string

Promoter Structure and Transcriptional Regulation

During postblastoderm embryogenesis cell cycles are differentially timed by bursts of stringtranscription. An analysis of string expression in 36 pattern-formation mutants shows that known patterning genes act locally to influence string transcription. Embryonic expression of string gene fragments shows that the complete pattern of string transcription requires extensive cis-acting regulatory sequence, but that smaller segments of this regulatory region can drive proper temporal expression in defined spatial domains. Apparently string upstream sequences integrate many local signals to direct string's transcriptional program. The spatiotemporal progression of string transcription is largely unaffected in mutant embryos specifically arrested in the G2 phase of cycles 14, 15, or 16, or the G1 phase of cycle 17. Thus, there is a regulatory hierarchy in which developmental inputs, not cell cycle inputs, control the timing of string transcription and hence cell cycle progression (Edgar, 1994a).

Genetic and molecular analyses of patterning in the Drosophila embryo have shown that the process of head segmentation is fundamentally different from the process of trunk segmentation. The cephalic furrow (CF), one of the first morphological manifestations of the patterning process, forms at the juxtaposition of these two patterning systems. The initial step in CF formation is a change in shape and the apical positioning of a single row of cells. The anteroposterior position of these initiator cells may be defined by the overlapping expression of the head gap gene buttonhead (btd) and the primary pair-rule gene even-skipped. The position of the furrow coincides with the second row of Even-skipped-expressing cells in stripe 1. Re-examination of the btd and eve phenotypes in live embryos indicates that both genes are required for CF formation. Activation of col requires btd. Conversely, in the absence of eve, collier expression is expanded posteriorly to overlap a region roughly corresponding to PS1, indicating that Eve acts as a repressor of col in this parasegment. Likewise, expression of string in mitotic domain 2, which also requires btd, is expanded posteriorly in eve mutant embryos. The current working model holds that the activation of eve by btd in anterior PS1 cells allows for differential gene expression between PS0 and PS1. In addition to the control of CF formation, the btd/eve interaction may thus assign separate gene expression and mitotic programs to cells on either side of the pro-cephalon/posterior head border (Vincent, 1997 and references).

collier expression at the blastoderm stage is restricted to a single stripe of cells corresponding to part of the intercalary and mandibular segment primordia, possibly parasegment 0. There is a striking similarity between the early stripe of collier expression and the position of a specific mitotic domain at cycle 14, mitotic domain 2 (MD2). Mitotic domains are defined as groups of cells that enter mitosis 14 both synchronously and out of synchrony with other groups of cells (Foe, 1989). The pattern of string (stg) transcription anticipates the pattern of cycle 14 mitoses. At the onset of gastrulation, string and collier are simultaneously expressed in a group of cells that correspond to MD2, suggesting that these cells not only share a mitotic fate, but also share a specific gene expression program. It is thought that col and stg respond to the same patterning information and act in parallel, with col assigning a specific gene-expression program in cells in MD2. stg and col expression in MD2 is concomitant and both require buttonhead (Crozatier, 1996).

Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of Prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the members of the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string (Ashraf, 2001).

Both snail and worniu have extensive expression in neuroblasts, while that of escargot is transient and sparse. Furthermore, based on genetic analysis, snail and worniu have more important role than escargot in the regulation of CNS development. The expression of snail and worniu in GMCs was carefully examined. In situ hybridization has revealed that worniu RNA, in contrast to its extensive expression in neuroblasts, is present in only a small number of GMCs. Even in later staged embryos, when there should be multiple GMCs surrounding each neuroblast, the staining in no more than one small cell next to each neuroblast could be detected. The limited staining in the GMCs is probably due to the segregation of some RNA from the parental neuroblast. Once the GMC is formed, the active transcription of worniu probably ceases. The protein and RNA expression of snail was also examined. The results showed that there is also very limited expression of snail in GMCs. snail RNA-containing GMCs were rarely detected next to neuroblast. Consistent with RNA expression, antibody staining revealed that the protein is predominantly in the neuroblasts (Ashraf, 2001).

One possibility that may explain the severe phenotype in snail family deletion mutants is additional defects in cell division. Neuroblasts are arrested at the G2/M transition at the embryonic cell cycle 14. After delamination, a pulse of string (which encodes a Cdc25 phosphatase homolog) expression in neuroblasts drives the cells to enter mitosis. The expression of string RNA was examined in whole-mount mutant embryos, but the result was ambiguous, owing to the dynamic, high level expression in ectoderm and other tissues, which obscures the signal in the neuroblast cell layer. Therefore tissue sectioning was used in order to better view the expression of string in neuroblasts. The sections clearly showed expression of string RNA in wild-type neuroblasts at stage 9 embryos. There are consistently three to four neuroblasts on each side of the midline that exhibit staining. This neuroblast expression appears very faint in the osp29 mutant embryos, and most sections do not show staining in neuroblasts while expression in ectoderm appears normal. The presence of wor and esg transgenes in the deletion mutant background led to accumulation of string RNA in some neuroblasts, suggesting a positive role for Snail family in regulating string expression (Ashraf, 2001).

If regulation of string is an important downstream event of Snail family of proteins, then cell division of neuroblasts should be affected in the absence of these proteins. The mitotic process was examined by staining for phosphorylated histone H3, which reveals condensed chromosomes. In wild-type embryos, although the neuroblasts do not exhibit highly synchronized mitosis, anti-phosphoH3 staining can be detected in multiple cells. In the osp29 mutant embryos, such staining is consistently reduced. The use of Prospero RNA to mark the neuroblast layer and the use of tissue sectioning has provided further support for the idea that the mutant embryos has reduced mitosis in neuroblasts (Ashraf, 2001).

The severe CNS defects are likely due to a combination of loss of inscuteable and string expression. Similar to the results obtained for inscuteable, transgenic expression of string alone has a weak and variable effect in the rescue of Prospero expression in GMCs. When both inscuteable and string are simultaneously expressed in neuroblasts of osp29 mutants using the UAS-Gal4 system, clear staining of Prospero in many cells resembling GMCs is observed. The staining is particularly apparent alongside the expanded midline, characteristic of mutant embryos with no Snail function in early mesoderm. The results support the idea that both inscuteable and string are relevant targets of the Snail family (Ashraf, 2001).

A clearly demonstrated in vivo function of Snail is transcriptional repression. The repression function is mediated through the recruitment of dCtBP (Drosophila C-terminal binding protein), which acts as a co-repressor for Snail to regulate target genes such as rhomboid, lethal of scute and single-minded. There are two conserved P-DLS-R/K motifs in Snail, as well as in Worniu and Escargot, and they have been shown to be critical for recruiting dCtBP. Mutations of these motifs abolish the repressor function of Snail in the blastoderm. To gain insight into the molecular mechanism of how Snail regulates CNS development, transgenic copies of snail, which had the dCtBP interaction motifs mutated were introduced into the osp29 deletion background. M1 contains the N-terminal motif mutation and M2 contains the C-terminal motif mutation. The expression of inscuteable and ftz was examined. The assay shows that the double mutant (M12) lost most of the ability to rescue, and M1 has lost some ability to rescue. However, M2 functioned quite efficiently, closer to that of the wild-type protein, to rescue inscuteable and ftz expression. These results demonstrate that the dCtBP interaction motifs are essential for the Snail function in the CNS, consistent with the idea that Snail acts as a repressor in neuroblasts to regulate gene expression. Thus, the activation of inscuteable and string by the Snail family may be indirect (Ashraf, 2001).

Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signaling from a specialized subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to secrete the EGF ligand, Spitz. The response to EGF signaling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signaling by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of pointedP2 is induced by Wingless signaling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. To understand how the proneural and neurogenic genes pattern the response to EGFR activation, the expression of genes involved in transduction of the pathway was analyzed. The orphan nuclear-receptor svp functions downstream of the EGF receptor to promote cell divisions in the tubules. In the absence of Svp function, cycE and stg transcription is abolished, with a consequent reduction in EGFR-driven cell divisions. These late divisions in the tubules of stage 12 wild-type embryos were followed and it was found that BrdU incorporation (and hence, cell division) is confined within the svp-lacZ domain. These results define the svp domain of expression as including those cells which will divide in response to Egfr activation. However, the expression of svp-lacZ is initiated in a group of cells surrounding the tip mother cell, before the birth of the TC. This early onset of svp expression occurs before the late divisions start (cycle 17 onwards), when neither Svp function nor Egfr activation is required for cell proliferation. The pattern of gene expression observed suggests that the Svp-positive cells surrounding the tip mother cell derive from the proneural cluster (Sudarsan, 2002).

Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, referred to collectively as Dorsocross, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).

One of the hallmarks of amnioserosa development is that the cells of this tissue never resume mitotic divisions after the blastoderm divisions. To a large extent, this cell cycle arrest is due to the absence of expression of cdc25/string in the prospective amnioserosa: this absence prevents the cells from entering M-phase and leads to G2 arrest. In addition, the expression of the Cdk inhibitor p21/Dacapo in the early amnioserosa is thought to contribute to the cell cycle arrest. Although a detailed description of the regulation of string and dacapo expression in dorsal embryonic areas is lacking, it has been reported that zen is required for repressing dorsal string expression -- this repression is expected to prevent further cell divisions. Notably, the observation that amnioserosa cells re-enter the cell cycle in Doc mutant embryos demonstrates that Doc genes are required for the cell cycle block in addition to zen. Whereas zen mutant embryos already feature ectopic cell divisions in dorsal areas from stage 8 onwards, in Doc mutants the amnioserosa cells resume mitosis only during and after stage 10, which is shortly after Zen protein disappears. Thus, it is hypothesized that the Doc genes take over the function of zen in repressing string and prevent cell divisions at later stages of amnioserosa development when Zen is no longer present. Overall, the phenotype of Doc mutant embryos suggests that amnioserosa differentiation, including cell cycle arrest and the development of squamous epithelial features, initiates in the absence of Doc activity but is not maintained beyond stage 11. Thereafter, cell division resumes and there is a reversal of the partially differentiated state. Apoptotic events are not observed prior to stage 11 in Doc mutants. However at later stages, many amnioserosa cells die prematurely and the remaining cells are difficult to distinguish morphologically from dorsal ectodermal cells (Reim, 2003).

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/Cdh^Fzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, Cdh^Fzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).

Mod can cooperate with String to promote growth

In gain of function and loss of function experiments, modulo has been demonstrated to be directly controlled by dMyc. Strikingly, in proliferative imaginal cells, mod loss-of-function impairs both cell growth and cell size, whereas larval endoreplicative tissues grow normally. In contrast to dMyc, over-expressing Mod in wing imaginal discs is not sufficient to induce cell growth. Taken together, these results indicate that mod does not possess the full spectrum of dMyc activities, but is required selectively in proliferative cells to sustain their growth and to maintain their specific size (Perrin, 2003).

In Mod⁺ clones, the cell size increase along with the G2 accumulation raises the possibility that Mod slows down cell division by delaying G2/M transition. To address this question, String (the rate limiting phosphatase for G2/M) was expressed alone or in combination with Mod using the `flip out' technique. Cell division was slightly faster in String⁺ cells as compare to wild type, and further accelerated upon co-expressing Mod and String together. If Mod functions as a brake of G2/M transition, co-expression of String might counteract this effect. However, expressing Mod and String together accelerates cell division rate more than String alone does. Hence, this cooperative effect rather excludes that Mod could antagonize String at G2/M transition. Strikingly, String over-expression promotes growth of Mod⁺ cells. No evident hypothesis can be proposed to explain how the overgrowth induced by Mod and String co-expression is accomplished. However, this observation indicates that Mod, while insufficient to direct growth alone, can nonetheless cooperate with Stg to promote growth (Perrin, 2003).

Phosphorylation of H2B-S33 by TAF1 is essential for transcriptional activation of stg/cdc25 and, consequently, cell cycle progression

TAF1 contains two kinase domains, an N-terminal (NTK, amino acids 1 to 496) and a C-terminal (CTK, amino acids 1496 to 2132) domain (Dikstein, 1996). In vitro, the NTK and the CTK autophosphorylate and the NTK transphosphorylates the RAP74 subunit of the GTF TFIIF. In contrast to the NTK, the CTK did not phosphorylate RAP74 but strongly phosphorylated H2B, indicating that the CTK possesses H2B-specific kinase activity. Protein kinases contain two essential functional motifs, an adenosine triphosphate (ATP) binding motif and an amino acid–specific kinase motif. Computational sequence comparison analyses identified a putative serine and threonine (S/T) kinase motif (amino acids 1534 to 1546) and two tandem ATP binding domains (amino acids 1747 to 1780) in the CTK. Interestingly, the S/T kinase motif is located in the first bromodomain of the double bromodomain module (DBD), which binds acetylated lysines. However, CTK(D1538A) retains the ability to bind acetylated H4 in vitro, indicating that the introduced mutation disrupts kinase activity rather than acetylated lysine-based substrate recognition. In addition to kinase domains, TAF1 has a histone acetyltransferase (HAT) domain that acetylates H3 Lys¹⁴ (H3-K14) and unidentified lysines in H4 in vitro (Maile, 2004).

Dynamic changes in chromatin structure, induced by posttranslational modification of histones, play a fundamental role in regulating eukaryotic transcription. Histone H2B is phosphorylated at evolutionarily conserved Ser³³ (H2B-S33) by the carboxyl-terminal kinase domain (CTK) of the Drosophila TFIID subunit TAF1. Phosphorylation of H2B-S33 at the promoter of the cell cycle regulatory gene string and the segmentation gene giant coincides with transcriptional activation. Elimination of TAF1 CTK activity in Drosophila cells and embryos reduces transcriptional activation and phosphorylation of H2B-S33. These data reveal that H2B-S33 is a physiological substrate for the TAF1 CTK and that H2B-S33 phosphorylation is essential for transcriptional activation events that promote cell cycle progression and development (Maile, 2004).

Transcription initiation in eukaryotes involves dynamic changes in chromatin structure that permit assembly of the transcription machinery at a gene promoter. The fundamental structural unit of chromatin is the nucleosome, which contains 146 base pairs of DNA wrapped around a histone octamer composed of two copies each of histones H2A, H2B, H3, and H4. Distinct patterns of histone modifications (e.g., acetylation, phosphorylation, and methylation) may act as 'modification cassettes' that facilitate DNA-dependent events. For example, in vertebrates phosphorylation of H2B Ser¹⁴ is associated with apoptotic chromatin, and in all eukaryotes phosphorylation of H3 Ser¹⁰ is associated with transcriptionally active and mitotic chromatin. Although all histones are phosphorylated in vivo, the function of many of these modifications and the kinases that carry them out are not known (Maile, 2004).

With the use of an in vitro kinase assay, it was found that the Drosophila general transcription factor (GTF) TFIID phosphorylates histone H2B but not H1, H2A, H3, or H4. TFIID is a multiprotein complex composed of the TATA box–binding protein (TBP) and numerous TBP-associated factors (TAFs). TFIID functions during transcription initiation by nucleating assembly of GTFs and RNA polymerase II at the promoter. TAF1 (formerly TAF_II250) is the only TFIID subunit that possesses kinase activity, suggesting that it phosphorylates H2B (Wassarman, 2001). In fact, recombinant TAF1 and denatured and renatured recombinant TAF1 phosphorylated H2B in vitro, demonstrating that TAF1 has intrinsic, H2B-specific kinase activity. Collectively, these results indicate that TAF1 alone and in the context of TFIID phosphorylates H2B (Maile, 2004).

TAF1 contains two kinase domains, an N-terminal (NTK, amino acids 1 to 496) and a C-terminal (CTK, amino acids 1496 to 2132) domain (Dikstein, 1996). In vitro, the NTK and the CTK autophosphorylate and the NTK transphosphorylates the RAP74 subunit of the GTF TFIIF. To determine which domain phosphorylates H2B, NTK and CTK were assayed separately in vitro. In contrast to the NTK, the CTK did not phosphorylate RAP74 but strongly phosphorylated H2B, indicating that the CTK possesses H2B-specific kinase activity (Maile, 2004).

Protein kinases contain two essential functional motifs, an adenosine triphosphate (ATP) binding motif and an amino acid–specific kinase motif. Computational sequence comparison analyses identified a putative serine and threonine (S/T) kinase motif (amino acids 1534 to 1546) and two tandem ATP binding domains (amino acids 1747 to 1780) in the CTK. To test whether the identified motifs mediate H2B phosphorylation, in vitro kinase assays were performed with the use of CTK polypeptides lacking the S/T kinase motif (CTKDelta1600) or the ATP binding motifs (CTKDeltaATP). Relative to the wild-type CTK, CTKDelta1600 and CTKDeltaATP weakly phosphorylated H2B. To confirm the role of the S/T kinase motif, a catalytically important aspartic acid was mutated to an alanine (D1538A) in the motif. Like CTKDelta1600, CTK(D1538A) exhibited weak autophosphorylation and H2B transphosphorylation activities. Interestingly, the S/T kinase motif is located in the first bromodomain of the double bromodomain module (DBD), which binds acetylated lysines. However, CTK(D1538A) retains the ability to bind acetylated H4 in vitro, indicating that the introduced mutation disrupts kinase activity rather than acetylated lysine-based substrate recognition. Thus, the identified S/T kinase and ATP binding motifs of the TAF1 CTK are essential for H2B phosphorylation (Maile, 2004).

To identify H2B residue(s) phosphorylated by the CTK, whether the CTK phosphorylates the N-terminal tail of Drosophila H2B (amino acids 1 to 39, H2BT) or the tailless H2B core domain (amino acids 40 to 123) was examined; the CTK phosphorylated H2BT but not the H2B core domain. Next, to pinpoint which residue(s) in H2BT is phosphorylated, mutant H2BT peptides were generated in which alanines replaced all or individual serines or threonines. The CTK did not phosphorylate peptides lacking all serines, suggesting that it phosphorylates either Ser⁵ (H2B-S5) or Ser³³ (H2B-S33). To test this, H2BT peptides were used as substrates that contained alanines in place of H2B-S5, H2B-S33, or both (H2BT-S5A, H2BT-S33A, and H2BT-S5/33A, respectively). The CTK phosphorylated H2BT-S5A but not H2BT-S33A or H2BT-S5/33A, indicating that H2B-S33 is the target of the CTK (Maile, 2004).

To investigate whether H2B-S33 is phosphorylated in vivo, a polyclonal antibody was raised recognizing phosphorylated H2B-S33 (H2B-S33P). On Western blots, the antibody recognized H2BT containing H2B-S33P but not recombinant, unphosphorylated H2B or an H3 peptide (amino acids 1 to 32) containing phosphorylated Ser¹⁰ and Ser²⁸. In addition, the H2B-S33P antibody recognized H2BT and recombinant H2B that was phosphorylated in vitro by the CTK or TFIID, indicating that the antibody specifically recognizes phosphorylated H2B-S33. The H2B-S33 antibody also recognized a protein with a molecular weight similar to that of H2B from histone preparations from Drosophila embryos or S2 cells, providing evidence that H2B-S33 is a target for phosphorylation in vivo. To determine whether TAF1 mediates H2B-S33 phosphorylation in vivo, RNA interference (RNAi) was used to eliminate TAF1 expression in S2 cells. As shown by Western blot analysis, both TAF1 expression and H2B-S33 phosphorylation were reduced in TAF1 RNAi cells compared with mock RNAi cells, suggesting that TAF1 is a major H2B-S33 kinase in vivo (Maile, 2004).

Flow cytometry analysis of TAF1 RNAi cells revealed that loss of TAF1 results in G2-M phase cell cycle arrest. To test the hypothesis that TAF1 controls the transcription of genes whose activities contribute to G2-M progression, microarray expression profiling and reverse transcription polymerase chain reaction (RT-PCR) were used to monitor transcription in mock and TAF1 RNAi cells. Both methods showed that transcription of string (stg), which encodes a Drosophila homolog of yeast Cdc25, was reduced. The Stg protein phosphatase is predominantly expressed during G2 and activates the cell cycle by dephosphorylating Cdc2. Because loss of stg from S2 cells by RNAi causes G2-M arrest, TAF1 may regulate G2-M progression by activating stg transcription (Maile, 2004).

Chromatin immunoprecipitation (XChIP) was used to establish whether there is a direct correlation between transcriptional activation of stg and TAF1-mediated phosphorylation of H2B-S33 at the stg promoter. Cross-linked chromatin was isolated from mock and TAF1 RNAi S2 cells and immunoprecipitated with TAF1 or H2B-S33P antibodies. Immunoprecipitated DNA was purified and used as a template for PCR to detect the stg promoter or coding region and actin5C promoter. In contrast, TAF1 is not essential for actin5C transcription, and H2B-S33P antibodies do not precipitate the actin5C promoter. Thus, the transcriptional dependence of a gene for TAF1 is correlated with H2B-S33 phosphorylation, not with TAF1 association (Maile, 2004).

To distinguish whether loss of H2B-S33 phosphorylation at the stg promoter is due directly to loss of TAF1 or indirectly to G2-M arrest, XChIP analysis was performed on S2 cells arrested in G2-M by RNAi of the SIN3 transcriptional corepressor. Stg transcription is repressed in SIN3 RNAi cells, yet the stg promoter remains associated with H2B-S33P and TAF1, indicating that loss of H2B-S33 phosphorylation in TAF1 RNAi cells is because of elimination of TAF1 rather than G2-M arrest (Maile, 2004).

In addition to kinase domains, TAF1 has a histone acetyltransferase (HAT) domain that acetylates H3 Lys¹⁴ (H3-K14) and unidentified lysines in H4 in vitro (Mizzen, 1996). XChIP analysis detected acetylated H3-K14 and H4 at the transcriptionally active stg promoter in mock RNAi cells but not at the inactive stg promoter in TAF1 RNAi cells. In contrast, TAF1-independent histone modifications did not correlate with activation of stg in mock and TAF1 RNAi cells. Taken together, these results indicate that TAF1-mediated phosphorylation of H2B-S33 and acetylation of H3 and H4 potentiate transcriptional activation in Drosophila cells (Maile, 2004).

To investigate the role of TAF1-mediated phophorylation of H2B-S33 during fly development, a recessive lethal TAF1 allele, TAF1^CTK, was used which contains a nonsense mutation at amino acid 1728 that truncates the CTK downstream of the DBD was used. The corresponding protein (TAF1DeltaCTK) is expressed in Drosophila but presumably does not have CTK activity, because it does not phosphorylate H2B in vitro. In situ hybridization was used to monitor transcription in embryos homozygous mutant for TAF1^CTK and heterozygous mutant for the maternal activator Caudal (Cad). In this genetic background, transcription of the gap gene giant (gt) was reduced. Gt is transcribed in two domains along the anterior-posterior axis of blastoderm-stage embryos. Transcription of the posterior gt domain (pgt) is Cad-dependent, whereas transcription of the anterior gt domain (agt) is Cad-independent. Relative to controls (cad/+ or TAF1^CTK), pgt transcription was reduced in cad/+;TAF1^CTK embryos (Maile, 2004).

XChIP analysis was used to examine whether TAF1-mediated phosphorylation of H2B-S33 contributes to pgt transcription. Cross-linked chromatin was isolated from the posterior halves of cad/+;TAF1^CTK and control embryos and immunoprecipitated with antibodies to H2B-S33P, acetylated histones, or TAF1. PCR analysis detected H2B-S33P at the transcriptionally active gt promoter in control embryos, but not at the transcriptionally repressed promoter in cad/+;TAF1^CTK embryos. To monitor TAF1 binding, two antibodies, TAF1-M and TAF1-C, where used that recognize the middle domain and the CTK of TAF1, respectively. Both antibodies precipitated the gt promoter from control embryos, indicating that TAF1DeltaCTK and maternally contributed, wild-type TAF1 are present at the gt promoter in the pgt. In contrast, although the TAF1-M antibody precipitated the gt promoter from cad/+;TAF1^CTK embryos, TAF1-C did not. Because TAF1DeltaCTK is present at a higher concentration in cad/+;TAF1^CTK embryos than maternal TAF1, this result indicates that TAF1DeltaCTK is preferentially recruited to the gt promoter in the pgt. This result is supported by the presence of TAF1-mediated histone acetylation at the transcriptionally silent gt promoter. Thus, TAF1-mediated phosphorylation of H2B-S33 contributes to transcriptional activation during Drosophila embryogenesis (Maile, 2004).

Ser³³ is the only evolutionarily conserved serine or threonine in the N-terminus of metazoan H2Bs. In the crystal structure of the Xenopus laevis nucleosome, the equivalent serine links the H2B DNA-binding N-terminal tail to the histone fold domain. Thus, replacing the hydroxyl group on Ser³³ with a bulkier, negatively charged phosphate group may drastically affect H2B tail interactions with DNA. This is important because the H2B tail regulates nucleosome mobility. Deletion of the tail bypasses the requirement for the SWI/SNF nucleosome-remodeling complex in yeast, and the tail is critical for maintaining the position of histone octamers in in vitro sliding assays. These findings support a model in which TAF1-mediated phosphorylation of H2B-S33 disrupts DNA-histone interactions, resulting in local decondensation of chromatin. Decondensation may trigger chromatin remodeling and formation of a chromatin structure that facilitates assembly of other GTFs at a promoter, a function that is primarily attributed to TFIID (Maile, 2004).

These data indicate that the S/T kinase motif of the CTK is located in the DBD. In the crystal structure of the DBD, the position of the S/T kinase motif does not overlap with the acetylated lysine-binding surface of the DBD, suggesting that it is an independent functional unit of the DBD. Members of the fsh/RING3 (BET) family of DBD proteins have kinase activity, suggesting that TAF1 is a member of a kinase family whose catalytic motif resides within the DBD (Maile, 2004).

Phosphorylation of H2B-S33 by TAF1 is essential for transcriptional activation of stg/cdc25 and, consequently, cell cycle progression. Similarly, depletion of yeast TAF5, human TAF2, or a twofold reduction in chicken TBP results in G2-M arrest. Like TAF1, TBP regulates stg/cdc25 expression, providing support for the finding that the H2B-S33 kinase activity of TAF1 occurs in the context of TFIID. Interestingly, depletion of yeast TAF1, which does not possess a CTK, and inactivation of TAF1 HAT activity induce G1 arrest because of reduced transcription of B- and D-type cyclins, respectively (Apone, 1996; Dunphy, 2001). Thus, loss of all TAF1 activities causes G2-M arrest whereas loss of TAF1 HAT activity causes G1 arrest, suggesting gene-specific requirements for TAF1 CTK and HAT activities. In contrast, the presence of phosphorylated H2B-S33 and acetylated H3 and H4 at the stg and gt promoters implies that TAF1 CTK and HAT activities can cooperate in transcriptional activation of some genes. This proposal is supported by the finding that loss of H2B-S33P from the gt promoter results in reduced transcription, despite the presence of TAF1-mediated histone acetylation. Thus, TAF1-mediated phosphorylation of H2B-S33 may work in concert with other TAF1-mediated histone modifications, H1 ubiquitination, and H3 and H4 acetylation to contribute to the chromatin-based mechanisms underlying transcription activation of eukaryotic genes (Maile, 2004).

Prospero, targeting stg, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2–5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10–11 embryos, approximately 4–7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

Hindsight mediates the role of Notch in suppressing Hedgehog signaling and cell proliferation

Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle (M/E switch). This study shows that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of cubitus interruptus. These studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation (Sun, 2007).

How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. These data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Sun, 2007).

Studies have shown that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature-cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed. The current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle (Sun, 2007).

The finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots, suggesting that an additional cell cycle also takes place. Further testing of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may not be the sole mediator of the Notch effect; for example, Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later, suggesting that Hnt function is also required for chorion-gene amplification (Sun, 2007).

The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells. Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots. The nuclear sizes of fu mutant cells were similar to those of the wild-type at the same developmental stage, and no fragmentation of the chromosomes was observed. Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus, Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of Ci-155 in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis. Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells (Sun, 2007).

Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells, but it is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt. Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Sun, 2007).

An interesting observation from these studies is that ttk clones have a phenotype similar to that of hnt clones. As in Notch regulation of Hnt, ttk is possibly downstream of Notch, but the current analysis of Notch mutants in stage-1 to stage-10 egg chambers showed no obvious change in Ttk expression. It was also found that Hnt has no role in regulating ttk expression. The findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis. The phenotypic similarity between hnt and ttk mutants suggests that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye. Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear (Sun, 2007).

Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt-deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process. Second, nurse-cell endoreplication does not require Hnt; no obvious defect was detected in hnt germline clones. The specific role of Hnt in follicle-cell-cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation (Sun, 2007).

Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on the cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway. Hh and Notch therefore act sequentially and positively during the SMW, whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis, but it is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling; mutation of the negative regulator of the Hh pathway, ptc, in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germline cells. These ptc mutant cells show no accumulation of Ci-155, consistent with the finding that Notch signaling suppresses ci transcription through Hnt. The ptc^S2 cells that were out of contact with germline cells remained in the mitotic cycle because they could not receive Dl signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state (Sun, 2007).

Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration. When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% of egg chambers showed defects in border-cell migration. Notch signaling, as well as ttk, has been reported to be required for border-cell migration. Hnt was found to be expressed in the border cells and depended on Notch signaling. The occasional hnt border-cell clones observed also showed defects in border-cell migration, so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells (Sun, 2007).

RNA Degradation

In Drosophila, maternal String mRNAs are stable for the first few hours of development, but undergo specific timed degradation at the cellularisation stage. Maternal mRNA degradation is unaffected by zygotic transcription. Therefore, the proteins required to activate the degradation of String mRNA are encoded on a maternally contributed mRNA (Myers, 1995).

Protein Degradation

Tribbles activity regulates cell cycle by directly and posttranscriptionally affecting String expression. During early embryonic development, string is transcribed in a spatial pattern controlled by the anterior-posterior and dorsoventral patterning systems. Expression of String mRNA in a given mitotic domain precedes mitosis by a few minutes. By analyzing the exception to this rule found in domain 10 on the ventral side at the embryo, the tribbles mode of regulation was uncovered. Although string is expressed in these cells, they do not divide until they are internalized. This delay depends on the activity of the tribbles gene (Seher, 2000) named after the small, round, fictional organisms (from the television series "Star Trek") that proliferate uncontrollably when they contact water. The tribbles effect is restricted to the ventral furrow, even though TRBL mRNA is also present outside of this domain and the trbl mutation can be rescued by uniform exogeneous expression. This suggests that trbl activity is triggered by an input which is present only in the ventral furrow region (Großhans, 2000). Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination (Mata, 2000).

String/CDC25 phosphatase is required for G2/M progression in imaginal disc cells. Cells homozygous for a hypomorphic string mutation can grow but they divide very slowly and thus become very large, similar to the tribbles overexpression phenotype. Given that string is a limiting regulator in G2 of imaginal disc cells, it was asked whether increased expression of string would affect the tribbles GOF phenotype. Expression of UAS-string under control of engrailed-GAL4 did not give an overt wing phenotype. When UAS-string is coexpressed with UAS-tribbles, the posterior compartment reverts to the normal cell number and density. Thus, overexpression of string suppresses the effect of tribbles overexpression in imaginal disc cells. In the germline of the ovary, overexpression of string also suppresses the extra cystocyte division phenotype caused by tribbles expression. Thus, despite the apparent differences in cell cycle control mechanisms, tribbles opposes or downregulates string/CDC25 in both the wing disc and the germline (Mata, 2000).

To see whether overexpression of tribbles affects String protein levels, discs were double-stained with anti-String and anti-Tribbles antibodies. Cells that overexpress tribbles do not show normal levels of endogenous String protein. String expression in third instar larval discs is nonuniform due to asynchronous cycling of the cells. By counting expressing and nonexpressing cells in several discs, it was verified that String-expressing cells do not express Tribbles. The lack of cells coexpressing String and Tribbles could not be explained by cells being in the wrong phase of the cell cycle to accumulate String, since overexpression of tribbles delays cells in G2, the phase of the cell cycle in which String protein normally accumulates. Expression of tribbles also affects the level of String protein when both were overexpressed by the GAL4/UAS system. The overall level of overexpressed String protein is clearly reduced by Tribbles, although not to background levels. The simplest interpretation of these results is that Tribbles directly and posttranscriptionally affects String expression (Mata, 2000).

To investigate the interaction between tribbles and string under more controlled conditions, tissue culture experiments were carried out. The CDC25 homologs String and Twine, and the mitotic Cyclins A and B, were epitope tagged at the N terminus and cloned into the metallothionine expression vector. When cotransfected into Schneider cells, expression of Tribbles severely reduced the level of HA-String. This reduction in protein level was observed without any change in HA-String mRNA levels. Tribbles has a similar effect on HA-Twine protein levels, although less severe, but has no effect on the mitotic Cyclins A and B. Given that the promoter, the 5'UTR and 3'UTRs as well as the initiator methionine and N-terminal epitope tag were identical in the HA constructs, it was reasoned that Tribbles might be acting by increasing String (and Twine) protein turnover. To test this hypothesis, the proteosome inhibitor lactacystin was used. Addition of lactacystin reverses the effect of Tribbles on HA-String protein. This indicates that Tribbles induces degradation of String protein via a proteosome-dependent pathway. To confirm this, a pulse-chase experiment was performed. As expected, metabolically labeled HA-String disappears more rapidly in the presence of Tribbles. Mitotic cyclins are also degraded via the proteosome, stimulated by anaphase-promoting complex (APC). Because Cyclin levels are unaffected by Tribbles, it has been concluded that Tribbles regulates CDC25 protein turnover in a specific manner, not just by generally increasing proteosome activity (Mata, 2000).

To determine whether endogenous tribbles acts by affecting CDC25/String turnover as proposed, String mRNA and protein accumulation were examined in tribbles mutant embryos. While String mRNA appears to accumulate normally, tribbles mutant embryos show a moderate, but reproducible, increase in String protein level at 2-4 hr of development. string mRNA and high level of tribbles mRNA coincide primarily in the ventral region in late stage 5 embryos. Since Western blots average the effect over the total String protein in the embryo, it is probable that the magnitude of the effect is higher in the presumptive mesodermal cells. This suggests that the main role of Tribbles is to delay accumulation of zygotic String protein. This view is supported by the observation that removing string suppresses the zygotic requirement for tribbles (Mata, 2000).

The delay in their mitosis suggests that ventral cells contain a factor lengthening the gap between appearance of String mRNA and entry into mitosis. This delay involves a subtle titration of string activity, since it can be shortened by addition of two more copies of the string chromosomal region, raising the copy number of string to four. Under these conditions the ventral cells divide at about the same time as the cells of domains 1 to 3, which matches the String mRNA pattern more closely than it does in wild-type embryos. Only the mitosis in domain 10 is shifted in these experiments, the order mitosis in the other mitotic domains is not changed (Großhans, 2000).

To examine more stringently whether the factor counteracting string is specific for ventral cells, exogeneous String mRNA was expressed at the same level in all cells of the embryo, using a UAS-String transgene driven by a maternally provided Gal4 in embryos otherwise homozygous for a string deletion. Four copies of maternally provided Gal4 produce high levels of string activity, indicated by the uniform entry of all cells into mitosis immediately at the beginning of gastrulation. In these embryos ventral furrow formation is inhibited. Using females with three or two Gal4 insertions, expression of string was gradually lowered. This shifts the onset of mitosis to a time when the first mitoses normally occur in wild-type embryos. Under these conditions differences in the behavior of the cells become apparent. In spite of the uniform string expression, the ventral cells undergoing cell shape changes to form the ventral furrow enter mitosis later than the other cells. This special behavior of the ventral cells is not observed in string heterozygous embryos that have endogeneous as well as exogeneous String mRNA. It is concluded from these experiments that ventral cells contain a dosage-sensitive factor, the ventral inhibitor, that counteracts string activity and that the delay of mitosis in domain 10 of wild-type embryos is due to this factor (Großhans, 2000).

In order to identify components that constitute the ventral inhibitor, a genome-wide screen was carried out for loci that are required for a delayed mitosis in domain 10. By screening 99% of the genome, two novel loci, frühstart and tribbles, were identified. In embryos deficient for either of these genes, cells in the ventral domain are the first to enter mitosis, such that their pattern of String mRNA expression and the mitotic pattern match one another. The order of the other mitotic domains is not altered, suggesting that frs and trbl act specifically in the ventral cells. The double mutant frs trbl shows the same phenotype as the single mutants, suggesting that frs and trbl are nonredundant genes in a common process (Großhans, 2000).

As a consequence of the early mitosis, the mesodermal precursors remain on the surface and do not form a proper ventral furrow. This defect is similar to that observed in embryos in which all cells have been forced into mitosis by increased string dosage or string overexpression. Although other zygotically active genes are known to affect formation of the ventral furrow, frs and trbl are unique in that their defects solely depend on the premature mitosis. In double mutant frs string and trbl string embryos, no mitosis takes place during gastrulation, and the ventral furrow forms as in wild-type. The premature mitosis in frs or trbl embryos is not caused by overexpression of String mRNA in the ventral region, since String mRNA is present in comparable amounts in mutant embryos and with a similar pattern as in their heterozygous siblings or wild-type embryos. Since String is the rate-limiting factor for entry into mitosis during gastrulation, this observation suggests that frs and trbl counteract string via a posttranscriptional mechanism (Großhans, 2000).

Drosophila Hfp negatively regulates stg to inhibit cell proliferation

Mammalian FIR has dual roles in pre-mRNA splicing and in negative transcriptional control of Myc. Half pint (Hfp), the Drosophila ortholog of FIR, inhibits cell proliferation in Drosophila. Hfp overexpression potently inhibits G1/S progression, while hfp mutants display ectopic cell cycles. Hfp negatively regulates dmyc expression and function: reducing the dose of hfp increases levels of dmyc mRNA and rescues defective oogenesis in dmyc hypomorphic flies. The G2-delay in dmyc-overexpressing cells is suppressed by halving the dosage of hfp, indicating that Hfp is also rate-limiting for G2-M progression. Consistent with this, the cycle 14 G2-arrest of stg mutant embryos is rescued by the hfp mutant. Analysis of hfp mutant clones revealed elevated levels of Stg protein, but no change in the level of stg mRNA, suggesting that hfp negatively regulates Stg via a post-transcriptional mechanism. Finally, ectopic activation of the wingless pathway, which is known to negatively regulate dmyc expression in the wing, results in an accumulation of Hfp protein. These findings indicate that Hfp provides a critical molecular link between the developmental patterning signals induced by the wingless pathway and dMyc-regulated cell growth and proliferation (Quinn, 2004).

The Drosophila stock EP(3)3058 (hfp^EP) harbors a recessive lethal P element insertion in the 5' UTR of hfp, 94 bp upstream of the initiating methionine codon. Homozygous hfp^EP larvae were of similar size to age-matched wild type third instar larvae. However, the pupariation of hfp^EP larvae was consistently delayed by approximately 2 days, and continued growth during this period resulted in wandering larvae and pupae ~20% larger than wild-type third instar larvae. The duration of the pupal stage was normal for hfp^EP mutant animals; however, they failed to eclose and died as pharate adults that were larger than wild type. The hfp^EP/hfp^EP terminal phenotype included duplication of superior scutellar macrochaete, and malformation of legs, wings and sex combs (Quinn, 2004).

The finding that hfp mutants do not phenocopy dmyc overexpression suggests that inhibition of dmyc expression is not the only role of Hfp. Although increased S phases are observed in hfp mutant wing discs, this is not associated with increased cell size, as occurs with dmyc overexpression in the wing disc. Rather, in hfp mutant wing discs the ZNC, which normally contains domains of G1- and G2-arrested cells, has ectopic S-phase and M-phase cells. Since cells in hfp mutant wing discs are of normal size and ectopically enter S phase, it is possible that progression through G2 may also be accelerated. Indeed, the increased number of mitotic cells observed in eye imaginal discs when the level of Hfp is reduced in a dmyc overexpression background, suggests that Hfp normally negatively regulates G2-M phase progression. Furthermore, the abnormal mitotic figures observed in hfp^EP mutant embryos are consistent with accelerated cell cycle progression. Most importantly, the hfp mutant rescues the cycle 14 G2-arrest that normally occurs in stg mutant embryos, and hfp mutant clones have increased levels of Stg protein, suggesting that Hfp normally exerts an inhibitory affect on G2-M progression via negatively regulating Stg translation or protein stability. Thus, Hfp may be required for negatively regulating both the G1-S phase transition by downregulating dmyc and the G2-M transition by negatively regulating stg (Quinn, 2004).

The Wg pathway is required to downregulate both dmyc and stg expression in order to limit cell proliferation in the ZNC during wing development. Activation of the Wg pathway, using either dominant negative Shaggy or by generation of axin clones, results in a strong and specific increase in Hfp protein, demonstrating that Wg pathway activation is sufficient to cause Hfp induction. These findings support a model in which Wg signalling causes induction of Hfp in the wing disc ZNC, which in turn inhibits dmyc expression (to elicit the posterior, G1 arrest) and stg expression or activity (to provide the anterior, G2-arrested domains). The involvement of Achaete and Scute in this process, which play a role in the negative regulation of stg remains to be elucidated. Previous studies have shown that Ras signalling through Raf/MAPK upregulates dmyc post-transcriptionally in wing disc cells and is required to maintain normal dMyc protein levels in the wing disc. In contrast, since hfp clones have increased dmyc mRNA, Hfp must normally inhibit dmyc mRNA accumulation. Furthermore, overexpression of Hfp inhibits cell proliferation in all wing and eye imaginal discs, suggesting that Hfp may normally override mitogenic signals and lead to cell cycle arrest during particular stages of development (Quinn, 2004).

In summary, these results suggested that Hfp negatively regulates cell proliferation by inhibiting dmyc transcription and Stg protein accumulation. Hfp is required for the developmentally regulated cell cycle arrest within the ZNC and is responsive to the Wg signalling pathway that regulates this arrest, suggesting that Hfp links patterning signals to cell proliferation during Drosophila development (Quinn, 2004).

Terminal mitoses require negative regulation of Fzr/Cdh1 by Cyclin A, preventing premature degradation of mitotic cyclins and String/Cdc25

Cyclin A expression is only required for particular cell divisions during Drosophila embryogenesis. In the epidermis, Cyclin A is strictly required for progression through mitosis 16 in cells that become post-mitotic after this division. By contrast, Cyclin A is not absolutely required in epidermal cells that are developmentally programmed for continuation of cell cycle progression after mitosis 16. These analyses suggest the following explanation for the special Cyclin A requirement during terminal division cycles. Cyclin E is known to be downregulated during terminal division cycles to allow a timely cell cycle exit after the final mitosis. Cyclin E is therefore no longer available before terminal mitoses to prevent premature Fizzy-related/Cdh1 activation. As a consequence, Cyclin A, which can also function as a negative regulator of Fizzy-related/Cdh1, becomes essential to provide this inhibition before terminal mitoses. In the absence of Cyclin A, premature Fizzy-related/Cdh1 activity results in the premature degradation of the Cdk1 activators Cyclin B and Cyclin B3, and apparently of String/Cdc25 phosphatase as well. Without these activators, entry into terminal mitoses is not possible. However, entry into terminal mitoses can be restored by the simultaneous expression of versions of Cyclin B and Cyclin B3 without destruction boxes, along with a Cdk1 mutant that escapes inhibitory phosphorylation on T14 and Y15. Moreover, terminal mitoses are also restored in Cyclin A mutants by either the elimination of Fizzy-related/Cdh1 function or Cyclin E overexpression (Reber, 2006).

Mitotic cyclins accumulate during the S and G2 phases of the cell cycle. Their C-terminal cyclin boxes mediate binding to cyclin-dependent kinase 1 (Cdk1). Their rapid degradation during late M and G1 phase depends on the D- and KEN-boxes in their N-terminal domains. These degradation signals are recognized by Fizzy/Cdc20 (Fzy) and Fizzy-related/Cdh1 (Fzr), which recruit the mitotic cyclins to the anaphase-promoting complex/cyclosome (APC/C) during M and G1, respectively. The ubiquitin ligase activity of the APC/C allows cyclin poly-ubiquitination and consequential proteolysis (Reber, 2006).

Metazoan species express three different types of mitotic cyclins: A, B and B3. The specific functions of these different cyclins are not understood in detail. The presence of single genes coding for either Cyclin A (CycA), Cyclin B (CycB) or Cyclin B3 (CycB3) has facilitated a genetic dissection of their functional specificity in Drosophila melanogaster. In this organism, development to the adult stage requires the zygotic function of CycA, but not of CycB or CycB3. Initial analysis of the embryonic cell proliferation program in CycA mutants revealed that epidermal cells fail to progress through the sixteenth round of mitosis. Cyclin A is also required for mitosis 16 in the epidermis of dup/Cdt1 mutant embryos, in which mitosis 16 is no longer dependent upon completion of the preceding S phase. The failure of mitosis 16 in CycA mutants therefore does not simply result from the activation of a DNA replication or damage checkpoint -- a possibility suggested by evidence obtained in vertebrate cells in which Cyclin A binds not only to Cdk1 but also to Cdk2, and provides crucial functions during S phase (Reber, 2006 and references therein).

The accumulation of Cyclin B and Cyclin B3 during cycle 16, which also occurs in CycA mutants, complicates the explanation of why mitosis 16 in the epidermis requires Cyclin A. In Xenopus egg extracts, Cyclin B can trigger entry into mitosis in the absence of Cyclin A. Conversely, mitosis is clearly inhibited in cultured human cells after the microinjection of antibodies against cyclin A. Cyclin A-Cdk1 complexes are thought to have special properties, important for starting up a positive-feedback loop that confers a switch-like behavior on the Cdk1 activation process. In this feedback loop, Cdk1 activity results in phosphorylation and suppression of the inhibitory Wee1 kinase, as well as in phosphorylation and activation of the String/Cdc25 phosphatase, which removes the inhibitory phosphate modifications from Cdk1. However, the analyses described in this study indicate that the Cyclin A requirement in Drosophila is not linked to this positive-feedback loop. Rather, it is linked to the fact that the sixteenth round of mitosis during embryogenesis is the last cell division for the great majority of the epidermal cells (Reber, 2006).

After mitosis 16, most epidermal cells enter a G1 phase and become mitotically quiescent. By contrast, all the previous embryonic divisions (mitoses 1-15) are followed by an immediate onset of S phase. The G1 phase after mitosis 16 is therefore the first G1 phase during development. Entry into this G1 phase is dependent upon a complete, developmentally controlled inactivation of Cyclin E-Cdk2 and Cyclin A-Cdk1, because both complexes can trigger entry into S phase. Cyclin E-Cdk2 inactivation results from transcriptional CycE downregulation and concomitant upregulation of dacapo, which encodes the single Drosophila CIP/KIP-type inhibitor specific for Cyclin E-Cdk2. Cyclin A-Cdk1 inactivation is dependent on Fzr, which is also transcriptionally upregulated. Moreover, Fzr is activated as a consequence of Cyclin E-Cdk2 inactivation. Importantly, this cell cycle exit program is initiated already during G2 of the final division cycle (Reber, 2006).

Although cycle 16 is the final division cycle for most epidermal cells, some defined regions do not activate the cell cycle exit program during cycle 16. Instead, they maintain CycE expression, enter S phase immediately after mitosis 16 and complete an additional division cycle 17. In these regions, mitosis 16 is not fully inhibited in CycA mutants. Cyclin A is therefore especially important for terminal mitoses preceding G1 and cell cycle exit. This study shows that the downregulation of Cyclin E-Cdk2 before terminal divisions, in preparation for the imminent cell cycle exit, converts Cyclin A from a redundant into an indispensable, negative regulator of Fizzy-related/Cdh1, preventing premature degradation of the mitotic inducers String/Cdc25 and the mitotic cyclins. The significance of the basic cell cycle regulator Cyclin A therefore depends on the developmental context (Reber, 2006).

The phenotypical characterization of mutations in the Drosophila genes encoding the A-, B- and B3-type cyclins have indicated that Cyclin A is the most crucial of these co-expressed mitotic cyclins. Although zygotic CycB or CycB3 function is not essential for cell proliferation and development to the adult stage, null mutations in CycA result in embryonic lethality. This study has clarified the molecular basis of the distinct importance of Cyclin A. The results indicate that the crucial role of Cyclin A is linked to its ability to inhibit Fzr-APC/C-mediated degradation. Moreover, this Cyclin A-dependent negative regulation of the Fzr-APC/C-degradation pathway is of particular importance for progression through the very last mitotic division preceding cell cycle exit and the proliferative quiescence of epidermal cells during embryogenesis. This particular Cyclin A requirement during terminal divisions is caused by a cell cycle exit program that is initiated already before the terminal mitosis. The cell cycle exit program includes downregulation of Cyclin E-Cdk2, which has a comparable ability to inhibit the Fzr-APC/C-degradation pathway to Cyclin A. The downregulation of Cyclin E-Cdk2 by the cell cycle exit program turns Cyclin A into an indispensable inhibitor of the premature degradation of mitotic cyclins and String/Cdc25 via Fzr-APC/C before the terminal mitosis. Accordingly, the terminal mitosis in the epidermis of CycA mutants can be restored by overexpression of Cyclin E, by genetic elimination of Fzr, or by simultaneous expression of the String/Cdc25-independent Cdk1^AF mutant and B-type cyclin versions that are no longer Fzr-APC/C substrates (Reber, 2006).

The fact that Cyclin A is also a substrate of Fzr-APC/C-mediated degradation complicates the interpretation of the results. Two findings, however, strongly suggest that Cyclin A functions not just downstream of Fzr, but also upstream as a negative regulator. The observed premature loss of B-type cyclins in CycA mutants is readily explained by a negative effect of Cyclin A on Fzr-APC/C activity and is difficult to explain if Cyclin A was only a Fzr-APC/C substrate. Moreover, the suppression of the UAS-fzr overexpression phenotype by co-expression of UAS-CycA, which is described here, includes the re-accumulation of B-type cyclins and not just the restoration of terminal mitosis 16 (Reber, 2006).

Work in mammalian cells has clearly established that Cyclin A functions as a negative regulator of Fzr/Cdh1. Human Cyclin A can bind directly to Cdh1. Moreover, Cyclin A-dependent Cdk activity phosphorylates Cdh1, resulting in the dissociation of Cdh1 from APC/C. Conversely, mutations in Cdk consensus phosphorylation sites of human CDH1 were reported to abolish inhibition by Cyclin A. The current findings point to alternative modes of Fzr-APC/C-inhibition by Cyclin A. Fzr^psm variant no longer contains canonical Cdk consensus phosphorylation sites (S/T P) and yet its activity is still suppressed by CycA overexpression. Fzr inhibition by CyclinA-dependent phosphorylation of non-consensus sites remains a possibility in Drosophila. However, it is pointed out that, apart from a potential control by Cdk phosphorylation, Fzr is inhibited by the Emi1-like Drosophila protein Rca1. Rca1 overexpression has been shown to prevent premature Cyclin B degradation and restore mitosis 16 in the epidermis of CycA mutant embryos. Based on these observations, the failure of mitosis 16 in CycA mutants was proposed to reflect premature Fzr activation, a suggestion fully confirmed by the current work. It is conceivable, therefore, that the Cyclin A-mediated suppression of Fzr^psm activity involves Rca1 or other unknown targets. The fact that not only Cyclin A, but also Cyclin E, effectively suppresses Drosophila Fzr and Fzr^psm provides further support of additional regulatory complexity. In vertebrate systems, only Cyclin A and not Cyclin E was shown to bind and inhibit Cdh1 (Reber, 2006).

The current findings demonstrate that the Cyclin A requirement in epidermal cells is maximal for progression through the last mitosis of Drosophila embryogenesis, which precedes cell cycle exit and proliferative quiescence. A prominent Cyclin A requirement for terminal mitoses appears to exist in neuroblast lineages during development of the embryonic CNS, although definitive proof will require further work. On the basis of this analysis in epidermal cells, a high Cyclin A requirement for entry into mitosis is expected whenever Fzr levels are high and Cyclin E levels low. During the comparatively slow postembryonic cell cycles of imaginal cells, the periodicity of Cyclin E expression is presumably far more pronounced than during the rapid embryonic cycles in which the persistent presence of maternally contributed Cyclin E eliminates G1 phases. In imaginal cell cycles, which have a G1 phase, Cyclin E expression might therefore be low before each mitosis, and not just before terminal divisions. In combination with Fzr expression, every imaginal mitosis might therefore be strongly dependent upon Cyclin A. By contrast, in the absence of Fzr, progression through mitosis appears to be almost completely independent of Cyclin A, as is evidenced by the observation that the epidermal cells in fzr CycA double mutant embryos not only progress successfully through mitosis 16, but also complete an extra division cycle 17. Nevertheless, 10% of the late mitosis 17 figures in these double mutants displayed lagging chromosomes, indicating that cell cycle progression is not entirely normal in the absence of Cyclin A (Reber, 2006).

The cell cycle exit program, which is activated during the final division cycle in the embryonic epidermis, includes the strong transcriptional upregulation of the CIP/KIP-type Cyclin E-Cdk2 inhibitor Dacapo, apart from the downregulation of Cyclin E and the upregulation of Fzr. Accordingly, genetic elimination of dacapo function should also restore progression through terminal mitosis 16 in CycA mutants. However, mitosis 16 was not observed in the epidermis of dacapo CycA double mutants. The contribution of Dacapo to Cyclin E-Cdk2 inhibition appears to be insignificant before mitosis 16. After the stage of mitosis 16, however, the epidermal cells in these double mutants entered an endoreduplication cycle, a behavior that is also displayed by some cells in the prospective anterior spiracle region of CycA single mutants. This region does not downregulate Cyclin E during cycle 16 in the wild type, it does not upregulate Dacapo, and it progresses through an additional cycle 17 instead of becoming postmitotic after mitosis 16, in contrast to the great majority of the other epidermal cells. The premature activation of Fzr in CycA mutants, therefore, appears to result in DNA replication origin re-licensing, perhaps as a result of B-type cyclin and geminin degradation. Cyclin E-Cdk2 activity might subsequently trigger endoreduplication in cells in which it is not effectively eliminated by both Cyclin E downregulation and Dacapo upregulation. Importantly, not all cells in the anterior spiracle region of CycA mutants endoreduplicate, some of the cells still manage to divide. This variability could reflect minor differences in the onset and strength of the zygotic Cyclin E expression. The outcome of insufficient Cyclin A levels appears to be highly dependent on the levels of Cyclin E and Fzr, which, in turn, are subject to developmental regulation, in particular during cell cycle exit. The significance of basic cell cycle regulators in vivo is therefore different in various tissues and developmental stages, and most likely in various cultured mammalian cell types as well (Reber, 2006).

Cell divisions in the Drosophila embryonic mesoderm are repressed via posttranscriptional regulation of string/cdc25 by HOW

Cell-cycle progression is tightly regulated during embryonic development. In the Drosophila early embryo, the levels of String/Cdc25 define the precise timing and sites of cell divisions. However, cell-cycle progression is arrested in the mesoderm of gastrulating embryos despite a positive transcriptional string/cdc25 activation provided by the mesoderm-specific action of Twist. Whereas String/Cdc25 is negatively regulated by Tribbles in the mesoderm at these embryonic stages, the factor(s) controlling string/cdc25 mRNA levels has yet to be elucidated. This study shows that the repressor isoform of the Drosophila RNA binding protein Held Out Wing [HOW(L)] is required to inhibit mesodermal cell division during gastrulation. Embryos mutant for how exhibit an excess of cell divisions, leading to delayed mesoderm invagination. The levels of the mitotic activator string/cdc25 mRNA in these embryos were significantly elevated. Protein-RNA precipitation experiments show that HOW(L) binds string/cdc25 mRNA. Overexpression of HOW(L) in Schneider cells reduces specifically the steady-state mRNA levels of a gfp reporter fused to string/cdc25 untranslated region (3'UTR). These results suggest that in wild-type embryos, string/cdc25 mRNA levels are downregulated by the repressor isoform HOW(L), which binds directly to string/cdc25 mRNA and regulates its degradation. Thus, this study proposes a novel posttranscriptional mechanism controlling cell-cycle progression in the Drosophila embryo (Nabel-Rosen, 2005; full text of article).

String/Cdc25 is a limiting factor that controls cell-cycle progression in early embryonic stages after cellularization. Both string/cdc25 mRNA and String/Cdc25 protein are extremely unstable (T_1/2 < 15 min). The instability of the mRNA and protein allows for a sensitive response of String/Cdc25 levels to transcriptional regulation by various transcription factors operating in pattern formation in the embryo. It has been reported that, in addition to the time of initiation of string/cdc25 transcription, accumulation of string/cdc25 mRNA is slower in mitotic domain 10 (MD10) than in MD2. This is consistent with lower mRNA levels detected in MD10 in relation to MD2 in wild-type embryos. This study provides a molecular basis for string/cdc25 mRNA instability. In situ hybridization with string antisense probe as well as RT-PCR experiments demonstrated that, in how mutant embryos, string/cdc25 is upregulated. Moreover, protein-RNA binding experiments showed a direct binding between HOW and string RNA, and in Schneider cells a gfp-string3'UTR reporter mRNA is specifically degraded in the presence of HOW(L). Collectively, these experiments are consistent with HOW(L) being the major factor responsible for string instability in the early embryo (Nabel-Rosen, 2005).

A consensus RNA binding site, (U>A/C/G)ACUAA, has been recently described for the binding of the STAR protein Gld-1. The same sequence has also been characterized in as being a consensus RNA binding site for HOW. Importantly, this sequence, GACUAA, is present in the string 3'UTR. These results are consistent with the idea that HOW binds the relaxed consensus sequence described for Gld-1, which is also present in string 3′UTR. Interestingly, this sequence appears also in the C. elegans cdc25/string homolog, suggesting that Gld-1 (similarly to HOW in Drosophila) may control cdc25/string in C. elegans (Nabel-Rosen, 2005).

The arrest of cell-cycle progression in the invaginating mesoderm must be transient because immediately after the invagination process the cells undergo a round of cell division. Thus, String/Cdc25 protein levels must be downregulated to a narrow time window to enable mesoderm invagination. This time frame may be achieved in the following manner: Twist, a regulator of mesoderm fate, activates the transcription of string/cdc25 and HOW. Maternal HOW, as well as zygotic HOW shown previously to be downstream of Twist, compromises string/cdc25 mRNA levels, and Tribbles (which requires Twist and Snail to perform its activity) reduces String/Cdc25 protein levels at this stage. Thus, in parallel to string/cdc25 transcriptional activation, Twist provides a double safe mechanism that silences string at the mRNA via HOW activity and String protein via Tribbles activity in MD10. The activity of both HOW and Tribbles should enable the eventual accumulation of String/Cdc25 protein at the end of the invagination process to allow cell-cycle progression at this stage. Therefore, both HOW's and Tribbles's inhibitory effect may not be highly efficient. This, together with the constitutive transcriptional activation of string by Twist, may lead to the eventual accumulation of string mRNA and protein levels, overcoming the negative control imposed by HOW and Tribbles. Alternatively, String accumulation may be caused by a more direct inhibition of both HOW and Tribbles activities, possibly by signaling pathways that operate in the mesoderm after its invagination (Nabel-Rosen, 2005).

Maternal HOW appears to reduce string mRNA levels in the lateral ectoderm in stage-5 and -6 embryos in addition to MD10. It is therefore possible that the extra cell divisions detected in these regions may have an additional indirect effect on mesoderm invagination (Nabel-Rosen, 2005).

It is instructive to ask whether HOW regulates additional processes during mesoderm development. Although zygotic how mutants do not exhibit mesoderm defects until late developmental stages, the how germline clone embryos do show significant mesoderm aberrations. The entire somatic muscle pattern of these embryos is severely disrupted, presumably owing to accumulation of defects. At this stage, it is impossible to distinguish between primary and secondary effects induced by the complete lack of HOW. The muscle defects detected in how germline clone embryos suggest that HOW has a broader function in the mesoderm and that it may regulate the levels of an array of essential genes necessary for appropriate mesoderm development. The identification of such genes should elucidate the full regulatory range of HOW activity (Nabel-Rosen, 2005).

Finally, regulation at the level of mRNA metabolism by STAR family proteins has been shown to occur in several developmental systems, for example, C. elegans gld-1 and mammalian quaking. These proteins exhibit a wide range of activities, affecting RNA splicing, mRNA nuclear export, mRNA stability, and possibly others. The advantage of such regulation is the ability to respond rapidly to external signals by controlling the mRNA levels of an array of target genes. The synchronization between muscle-cell differentiation and cell-cycle progression may be based on the activities of both HOW and Tribbles, but the molecular link between both processes has yet to be elucidated (Nabel-Rosen, 2005).

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