoic acid receptor-dependent transcription activation. To investigate the potential role of this gene in vertebrate development, cDNA clones of the murine TGIF (mTGIF) gene were isolated and its expression pattern during mouse embryogenesis and postnatal development was analyzed by Northern analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization histochemistry. mTGIF transcripts were detected at day E16 in the emerging external granular layer (EGL), the cells of which arise from the proliferating cerebellar neuroepithelium. Expression of mTGIF transcripts was also detected at day E16 in the proliferating cells in the neuroepithelium of the hippocampal formation. Following gestation, mTGIF expression increases to a maximum between postnatal days 5 and 10 (PN5 and PN10) in the rapidly expanding cerebellar EGL. mTGIF transcripts are no longer detectable when EGL proliferation ceases on approximately day PN15. Throughout embryo development and in the adult mice, TGIF is detected in a restricted number of tissues, mostly in proliferating and differentiating cell lineages, such as tongue and testis. These results suggest that the TGIF gene regulates target genes involved in the proliferation, migration, and/or differentiation of particular neuronal cell lineages in the developing brain (Bertolino, 1996).

A novel homeobox gene designated Tex1 (see NCBI BLAST) has been isolated from a mouse testis cDNA library by degenerate oligonucleotide polymerase chain reaction (PCR) screening. Three extra amino acid residues, AYP, are inserted between the first and second helices of the homeodomain, which makes Tex1 a TGIF subclass homeobox gene. Tex1 was expressed specifically in the testis as seen by both Northern blot and RT-PCR analysis. Tex1 expression is initiated at around day 20 of testes. Furthermore, the results of in situ hybridization analysis reveal that the expression of Tex1 in testis is restricted in the germ cells at spermatid stage. Based on these data, it is suggested that Tex1, a novel testis homeobox gene, may play a crucial role during spermatogenesis (Lai, 2002).

Since achi/vis mutants in Drosophila primarily affect spermatogenesis it was of interest to examining the potential for a similar role of vertebrate TGIF. The expression profile of TGIF in the mouse indicates widespread expression including strong expression in the testes (Bertolino, 1996). An antibody raised against human TGIF was used to examine the protein localization of TGIF in the mouse testis. In the mouse seminiferous tubules the spermatogonial stem cells and mitotic spermatogonial cells are found at the periphery of the tubules. Older cells are displaced towards the center of the tubule so that a transect across the tubule gives a time-line of development with more mature meiotic stages towards the center. TGIF is most prominently expressed in cells in more peripheral regions of the tubules, including spermatogonia and primary spermatocytes, and the labelling is restricted to cell nuclei. Therefore TGIF is available in the mouse in appropriate cells for the activation of the spermatogenesis transcriptional program, as in the fly (Ayyar, 2003).

Physical interactions of TGIFs

Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. The homeodomain protein TGIF has been identified as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell (Wotton, 1999a).

TGIF is a DNA-binding homeodomain protein that has been demonstrated to play a role in transforming growth factor beta-regulated transcription and implicated in the control of retinoid-responsive transcription. The intrinsic transcriptional activity of TGIF fused to a heterologous DNA-binding domain has been investigated. TGIF is shown to be a transcriptional repressor able to repress transcription from several different promoters. Repression by TGIF is insensitive to the distance at which it is bound from the promoter. Moreover, the wild type TGIF effectively represses transcription when bound to its cognate DNA-binding site via its homeodomain. Deletion analysis reveals the presence of at least two separable repression domains within TGIF. Repression by one of these is dependent on the activity of histone deacetylases (HDACs), whereas the other appears not to require HDAC activity. Finally, it has been demonstrated that TGIF interacts with HDACs via its carboxyl-terminal repression domain. Together, these results suggest that TGIF is a multifunctional transcriptional repressor, which acts in part by recruiting HDAC activity (Wotton, 1999b).

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and TGIF is shown to be able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF (Melhuish, 2000).

The homeodomain protein TG-interacting factor (TGIF) represses transcription by histone deacetylase-dependent and -independent means. Heterozygous mutations in human TGIF result in holoprosencephaly, a severe genetic disorder affecting craniofacial development, suggesting that TGIF is critical for normal development. After transforming growth factorbeta (TGFbeta) stimulation, Smad proteins enter the nucleus and form transcriptional activation complexes or interact with TGIF, which functions as a corepressor. The relative levels of Smad corepressors and coactivators present within the cell may determine the outcome of a TGFbeta response. TGIF interacts directly with the paired amphipathic alpha-helix 2 domain of the mSin3 corepressor, and TGIF recruits mSin3 to a TGFbeta-activated Smad complex. The mSin3 interaction domain of TGIF has been shown to be essential for repression of a TGFbeta transcriptional response. Thus, TGIF represents a targeting component of the mSin3 corepressor complex (Wotton, 2001a).

Smad transcription factors mediate the actions of transforming growth factor-beta (TGF-beta) cytokines during development and tissue homeostasis. TGF-beta receptor-activated Smad2 regulates gene expression by associating with transcriptional co-activators or co-repressors. The Smad co-repressor TGIF competes with the co-activator p300 for Smad2 association, such that TGIF abundance helps determine the outcome of a TGF-beta response. Small alterations in the physiological levels of TGIF can have profound effects on human development, as shown by the devastating brain and craniofacial developmental defects in heterozygotes carrying a hypomorphic TGIF mutant allele. TGIF levels modulate sensitivity to TGF-beta-mediated growth inhibition. TGIF is a short-lived protein. Epidermal growth factor (EGF) signaling via the Ras-Mek pathway causes the phosphorylation of TGIF at two Erk MAP kinase sites, leading to TGIF stabilization and favoring the formation of Smad2-TGIF co-repressor complexes in response to TGF-beta. These results identify the first mechanism for regulating TGIF levels and suggest a potential link for Smad and Ras pathway convergence at the transcriptional level (Lo, 2001).

TG-interacting factor (TGIF) is a transcriptional repressor that represses transcription by binding directly to DNA or interacts with transforming growth factor beta (TGF beta)-activated Smads, thereby repressing TGF beta-responsive gene expression. Mutation of TGIF in humans causes holoprosencephaly, a severe genetic disorder affecting craniofacial development. Searching human expressed sequence tag data bases revealed the presence of clones encoding a TGIF-related protein (TGIF2), which contains two regions of high sequence identity with TGIF. Like TGIF, TGIF2 recruits histone deacetylase, but in contrast to TGIF, is unable to interact with the corepressor CtBP. TGIF2 and TGIF have very similar DNA-binding homeodomains, and TGIF2 represses transcription when bound to DNA via a TGIF binding site. TGIF2 interacts with TGF beta-activated Smads and represses TGF beta-responsive transcription. TGIF2 appears to be a context-independent transcriptional repressor, which can perform similar functions to TGIF and may play a role in processes, which, when disrupted by mutations in TGIF, cause holoprosencephaly (Melhuish, 2001).

Effects of TGIF mutation

Holoprosencephaly (HPE) is the most common structural defect of the developing forebrain in humans (1 in 250 conceptuses, 1 in 16,000 live births). HPE is aetiologically heterogeneous, with both environmental and genetic causes. So far, three human HPE genes are known: SHH at chromosome region 7q36; ZIC2 at 13q32; and SIX3 at 2p21. In animal models, genes in the Nodal signalling pathway, such as those mutated in the zebrafish mutants cyclops, squint and one-eyed pinhead (oep), cause HPE. Mice heterozygous for null alleles of both Nodal and Smad2 have cyclopia. The involvement of the TG-interacting factor (TGIF), a homeodomain protein, in human HPE is described. TGIF maps to the HPE minimal critical region in 18p11.3. Heterozygous mutations in individuals with HPE affect the transcriptional repression domain of TGIF, the DNA-binding domain or the domain that interacts with SMAD2. (The latter is an effector in the signalling pathway of the neural axis developmental factor NODAL, a member of the transforming growth factor-beta (TGF-beta) family.) Several of these mutations cause a loss of TGIF function. Thus, TGIF links the NODAL signalling pathway to the bifurcation of the human forebrain and the establishment of ventral midline structures (Gripp, 2000).

Regulatory interactions of TGIFs

A novel homeobox gene, denoted TGIF (5'TG3' interacting factor), has been identified that belongs to an expanding TALE (three amino acid loop extension) superclass of atypical homeodomains. The TGIF homeodomain binds to a previously characterized retinoid X receptor (RXR) responsive element from the cellular retinol-binding protein II promoter (CRBPII-RXRE), which contains an unusual DNA target for a homeobox. The interactions of both the homeprotein TGIF and receptor RXR alpha with the CREBPII-RXRE DNA motif occur on overlapping areas and generate a mutually exclusive binding in vitro. Transient cellular transfections demonstrate that TGIF inhibits the 9-cis-retinoic acid-dependent RXR alpha transcription activation of the retinoic acid responsive element. TGIF transcripts were detected in a restricted number of tissues. The canonical binding site of TGIF is conserved and is an integral part of several responsive elements that are organized like the CRBPII-RXRE. Hence, a novel auxiliary factor to the steroid receptor superfamily may participate in the transmission of nuclear signals during development and in the adult, as illustrated by the down-modulation of the RXR alpha activities (Bertolino, 1995).

Three-amino acid extension loop (TALE) homeobox proteins are highly conserved transcription regulators. Two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in opposite orientations, can function competitively. For example, in the D(1A) gene, which encodes the predominant dopamine receptor in the striatum, Meis2 and TGIF bind to the activator sequence ACT (-1174 to -1154) and regulate transcription differentially in a cell type-specific manner. Among the five cloned splice variants of Meis2, isoforms Meis2a-d activate the D(1A) promoter in most cell types tested, whereas TGIF competes with Meis2 binding to DNA and represses Meis2-induced transcription activation. Consequently, Meis2 cannot activate the D(1A) promoter in a cell that has abundant TGIF expression. The Meis2 message is highly co-localized with the D(1A) message in adult striatal neurons, whereas TGIF is barely detectable in the adult brain. These observations provide in vitro and in vivo evidence that Meis2 and TGIF differentially regulate their target genes. Thus, the delicate ratio between Meis2 and TGIF expression in a given cell type determines the cell-specific expression of the D(1A) gene. Splice variant Meis2e, which has a truncated homeodomain, cannot bind to the D(1A) ACT sequence or activate transcription. However, Meis2e is an effective dominant negative regulator by blocking Meis2d-induced transcription activation. Thus, truncated homeoproteins with no DNA binding domains can have important regulatory functions (Yang, 2000).

Like other nuclear receptors, the AR exerts its transcriptional function by binding to cis elements upstream of promoters and interacting with other transcriptional factors (e.g. activators, repressors, and modulators). Among them, histone acetyltransferases (HATs) and histone deacetylases (HDACs) play critical roles in altering the acetylation state of core histones, thereby regulating nuclear hormone receptor-mediated transcription. The nuclear receptor corepressor can repress the TR and RAR in the absence of ligand through either a Sin3A-dependent or Sin3A-independent manner by recruiting HDACs. AR and some other steroid hormone receptors cannot silence transcription through a similar mechanism in that they are located in the cytoplasm as complexes with heat-shock proteins before exposure to ligand. It has been shown that AR can bind to p160/SRC, cAMP response element-binding protein-binding protein (CBP)/P300 and other coactivators to increase the AR-mediated transcription. However, the molecular mechanism for turning AR from transcriptionally active into silent states is unknown. The transcription repressor, 5'TG3' interacting factor (TGIF), selectively represses AR-mediated transcription from several AR-responsive promoters. The repression is mediated through binding of TGIF to the DNA binding domain of AR and is trichostatin sensitive. A direct protein-protein interaction has been detected between TGIF and a transcription corepressor, Sin3A, which suggests a novel pathway for TGIF recruiting HDAC1 to the repression complex. These results provide fresh insight into understanding the mechanism for repressing AR-, and perhaps other steroid hormone receptor-mediated transcriptions (Sharma, 2001).

TG-interacting factor (TGIF) is a transcriptional co-repressor that directly associates with Smad (Sma- and Mad-related protein) proteins and inhibits Smad-mediated transcriptional activation. By using Affymetrix oligonucleotide microarray analysis, it was found that TGIF mRNA level is elevated by transforming-growth-factor-beta (TGF-beta) treatment in a human T-cell line, HuT78. Subsequent reverse-transcription PCR assays indicate that TGF-beta1 and activin are able to induce a rapid and transient increase in the level of TGIF in both HuT78 and HepG2 hepatoma cells. To analyse whether or not the regulation of TGIF mRNA occurs at the transcriptional level, a 2.4 kb human TGIF promoter was isolated. A primer extension assay was performed to localize the putative transcription initiation site of the promoter. When transiently expressed in HepG2 cells, this promoter is stimulated by TGF-beta1 and activin treatment in a time-dependent manner. A series of deletion mutants of the TGIF promoter were also generated to further characterize the TGF-beta responsive region of the promoter. In addition, expression of TGIF is able to cause a dose-dependent inhibition of TGF-beta and activin signalling. Taken together, these experiments indicated that TGIF is a novel transcriptional target of TGF-beta and activin signalling and is likely involved in a negative feedback loop to desensitize TGF-beta/activin action (Chen, 2003).

Search PubMed for articles about Drosophila Achintya and Vishmay

Aoyagi, N. and Wassarman, D. A. (2000). Genes encoding Drosophila melanogaster RNA polymerase II general transcription factors: diversity in TFIIA and TFIID components contributes to gene-specific transcriptional regulation. J. Cell Biol. 150: F45-F49. 10908585

Aoyagi, N. and Wassarman, D. A. (2001). Developmental and transcriptional consequences of mutations in Drosophila TAF(II)60. Mol. Cell Biol. 21: 6808-6819. 11564865

Ayyar, S. and White, R. A. H. (2001). achintya and vismay: two novel, tandem-repeated TALE-class homeobox genes. A. Dros. Res. Conf. 42: 504C

Ayyar, S., Jiang, J., Collu, A., White-Cooper, H. and White, R. A. (2003). Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis. Development 130(13): 2841-2852. PubMed ID: 12756169

Beall, E. L., Lewis, P. W., Bell, M., Rocha, M., Jones, D. L. and Botchan, M. R. (2007). Discovery of tMAC: a Drosophila testis-specific meiotic arrest complex paralogous to Myb-Muv B. Genes Dev 21(8): 904-919. PubMed ID: 17403774

Beitel, G. J., Lambie, E. J. and Horvitz, H. R. (2000). The C. elegans gene lin-9, which acts in an Rb-related pathway, is required for gonadal sheath cell development and encodes a novel protein. Gene 254: 253-263. 10974557

Bertolino, E., Reimund, B., Wildt-Perinic, D. and Clerc, R. G. (1995). A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoid-responsive motif. J. Biol. Chem. 270: 31178-31188. 8537382

Bertolino, E., Wildt, S., Richards, G. and Clerc, R. G. (1996). Expression of a novel murine homeobox gene in the developing cerebellar external granular layer during its proliferation. Dev. Dyn. 205: 410-420. 8901052

Blanco-Arias, P., Sargent, C. A. and Affara, N. A. (2002). The human-specific Yp11.2/Xq21.3 homology block encodes a potentially functional testis-specific TGIF-like retroposon. Mamm. Genome 13: 463-468. 12226713

Burglin, T. R. (1997). Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucleic Acids Res. 25: 4173-4180. 9336443

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date revised: 15 April 2020

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