Myb oncogene-like


Transcriptional Regulation

Attempts to demonstrate trans-activation activity by the Drosophila Myb gene product have been unsuccessful so far. Co-transfection of Schneider cells with a plasmid expressing the Drosophila homolog of transcriptional co-activator CBP (dCBP or nejire) results in transactivation by Myb. Using this assay system, the functional domains of Myb have been analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution (Hou, 1997).

The DNA replication-related element-binding factor (DREF) is a transcriptional regulator of the Drosophila myb gene

Drosophila melanogaster possesses a single gene, Dm myb, that is closely related to the vertebrate proto-oncogene c-Myb, and its other family members (A-Myb and B-Myb), all of which encode transcription factors. Dm myb is expressed in all proliferating cells throughout development, and previous studies demonstrate that Dm myb promotes both S-phase and M-phase in proliferating cells, while preserving diploidy by suppressing endoreduplication. A characterization of the mechanisms that regulate Dm myb expression has been initiated, and the transcriptional activator DREF was found to activate Dm myb transcription via two binding sites located in the 5' flanking region. The Dm myb promoter lacks a prototypical TATA box sequence and instead appears to use an initiator/downstream promoter element (Inr/DPE) type promoter. Dm myb expression is regulated at the translational as well as transcriptional level (Sharkov, 2002).

Targets of Activity

the Drosophila Myb homolog retains as its presumptive DNA binding domain an evolutionarily conserved typical sequence of 51-53 amino acids, comprising three imperfect tandem tryptophan repeat units (R1-R2-R3). These repeats are located towards the N-terminus of the Myb sequence. Using PCR amplification and the T7 expression vector pET 11d, this tryptophan repeat domain of Drosophila Myb has been expressed in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence (TAACGG) as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 is quenched when synthetic duplex DNA oligomers are added to the protein. The oligomers containing specific Myb target sites quench the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets are also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicate that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues are affected by DNA binding whereas in the latter only the solvent-exposed residues are involved (Madan, 1996).

The c-myb proto-oncogene product (c-Myb) is a transcriptional activator. Vertebrate c-Myb is a key regulator of the G1/S transition in cell cycle, while Drosophila Myb (dMyb) is important for the G2/M transition. dMyb induces expression of cyclin B (a critical regulator of the G2/M transition) in the Drosophila eye imaginal disc. In the wild-type eye disc, dmyb mRNA is expressed in the stripes both anterior and posterior to the morphogenetic furrow. Ectopic expression of C-terminal-truncated dMyb in the eye disc causes ectopic expression of cyclin B and the rough eye phenotype. This rough eye phenotype correlates with prolonged M phase, caused by overexpression of cyclin B. Cyclin B expression is lost in dmyb-deficient clones. In Schneider cells, the activity of the cyclin B promoter is dramatically reduced by loss of dMyb using the RNA interference method. Mutations of the multiple AACNG sequences in the cyclin B promoter also abolish the promoter activity. These results indicate that dMyb regulates the G2/M transition by inducing cyclin B expression via binding to its promoter (Okada, 2002).

To investigate the effect of overexpression of dmyb in Drosophila, transgenic flies were generated carrying a transgene encoding wild-type dMyb expressed from the eye-specific expression vector, Glass multimer reporter (pGMR). The pGMR P-element vector contains a pentamer of the Glass-binding site derived from the Drosophila Rh1 promoter. Since Glass is expressed in the morphogenetic furrow (MF) and the whole region posterior to the MF of the third instar eye imaginal disc, expression of dMyb from this transgene was expected in the MF and the posterior region of the eye disc. Forty independent transgenic lines expressing wild-type dMyb were generated, but none of them exhibited any morphological abnormality in the adult eye. The C-terminal portion of vertebrate c-Myb negatively regulates c-Myb activity. However, it remains unclear whether dMyb also contains a negative regulatory domain (NRD) in its C-terminal region, since a dMyb protein lacking the C-terminal 241 amino acids activates a promoter containing tandem repeats of the Myb-binding site to almost the same extent as wild-type dMyb in co-transfection assays using Drosophila cultured cells. In spite of this, it might be possible that the C-terminal portion of dMyb negatively regulates the dMyb activity in the eye disc. Therefore, transgenic flies were generated carrying a transgene encoding a C-terminal-truncated dMyb lacking the C-terminal 241 amino acids (dMybDeltaC) expressed from the pGMR vector. In contrast to the transgenic flies overexpressing wild-type dMyb, 32 independent transgenic lines expressing the C-truncated dMyb showed a variety of dominant morphological disorders of the adult compound eye (rough eye phenotypes), probably due to the position effect. These results suggest that the C-truncated dMyb has a stronger activity than wild-type dMyb in eye imaginal disc cells. In the present study, two of the transgenic lines were used, GMR–dMybDeltaC-F25 and GMR–dMybDeltaC-F54, which exhibited severe and mild rough eye phenotypes, respectively. As expected, in the transgenic flies (GMR–dMybDeltaC-F25), dmyb mRNA was ectopically expressed in the MF and the whole posterior region to the MF in the developing eye imaginal disc (Okada, 2002).

Since it has been demonstrated that dMyb plays an important role in the G2/M cell cycle transition, the expression of regulators of cell-cycle control was investigated. Ectopic expression of cyclin B, which is a key regulator of the G2/M transition, is seen in the transgenic flies expressing the C-truncated dMyb. Imaginal disc cells in the developing wild-type eye become synchronized at the G1 phase of the cell cycle within the MF and cyclin B is strongly expressed in the stripe posterior to the MF. The width of the cyclin B-expressing region in the GMR–dMybDeltaC-F25 eye imaginal discs is broader than in wild-type control discs. Since the level of cyclin B is tightly regulated by protein degradation, in situ hybridization was used to confirm that ectopic expression of cyclin B in the GMR–dMybDeltaC-F25 eye imaginal discs takes place at the transcriptional level. In the wild-type eye disc, the cyclin B (cycB) mRNA is expressed in the whole region anterior to the MF at a high level and the whole region posterior to the MF at a low level, suggesting that the high expression of cyclin B protein in the stripe posterior to the MF of the wild-type eye disc may be due to protein stabilization. In contrast, cycB mRNA is strongly expressed in the broad stripe posterior to the MF of GMR–dMybDeltaC-F25 eye imaginal discs. To confirm the upregulation of the cycB gene in the GMR–dMybDeltaC-F25 eye imaginal discs, RT–PCR analysis was performed using a series of decreasing amount of RNA prepared from the wild-type and the GMR–dMybDeltaC-F25 eye imaginal discs. The results indicate that the level of cycB mRNA in the GMR–dMybDeltaC-F25 eye imaginal discs is~2.2-fold higher than that in wild-type discs. Since the whole eye disc was used as a source of RNA, the degree of increase in cycB mRNA levels in the region posterior to the MF may be higher than 2.2-fold. Thus, overexpression of C-truncated dMyb in the whole region posterior to the MF of eye imaginal discs causes the rough eye phenotype and ectopic expression of cycB mRNA in the stripe posterior to the MF. No induction of cycB mRNA in the posterior region other than in the stripe suggests that activation of the cycB promoter requires not only dMyb but also other transcription factor(s) expressed in the stripe posterior to the MF (Okada, 2002).

The kinase activity of cyclin B–Cdk1 complex promotes mitosis, whereas the destruction of cyclin B and loss of kinase activity is associated with and required for exit from mitosis. Therefore, ectopic expression of cyclin B in the posterior region of GMR–dMybDeltaC eye imaginal discs might be expected to slow down progression through M phase. To investigate whether the M-phase population in the posterior region of GMR–dMybDeltaC eye imaginal discs is in fact higher than that in wild-type discs, immunostaining was performed using an antibody specific for phosphorylated histone H3. Phosphorylation of histone H3 is induced at M phase and phosphorylated histone H3 can be used as a marker of M-phase cells. The entire region in the anterior and posterior portion of MF was investigated. In order to count the stained cells that are located both at the apical and basal surface, discs were visualized with a fluorescent microscope at the lower magnification. The numbers of cells stained by anti-phospho-histone H3 antibody in the whole region posterior to the MF of the GMR–dMybDeltaC-F25 and the wild-type eye discs were 93 and 59 (average of 11 discs), respectively. Thus, the population of M-phase cells in the posterior region of GMR–dMybDeltaC-F25 eye discs was 58% larger than the corresponding population in wild-type discs. In contrast, the numbers of cells stained by anti-phospho-histone H3 antibody in the whole region anterior to the MF of the GMR–dMybDeltaC-F25 and the wild-type eye discs were 44 and 49 (average of 11 discs), respectively, indicating that the populations of M-phase cells are similar sizes in the anterior region of GMR–dMybDeltaC-F25 and wild-type eye discs (Okada, 2002).

dMyb-deficient clones are not tiny, suggesting that the dmyb-deficient cells keep proliferating for a while to produce clones of reasonable size. This may suggest that loss of dMyb function slows down cell-cycle progression through mitosis but does not stop the cell cycle. In fact, the presence of M-phase cells stained with anti-phospho-histone H3 antibody was observed in the dmyb-deficient clone, suggesting that loss of dMyb does not block the G2/M transition. This is consistent with the report that loss of either cyclin A or cyclin B does not block the cell-cycle progression in Drosophila embryos and that loss of both cyclins blocks the cell-cycle progression. Probably, dMyb is required for expression of cyclin B but not cyclin A. However, the possibility that the dMyb protein is relatively stable and persists in mutant cells for several generations after mitotic recombination events so that mutant cells can continue to divide for a while cannot be excluded. Further analyses are required for understanding the precise role of dMyb in cell-cycle regulation (Okada, 2002).

The results suggest that overexpression of the C-truncated dMyb retains the cells in M phase and that the abnormal cell-cycle regulation causes apoptosis. These two events result in the rough eye phenotype. Since R2-5 and R8 are already determined by the time that the GMR promoter is activated, these cells presumably are not affected by overexpression of dMyb. This suggests that R1, 6 and 7 and later cell types are specifically affected in these discs. One possibility is that, since mitosis may be prolonged, cells are unable to respond to differentiation signals in the appropriate temporal window and this may also lead in part to cell death (Okada, 2002).

Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells

Myb-MuvB (MMB)/dREAM is a nine-subunit complex first described in Drosophila as a repressor of transcription, dependent on E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent on DNA sites and developmental context. This study used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNA interference and microarray expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupied 3538 chromosomal sites and was promoter-proximal to 32% of Drosophila genes. MMB contains multiple DNA-binding factors, and the data highlighted the combinatorial way by which the complex was targeted and utilized for regulation. Interestingly, only a subset of chromatin-bound complexes repressed genes normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression, whereas at other nonoverlapping sites, Myb was critical for repression. Sites were also found where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants (Georlette, 2007).

MMB is ~700 kDa, and contains a unique ensemble of nine proteins, of which five are capable of binding to DNA: Myb, E2F2/DP, and Mip120 are site-specific DNA-binding factors, and a fifth protein, Mip130, contains an A-T hook domain capable of binding to AT-rich DNA. Although E2F2 and Myb have been widely studied individually, nothing has yet been explored as to how these DNA-binding proteins behave in an ensemble. Perhaps the more biologically relevant question is: How do the different site-specific DNA-binding proteins choose where to bind among the potentially large number of binding sites within the genome of a cell? It is likely that all or some combination of the DNA-binding activities may participate in MMB targeting to specific DNA sites. The other MMB factors (RBF1 or RBF2, Caf1/p55, Mip40, and Lin-52) are not known as DNA-binding proteins, but may contribute indirectly to DNA targeting through association with histones (Georlette, 2007).

The major findings reported here revealed the extraordinary diversity of use and combinatorial requirements for the factors of the MMB/dREAM complex in regulating gene expression. The Venn diagrams and subclasses for each set of genes regulated by MMB members illustrate this point for both the transcriptional repression and activation of gene expression by MMB. In the initial characterization of the complex, E2F2 and the RBFs but not Myb were found central for transcriptional repression. A more complete description of the network of genes regulated by MMB, and the genome-wide location of the individual MMB members analyzed in this study revealed a more complex picture of the MMB regulatory network than previously thought. Some of this complexity may be the result of complexes containing subsets of individual MMB members. However, this scenario would not significantly change the present prospective. This is so because >50% of all genes in each of the classes had proximal intact MMB complexes as determined by ChIP-chip analysis. For example, the genes involved in the G2/M transition and chromosome maintenance contained all five MMB members tested at sites proximal to the expressed gene, irrespective of the class into which the gene was classified by RNAi and microarray analysis (Georlette, 2007).

Levels of Myb or E2F2 were dependent on Mip120 and Mip130 but not vice versa, and hence one would not anticipate classes of regulated genes to be dependent solely on Myb or E2F2. A number of confounding issues may contribute to this paradox, including incomplete RNAi and the possibility that partial complexes, arising after loss of a factor after ablation, may remain bound to a site. Such limitations might lead to misclassification of a certain gene(s). Focusing on Myb as an example, removal of Myb may critically limit regulation of a class of genes with different kinetics than would be observed following depletion of Mip120 or Mip130. Class Q is one such class for which the depletion of only Myb (and not any other MMB member) resulted in lowered transcript levels for Class Q members. One hypothesis is that RNAi treatment to remove either Mip120 or Mip130 resulted in incomplete loss of Mip120 and Mip130 such that just enough of the MMB complex remained targeted to such sites where MMB levels were sufficient to produce normal transcript levels for that gene. It is inferred that for such sites in the Q class, Myb loss would be most limiting (Georlette, 2007).

Previous genetic and biochemical studies that linked the MMB members as one entity have been substantiated and extended by the data presented in this study. The five MMB members tested (Myb, Mip120, Mip130, E2F2, and Lin-52) had strikingly coincident genomic binding site profiles. However, among the 3538 MMB-binding sites where the five proteins colocalized, there were significant differences in the enrichment signals. The observed variations in signal strengths at different sites for MMB members by ChIP-chip provided some intriguing correlations. For Class A repressed genes, the Myb ChIP-chip signal was lower than the ChIP-chip signal for E2F2, and this relationship was reversed for the Class B repressed genes. There are many potential reasons for such differences in signal strengths including: different cofactor associations with MMB bound to a particular class of gene promoters, different subunit conformations/accessibility (resulting in altered pull-down antibody affinities), or perhaps because the formaldehyde cross-linking efficiency was highest for a protein that had a high affinity for a proximal DNA site. Moreover, MMB subunit composition in vivo may be dynamic, and off-rates for a DNA-binding factor may be lowest for those MMB factors that were tightly bound to a DNA site. Hence, for Class A genes that required E2F2 for repression, the cross-linking efficiency and potential targeting of E2F2 might dominate, whereas for Class B genes that required Myb for repression, the reverse would be found (Georlette, 2007).

Consistent with this view, a statistically significant enrichment of Myb consensus binding sites was found nearby Class B genes and E2F2 consensus binding sites nearby Class A genes. MMB targeting to one example was directly examined for both Class A and Class B genes, and for the Class A gene, E2F2 was critical for DNA binding whereas for the Class B gene, Myb was found to be a key targeting factor. Furthermore, for Class C repressed genes, where neither E2F2 nor Myb were required for repression, equivalent ChIP-chip enrichment signals were found (Georlette, 2007).

The genes repressed by MMB for both Class A and Class B were not cell cycle regulated and there was no biochemical evidence in Drosophila for the existence of two separate Myb- or E2F2-containing MMB complexes. Anti-Myb and anti-E2F2 antibodies coimmunopreciptated the entire set of MMB factors and hence each other. Furthermore, both E2F2 and Myb were stoichiometric in the defined complex - even after many different biochemical purification protocols of MMB. This point was tested in DNA binding experiments with purified MMB complex and biotinylated DNA fragments from either a Class A or B gene with a strong E2F2 site or strong Myb site, respectively. Regardless of the DNA fragment used in pulldown experiments, E2F2 and Myb stayed together. It is thus concluded that the complexity of both the targeting of MMB to DNA and the transcriptional function(s) of MMB are determined by DNA context and other proteins that may have associated with MMB. In this sense, MMB behaves like the multisubunit TFIID complex, where different TAF subunits determine DNA targeting at a specific promoter (e.g., TAFII2 at an Inr site; TAFII6 at a DPE site, and TBP at a TATA box) and the different TAF subunits interact with different coactivators (Georlette, 2007).

Many genes required for the G2/M transition were regulated by MMB. Reduced levels for many of the genes could readily account for the chromosome phenotypes that were characterized after RNAi depletion of Myb, Mip120, or Mip130 including: impaired sister chromatid cohesion, chromosome fragmentation, and condensation defects. Furthermore, transcript levels for regulatory checkpoint genes involved in spindle assembly that might indirectly lead to chromosome instability were also affected by loss of MMB. In a recent study, Goshima (2007) conducted a 'genome- wide' RNAi screen to identify factors contributing to spindle assembly in Drosophila. Among the unexpected genes revealed by this screen were Myb, Mip130, Lin-52, Mip40, and Caf1/p55. It is suggested that the MMB member genes were identified in their study, at least in part, because MMB regulates the levels of other 'expected' spindle assembly genes such as Klp61F (a kinesin) or Ial (Aurora-B kinase) or the 'unexpected' spindle assembly genes such as RacGap50C - all of which are MMB-regulated Class D genes. It will be interesting to learn if the human homologs of MMB also regulate genes required for G2/M in humans, because the oncogenic role of Myb in certain cell types may involve misregulation of spindle assembly genes that ensure normal karyotypes. The human repressor E2F4 has been shown to bind to genes involved in chromosomal stability, and one might suspect that this activity is functioning in the context of the paralogous human MMB/dREAM complex (Georlette, 2007).

Many of the genes regulated by MMB are essential, and in particular, the Class D genes are prominent in this regard. It is possible that the lethality observed for myb-null mutants is the result of misexpression of one or a set of these genes. However, even with a reduction of MMB factors >95% after sustained RNAi treatment, proliferation of Kc cells is still seen in culture. The transcript levels for genes requiring MMB for activation (Class D genes) were only modestly reduced (two- to fourfold) in the absence of MMB. Thus, the regulation of these genes by Myb may not be profound or responsible for the lethality of myb mutations in flies. Nevertheless, there are reasons to suspect that the regulation by MMB of these target genes may be relevant at least in part to the essential requirements for Myb in vivo. Recall that myb-mutant lethality is suppressed by loss of mip40, mip120, or mip130. Following along the lines of the model derived from these genetic studies one might suggest that the critical regulatory step dependent on Myb involves derepression from a quiescent state where cells need to switch on such essential genes for mitotic functions. Such a switching mechanism may be nonessential in cell culture. Hence, repression in a quiescent cell in the developing fly, perhaps mediated by Mip120 and Mip130, may require Myb for induction at a later time or in a specific tissue. In vivo, but not in cell culture, loss of Myb alone (as in myb null mutants) could result in a 'permanently repressed' essential gene whereas loss of the entire MMB complex (as in myb; mip120 mutants) may allow for suboptimal expression levels of an essential gene(s); for instance, at a two- to fourfold reduced level. Thus, a presently scored Class D gene may behave as a Class C repressed gene in another cell type, where loss of Myb would leave a repressive MMB complex that is unable to be induced (Georlette, 2007).

One important issue that needs to be explored is how MMB is targeted to such essential genes. From the genetic suppression data, it is inferred that MMB is still targeted to essential genes even in the absence of Myb, and therefore does not require Myb for targeting to these gene promoters. If the vital function of Myb is to somehow induce a MMB-repressed vital gene(s) where loss of Myb was not critical for repression, then the essential activity of Myb may not require the Myb DNA-binding domain at all. In fact, recent data showed that a transgene containing a complete deletion of the DNA-binding domain of Myb is sufficient for myb-mutant viability (Georlette, 2007).

The number of genomic binding sites for MMB far exceeded what was expected from the MMB gene regulatory network defined by the RNAi analysis. While >80% of the 3538 MMB-binding sites were proximal to promoters, only 25% of proximal genes showed any change is expression when MMB members were depleted following RNAi treatment. Similar observations have been made for other proteins; for example, the number of genes regulated by the Myc, Max, and Mad/Mnt transcriptional network is far lower than the measured number of binding sites for these factors. This type of phenomenon may be a simple consequence of biological noise. It is possible that many of the complexes that are bound to sites not regulating transcription of a nearby gene are simply 'junk' or vestigial in nature. A majority of such sites would have little selective advantage, or they may simply serve as a nonspecific binding pool to keep the levels of non-DNA-bound MMB low. Alternatively, these sites may play some role in other chromosome function(s) apart from gene expression. For instance, some of these 'silent' MMB-binding sites may be directly modulating the selection of replication initiation sites, a point suggested by the role played by the complex in follicle cell gene amplification (Georlette, 2007).

Interestingly, many of the genes that contain 'silent' promoter-proximal MMB-binding sites are expressed in Kc cells and transcript levels are unaffected following removal of MMB. Perhaps, at some of these occupied sites, MMB is simply poised to respond to signals that are absent in the culture media. Prominent among this list are the genes encoding DNA replication proteins such as Chiffon, the ORCs, and MCMs. An evolutionarily conserved multisubunit complex in human cells that contains homologs of many of the MMB/dREAM subunits, represses cell cycle-dependent genes during quiescence. It is, of course, possible that similar control in resting cells will be found for the fly complex. This prospect would then add to the established functions of MMB in repressing differentiation-specific genes, and promoting transcription of Mphase genes. Extending this general point, the Kc cells are fixed in one state through their isolation in culture and well-defined passage conditions. It is possible that some of the 'silent' MMB-binding sites may function in a developmental pathway that is dependent on the action of a new factor or signal not normally seen in Kc cells in culture (Georlette, 2007)

Protein Interactions

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-ß, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-ß. The Drosophila homolog of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication (Beall, 2002).

To identify proteins that bind to either ACE3 or Ori-ß, Drosophila tissue culture nuclear extracts were fractionated. DNase I protection was used to assay site-specific ACE3- and Ori-ß-binding proteins, and to follow their purification. The final glycerol gradient fractions were found to contain five polypeptides that co-elute with binding activity for both DNAs in multiple independent fractionation schemes from either Schneider L2 or Kc cell lines. Utilizing peptide sequences from proteolysed purified protein, database searches identified Drosophila Myb (p85) and Caf1 p55 proteins, as well as three new Drosophila proteins (p40, p120, p130; Berkeley Drosophila Genome Project CG15119, CG6061 and CG3480) (Beall, 2002).

Drosophila Myb recognizes a highly conserved DNA sequence, but the specific binding properties of the glycerol gradient fractions might be more complex than that of Myb alone. Therefore, whether any of the other proteins in the footprinting fractions might interact site-specifically with DNA was tested. Each protein was produced individually and purified to homogeneity as either a (His)6- or Flag-tagged protein using the baculovirus system. Only recombinant (r) Myb and rp120 bind to ACE3. rMyb protects nucleotides -471 to -511, and at higher concentrations protected -525 to -541, whereas rp120 protects -413 to -445, -525 to -541, and -575 to -603. However, the protection from the glycerol gradient fractions was more complex than the simple sums of the protections observed for these two purified proteins. Moreover, rMyb on its own does not bind to Ori-ß, whereas p120 does bind (Beall, 2002).

All five proteins co-immunoprecipitate together when any of the five antibodies are used. This association was not mediated through DNA, because ethidium bromide does not disrupt the interactions. Identical results were obtained using either 0–12-h embryo or ovary nuclear extracts. Myb co-fractionates with only the four other complex members. No indication of free or other Myb forms was found. It is therefore concluded that most of the Myb in these extracts is in a tight complex with the four additional proteins (Beall, 2002).

Since the Drosophila Myb complex binds to both ACE3 and Ori-ß in vitro, whether the Myb complex directly interacts with Orc was tested. Immunoprecipitation from Kc cell nuclear extracts shows that anti-Orc1 or anti-Orc2 antibodies co-immunoprecipitate Orc1, 2 and 6. Immunoprecipitations with anti-Myb antibodies co-immunoprecipitate Orc. Reciprocal experiments showed that anti-Orc2 antibodies co-immunoprecipitate the Myb complex (Beall, 2002).

As a first step in exploring the role of the Drosophila Myb complex in vivo, chromatin immunoprecipitation assays were performed on whole ovaries dissected from females that were aged to maximize the number of stage-10 egg chambers. Antibodies against Myb, Orc2 and p120 specifically precipitate ACE3-containing chromatin. Thus, Myb, p120 and Orc are bound to ACE3 in ovaries enriched for egg chambers undergoing chorion gene amplification. E2F-containing complexes can bind to Orc and are associated with ACE3 in ovaries, but the location of this E2F cis-element is unknown. The interactions between the Myb complex, Orc and E2F proteins in sculpting the properties of the ACE3 element will be critical to understanding how this element functions as a replication enhancer (Beall, 2002).

Small P element transgenes containing ACE3 and Ori-ß amplify efficiently at ectopic genomic sites only when both elements are present. A minimal replication reporter was used to assess the role of protein binding sites within ACE3 to minimize the complications of redundant cis-elements. 'Suppressor of hairy wing binding sites' (SHWBS) were used to insulate the transgenes from chromosomal position effects. Such reporters allow for investigation, at various chromosomal positions, whether the binding sites in ACE3 for Myb and p120 are important cis-acting elements for amplification. Transgenes were constructed that contain deletions of each of the binding sites identified by DNase I protection and several transgenic lines were generated for each deletion. Mutations abolished DNA binding of both recombinant Myb and the entire complex to the regulatory sequences in electrophoretic mobility shift assays (EMSAs) experiments. Deletion of either the Myb (-471 to -511) or one of the p120 binding sites (-413 to -445) resulted in severely reduced amplification in stage-13 egg chambers. Deletion of the other two p120 binding sites resulted in reduced levels of amplification, but to a less severe degree (Beall, 2002).

The mature somatic follicle cells that surround the developing oocyte derive from a series of mitotic cell divisions followed by genome-wide endocycles. At stage 10B, global DNA replication shuts down and the chorion loci on the X and third chromosomes (and two additional unidentified loci) begin locus-specific amplification. Amplification can be visualized by the incorporation of bromodeoxyuridine (BrdU) at four sub-nuclear foci using immunofluorescence microscopy, where the two largest foci represent incorporation on the X and third chromosomes (Beall, 2002).

Since Orc2 also localizes to these foci, ovaries were stained with either anti-Myb or anti-Orc2 antibodies. It was found that Myb is diffusely nuclear and not localized to the distinct sub-nuclear foci that contain Orc2. Identical results were observed with the four other complex members (Beall, 2002).

Drosophila Myb has been suggested to have a general role in S phase in several different tissues. However, a direct role for Drosophila Myb in replication at a specific location has not been demonstrated. To test the need for Drosophila Myb in trans for replication at the chorion loci, a directed mosaic system was used to generate green fluorescent protein (GFP)-negative, homozygous Drosophila Myb mutant clones in a heterozygous ovary. In the absence of Drosophila Myb, members of the pre-replication-complex (RC) are present at the sub-nuclear foci but are unable to initiate detectable replication. One prediction from these results is that in late-stage mosaic egg chambers with Myb clones, patches of thin, fragile eggshell and thin dorsal appendages should result from reduced chorion gene expression. Myb mutant patches in the regions that are normally responsible for dorsal appendage formation results in greatly reduced appendages. As egg chambers age, the Myb mutant nuclei appear more compact. There was no underlying chorion beneath the follicle cells in mutant patches (Beall, 2002).

Studies of Myb family members have largely focused on their functions as transcription factors, though important targets for gene activation that might explain the role of these proteins in the cell cycle remain unclear. Drosophila Myb has been shown in this study to play a direct role in DNA replication. These biochemical experiments imply that this protein functions in tight complex with four other proteins. Recent studies suggest that in a variety of tissues, but not all, Drosophila Myb seems to be important for S-phase progression. These findings support a role for Drosophila Myb in tissue-specific and temporally defined DNA replication, much as enhancer proteins define site-specific transcription (Beall, 2002).

The genetic studies and, in particular, the mosaic analyses indicate that Drosophila Myb probably functions at a late step in the replication process, since Orc2 and Cdt1 were both localized at discrete foci within Drosophila Myb mutant stage-10B follicle cells. It is inferred that Orc and other general replication proteins are localized at ACE3. However, it is known when in the developmental process Orc appears at ACE3 with regard to Drosophila Myb loss. Thus, Drosophila Myb perdurance after genomic deletion could certainly complicate any conclusions about the role of Drosophila Myb in establishing a pre-RC at the amplification foci. In any case, Myb family members interact with both acetylases and deacetylases; thus, it is intriguing to consider the potential roles of this modification in either early or late steps of DNA replication initiation (Beall, 2002).

Dm-myb mutant lethality in Drosophila is dependent upon mip130: positive and negative regulation of DNA replication

Gene amplification at the chorion loci in Drosophila ovarian follicle cells is a model for the developmental regulation of DNA replication. It has been shown that the Drosophila homolog of the Myb oncoprotein family (DmMyb) is tightly associated with four additional proteins and DmMyb is required for this replication-mediated amplification. Targeted mutagenesis was used to generate a mutant in the largest subunit of the DmMyb complex, the Aly and Lin-9 family member, Myb-interacting protein 130 (Mip130). mip130 mutant females are sterile and display inappropriate bromodeoxyuridine (BrdU) incorporation throughout the follicle cell nuclei at stages undergoing gene amplification. Whereas mutations in Dm-myb are lethal, mutations in mip130 are viable. Surprisingly, Dm-myb mip130 double mutants are also viable and display the same phenotypes as mip130 mutants alone. This suggests that Mip130 activity without DmMyb counteraction may be responsible for the Dm-myb mutant lethality. RNA interference (RNAi) to selectively remove each DmMyb complex member revealed that DmMyb protein levels are dependent upon the presence of several of the complex members. Together, these data support a model in which DmMyb activates a repressive complex containing Mip130 into a complex competent to support replication at specific loci in a temporally and developmentally proscribed manner (Beall, 2004).

Drosophila contains a single Myb-related gene (Dm-myb) that appears to be most closely related to the vertebrate B-Myb. Dm-myb is essential, and studies of Dm-myb mutants suggest nontranscriptional roles for DmMyb in S phase and in the prevention of genomic instability, as well as transcriptional roles at the cyclin B promotor. A DmMyb-containing complex binds site-specifically to the two critical control elements required for chorion gene amplification, ACE3 and Ori-ß, on the third chromosome. Reporter transgenes harboring deletions in regions of the DNA important for complex binding were defective for amplification, supporting the notion that the DmMyb complex is required for gene amplification in both cis and trans. Furthermore, somatic clones in the follicle cell layer surrounding the developing oocyte that are mutant for Dm-myb are defective for amplification. The characteristic thin eggshell phenotype of female-sterile mutations and loss of bromodeoxyuridine (BrdU) incorporation at the amplification foci were found in Dm-myb mutant patches despite proper DmOrc2 and Cdt1 localization at the amplification foci. Taken together, these results suggest that DmMyb is required for replication at the chorion origins at a step postprereplication complex (pre-RC) assembly. Because both DmMyb and one other complex member, Mip120, bind to ACE3 in vivo (as assayed by chromatin immunoprecipitation), and no 'free' DmMyb can be detected biochemically, it seemed likely that DmMyb was acting at the chorion origins in association with the other complex members, Mip40, Caf1 p55, Mip120, and Mip130. These data left open important questions: what is DmMyb's function at the chorion origins and in replication in general? Does DmMyb always work in association with the four other complex members, or does DmMyb have functions that are independent of the DmMyb complex? Stated another way: While DmMyb's essential function(s) for viability are undetermined, it was of interest to know if DmMyb's activity was executed coordinately with the other complex members (Beall, 2004 and references therein).

The genetic interaction between Dm-myb and mip130, together with the protein accumulation data, suggests that the primary function(s) carried out by DmMyb is in close association with the other complex members. The key observation that helped unravel this genetic complexity was that loss of Mip130 leads to un-scheduled DNA replication throughout the follicle cell nuclei. A simple hypothesis that can explain the present data is that DmMyb itself activates a repressive complex that contains Mip130 as an important member. By extension, Mip130, therefore, may function as a member of a complex that can serve as either a repressor or activator of chromosomal activity. Repression and activation carried out by the DmMyb complex must be vitally important only in some tissues, since follicle cell development is not required for female viability. In tissues critical for viability, the inability to activate the repressor is more deleterious (i.e., in Dm-myb mutants alone) than when both components are lost (i.e., in Dm-myb mip130 double mutants). Clearly in the ovarian follicle cells, proper development requires both components. However, before discussing the model in greater detail, other data will be summarized and parallels will be drawn to another constellation of DNA-binding factors that have a similar functional relationship to DmMyb and Mip130 (Beall, 2004).

The data showing that (1) DmMyb is bound to ACE3 in vivo; (2) DmMyb-binding sites are required in cis for amplification; (3) the DmMyb complex is associated with DmORC, and (4) Dm-myb mutant follicle cell clones show pre-RC assembly at the amplification foci without BrdU incorporation, suggest that DmMyb is required as a positive regulator for replication initiation at the chorion origins. The observation that in mip130 mutants, BrdU incorporation occurs throughout the follicle cell nuclei at stages normally undergoing site-specific amplification suggests that Mip130 acts as an inhibitor of replication at regions of the genome not normally targeted for replication. Interestingly, these Dm-myb and mip130 mutant phenotypes are strikingly similar to those of e2f1 and e2f2 mutants (See E2f and E2f2). For e2f1i1 mutants that contain a mutation in the DNA-binding domain of E2F1, there is severely reduced or no BrdU incorporation in follicle cell nuclei at amplification stages. In contrast, in e2f2 null mutants, genome-wide BrdU incorporation occurs throughout the follicle cell nucleus at amplification stages. Thus E2F1 works as an activator of site-specific amplification, whereas E2F2 clearly participates in repression. Further drawing a parallel with the Dm-myb/mip130 data presented in this study, even though e2f1 null mutants are lethal at late larval/early pupal stages, e2f1 e2f2 double mutants survive to near adulthood. The complicated genetic interaction between the Drosophila E2Fs suggests that the observed lethality of e2f1 mutants is in part due to unchecked E2F2 activity (Beall, 2004 and references therein).

E2F1 has been primarily studied as a transcription factor, required for gene expression of some of the key proteins required for the G1/S transition. Nevertheless, E2F1 directly binds to ACE3 and is associated with DmOrc2 in ovary extracts, providing evidence that E2F1 has roles at the chorion loci that are independent of transcription. These data together imply that both the E2F and DmMyb factors site-specifically coregulate replication at the ACE3 locus and may also carry out parallel roles in repression. Data to be presented elsewhere (P. Lewis and M. Botchan, in prep., cited in Beall, 2004) directly support these arguments: Fractionation of embryo extracts with a final affinity step using anti-Mip120 antibodies has shown that Mip120 and the other DmMyb complex members are in tight association with E2F2, DP1, Rbf1, Rbf2, and the histone deacetylase Rpd3. These biochemical results link the repressive activities of the Mip complex and E2F2, first brought to light by genetic and cytological experiments. A reasonable extrapolation from these data is that at ACE3, the E2F2 and Mip activities coordinate repression before stage10 and subsequently, E2F1 and DmMyb act coordinately to activate replication site-specifically during amplification. A proposed switch between E2Fs has been suggested previously. Although both the DmMyb and E2F complexes are cis-acting at the ACE3 enhancer there are no direct data as yet that link the complexes to other specific chromosomal replication origin control elements (Beall, 2004).

Focusing solely on the Mips and DmMyb, a model is presented to show how these factors might function as regulators of chromosomal activity. It is posited that at some stage prior to stage 10 in egg chamber development, the DmMyb complex is bound to many sites in the genome and is required for inactivating potential replication initiation sites and perhaps inhibiting gene expression. In this configuration, DmMyb acts as a silent partner and is not required for repression. Through unknown mechanisms, the ACE3 locus is targeted as a site for replication initiation and is subsequently bound by members of the pre-RC. In a late step of origin activation, it is suggested that DmMyb somehow converts an inactive replication origin to one that is competent for initiation, possibly through posttranslational modification of DmMyb. Such a switch in mammalian B-Myb transcriptional activity after phosphorylation has been suggested. In the absence of Mip130, as exemplified in the mip1301-723 + 1-36 strain, repression (both transcriptional and replicative) is relieved, pre-RC protein transcript levels may increase, and pre-RC assembly at chromosomal positions may no longer be restricted. This could lead to pre-RC association at sites spread through different regions along the entire chromosome. The model stipulates that neither DmMyb nor Mip130 targets the pre-RC to an origin, but rather suggests that DmMyb and Mip130 participate in origin activation and repression directly. It is further emphasized that the data have shown a cis-acting function for the DmMyb complex only at ACE3, and it seems entirely likely that the DmMyb complex has functions both in cis and in trans in the developmental pathway under focus in this study (Beall, 2004).

Microarray analyses of e2f2 mutant follicle cells reveal an increase in transcription of several key replication factors, including DmOrc5, which may in part explain the genome-wide replication observed in e2f2 mutants. Thus by analogy, it is posited that the DmMyb complex has roles both in cis and in trans for gene expression (and other chromosomal activities), perhaps in some cases acting antagonistically with E2F family members. It is clear that the DmMyb complex is not required for replication from every origin, as mutants lacking both DmMyb and Mip130 are viable. Rather it appears that the DmMyb complex is required for activating replication of a set of origins at certain developmental stages or tissues, such as in the follicle cells undergoing chorion gene amplification (Beall, 2004).

Certain facts about the members of the DmMyb complex also provide some hints about the mechanistic functions of the complex. Caf1 p55 is a member of several different complexes involved in histone metabolism in Drosophila, including the chromatin-remodeling complex NURF and the chromatin assembly complex Caf 1. Mip130 is a member of a new family of proteins that is conserved from plants to humans and includes the Drosophila always early (aly) gene and the Caenorhabditis elegans SynMuvB gene, lin-9. Both the aly and SynMuvB genes encode chromatin-binding proteins and may have roles in DNA remodeling complexes for both transcription and DNA replication. Furthermore, Mip120 contains a C-terminal cysteine-rich domain that is found in a number of chromatin-binding proteins including members of the polycomb group. Myb family members have been shown to interact with both histone acetyltransferases and deacetylases, and DmMyb is known to interact with the acetyltransferase p300. The nucleosome structure surrounding eukaryotic origins is usually in a nuclease-sensitive state similar to that found at promoters and enhancers, and an ORC-dependent nucleosome configuration at ARS1, in Saccharomyces cerevisiae, is required for pre-RC assembly and origin firing. Together, these data support a role for chromatin modifying and/or remodeling complexes in promoting DNA replication initiation. Therefore, it is tempting to speculate that one function for the DmMyb complex is to regulate chromatin structure in such a way as to permit replication initiation and/or transcription through association of a chromatin-remodeling and/or modifying complex (Beall, 2004).

Native E2F/RBF complexes contain Myb-interacting proteins and repress transcription of developmentally controlled E2F target genes

The retinoblastoma tumor suppressor protein (pRb) regulates gene transcription by binding E2F transcription factors. pRb can recruit several repressor complexes to E2F bound promoters; however, native pRb repressor complexes have not been isolated. E2F/RBF repressor complexes have been isolated from Drosophila embryo extracts and their roles in E2F regulation have been characterized. These complexes contain RBF, E2F, and Myb-interacting proteins that have previously been shown to control developmentally regulated patterns of DNA replication in follicle cells. The complexes localize to transcriptionally silent sites on polytene chromosomes and mediate stable repression of a specific set of E2F targets that have sex- and differentiation-specific expression patterns. Strikingly, seven of eight complex subunits are structurally and functionally related to C. elegans synMuv class B genes, which cooperate to control vulval differentiation in the worm. These results reveal an extensive evolutionary conservation of specific pRb repressor complexes that physically combine subunits with established roles in the regulation of transcription, DNA replication, and chromatin structure (Korenjak, 2004).

The Drosophila genome encodes two pocket proteins, RBF1 and RBF2, and two E2F proteins, dE2F1 and dE2F2, that act in heterodimers with a common partner, dDP. It was reasoned that this streamlined version of the E2F/pRb network would greatly simplify the chromatographic separation of native complexes. Drosophila embryo nuclear extracts were subject to gel filtration to verify the presence of RBF complexes. RBF1 was detected by Western blot in many fractions ranging in apparent molecular weight from 66 kDa to 1.2 MDa. RBF2 was detected in a narrower peak with an apparent molecular weight of 669 kDa to 1.2 MDa. dDP was likewise detected in fractions ranging in molecular weight from 443 kDa to 1.2 MDa. These findings suggest that Drosophila embryos contain multisubunit dE2F/RBF complexes (Korenjak, 2004).

Next, extracts were subjected to ion exchange chromatography. This resolved three peaks of RBF1 activity. Peak I contained RBF1 but did not contain RBF2, dE2F, or dDP. When peak I was subjected to gel filtration, RBF1 eluted with an apparent molecular weight of 100 kDa, close to its theoretical molecular weight (91.8 kDa), suggesting that peak I contains monomeric RBF1. RBF1, dE2F1, and dDP coeluted in peak II. During subsequent gel filtration, these three proteins coeluted with an estimated molecular weight of 500 kDa. Analysis of peak III revealed the presence of RBF1, RBF2, dE2F2, and dDP. These four proteins coeluted during gel filtration with an apparent molecular weight of 669 kDa to 1.2 MDa (Korenjak, 2004).

Previous studies have shown that RBF1 associates with both dE2F1 and dE2F2, whereas RBF2 interacts exclusively with dE2F2, and these binding specificities are reflected in the elution profile. Interestingly, peak III fractions contain both RBF1 and RBF2 even though they do not interact with each other. This suggests that peak III contains two separate dE2F2/RBF1 and dE2F2/RBF2 complexes with similar subunit composition. The molecular weight of these complexes (669 kDa to 1.2 MDa) indicates that they contain additional subunits. Therefore dE2F2/RBF complexes present in peak III were purified (Korenjak, 2004).

dE2F2/RBF complexes were purified by classical chromatography. dE2F2, dDP, RBF1, and RBF2 coeluted from the final gel filtration column with an apparent molecular weight of 669 kDa to 1.2 MDa. Silver staining detected seven bands that perfectly coeluted with the Western signals. These polypeptides were present in similar stoichiometric amounts, with the exception of one (55 kDa) that was stained more intensely. Peptide mass fingerprinting revealed that the 55 kDa band comprised two distinct polypeptides (Korenjak, 2004).

Identification of copurifying polypeptides revealed Twilight (also known as Mip130; from here on referred to as Mip130/TWIT), RBF1, RBF2, dMyb, dDP, dE2F2, CAF1p55, and Mip40. The identity of these polypeptides was confirmed by Western blot. Intriguingly, Mip130/TWIT, dMyb, CAF1p55, and Mip40 have recently been identified as components of a dMyb complex that regulates chorion gene amplification in follicle cells. The fifth subunit of the dMyb complex, Mip120, was apparently absent from the final preparation as judged by silver staining. However, Western analysis with Mip120-specific antibody has demonstrated that Mip120 coelutes with other complex subunits throughout the fractionation. The Mip120 signal became progressively weaker during purification but was still detectable in fractions eluting from the final gel filtration column, suggesting that Mip120 might have been progressively lost or degraded. Indeed, several results presented below suggest that Mip120 is a bona fide complex subunit. Since these complexes are a composite of known transcriptional regulators, they go by the acronym dREAM (Drosophila RBF, E2F, and Myb-interacting proteins) (Korenjak, 2004).

The idea that E2F proteins have tissue-specific, developmentally regulated functions is supported by the identification of novel E2F regulated genes in human, mouse, and fly. In addition to cell cycle-related E2F targets, these studies reveal numerous genes that have developmental functions or display a strictly tissue-specific expression pattern. Analysis of the E2F transcriptional program in Drosophila indicates that there are at least two different types of E2F regulation (Dimova, 2003): expression of cell cycle-regulated E2F targets is primarily dependent on dE2F1/dDP-mediated activation and is repressed by RBF1 (A group genes). In contrast, other E2F targets are actively repressed in proliferating cells by dE2F2, dDP, and either RBF1 or RBF2, and these genes are expressed in developmentally regulated patterns (E group genes). These two types of regulation appear to be combined in differing proportions over the spectrum of E2F targets, generating a broad variety of E2F control (Korenjak, 2004).

dREAM repressors are required for a recently discovered aspect of dE2F transcriptional regulation. RNAi-mediated disruption of dREAM complexes by depletion of Mip130/TWIT and Mip120 specifically derepresses E group genes, genes that have previously been shown to be repressed in a cell cycle-independent manner by dE2F2, dDP, and a function that is redundant between RBF1 and RBF2. Although depletion of Mip130/TWIT and Mip120 has no effect on expression of A group genes, it is probable that dREAM complexes also repress cell cycle-related targets in other situations: ChIP experiments show that dE2F2, RBF1, and RBF2 are normally present at almost all dE2F-regulated genes, including A group genes; the distinction between A and E group genes lies, therefore, not in the binding of the repressor proteins but in the binding of dE2F1 (Dimova, 2003). Accordingly, in cells lacking dE2F1, dE2F2-mediated repression prevents the expression of both cell cycle-dependent and -independent targets (Dimova, 2003). The extensive colocalization of dE2F2, RBF1, Mip120, and Mip130/TWIT on polytene chromosomes suggests that dREAM complexes are present at most sites of dE2F action (Korenjak, 2004).

The fact that dMyb is a stoichiometric subunit of dREAM complexes hints at an extensive collaboration between dE2F and dMyb. However, depletion of dMyb has no effect on expression of the A and E group genes tested. It is clear, therefore, that dMyb is not required for all aspects of dREAM complex function. However, it is possible that dE2F and dMyb cooperate to regulate transcription of other genes that have not been investigated. Moreover, as will be discussed below, dE2F2 and dMyb appear to converge on the regulation of chorion gene amplification (Korenjak, 2004).

The mechanism of E2F regulation provided by dREAM appears to be highly conserved during evolution. Strikingly, with the exception of dMyb, all components of dREAM are either homologs of previously described C. elegans synMuv class B genes (mip130/twit/lin-9, rbf1 and rbf2/lin-35, de2f2/efl-1, ddp/dpl-1, and caf1p55/lin-53), contain regions of sequence conservation (Mip40/lin-37), or produce a synMuv phenotype when the corresponding C. elegans gene is inactivated (Mip120/JC8.6). Genetic studies have shown that synMuv class B genes are required for development of the worm's male and female reproductive systems, and it has been suggested that some encode subunits of a hypothetical complex that represses vulva-specific gene transcription; however, the precise transcriptional changes underlying the synMuv phenotype are unknown (Ceol, 2001). The discovery of dREAM complexes suggests an intriguing model for synMuv class B gene function: it is proposed that at least seven synMuv class B gene products physically associate to form a complex that, like its Drosophila counterpart, represses sex-related targets and that misexpression of these genes causes a change in cell fate. Given the vast differences between C. elegans and Drosophila embryogenesis, it is considered unlikely that REAM complexes will regulate the exact same set of genes in both species. However, it is proposed that, in both organisms, REAM complexes control transcriptional programs required for development of the reproductive system. In agreement with this model, dE2F2 has been shown to be needed to repress genes like vasa and spn-E that are important for Drosophila gametogenesis (Dimova, 2003) and that dE2F2 mutants have both male and female fertility defects (Korenjak, 2004).

Do mammalian cells contain similar complexes? Mammalian homologs exist for all dREAM subunits. Intriguingly, B-Myb associates with the N terminus of p107. RbAp48/p46, human orthologs of CAF1p55, were first isolated through their ability to bind a pRb-affinity column but are now known as components of several chromatin-associated complexes, including a putative pRb-histone deacetylase and the NuRD complex. Human homologs of Mip130/TWIT, Mip120, and Mip40 had not previously been linked to pRb. All three interact with pRb in vitro. Furthermore, endogenous hMip130/TWIT associates specifically with pRb, p107, and p130 fusion proteins. In agreement with these results, interaction has been demonstrated between pRb and Mip130/TWIT in human cells in vivo (S. Gaubatz, personal communication to Korenjak, 2004). Clearly, further studies are needed to define the properties and biological roles of pRb/hMip complexes. Nevertheless, these preliminary findings suggest that such complexes may well exist in mammalian cells, and, if studies in C. elegans and Drosophila are a guide, it might be expected that they function in developmentally regulated aspects of E2F/pRB function (Korenjak, 2004).

What is the biochemical function of dREAM complexes? dREAM complexes lack known chromatin-modifying enzymes. Studies of mammalian E2F targets show that activation and repression correlate with histone acteylation and deacetylation, respectively. The finding that dREAM complexes associate specifically with unmodified histone H4 tails but fail to bind hyperacetylated tails implies that they bind specifically to deacetylated histones that are characteristic of repressed chromatin. Consistent with this, dE2F2, RBF, Mip120, and Mip130/TWIT colocalize at chromosomal sites that are not actively transcribed. It is proposed that dREAM complexes bind deacteylated nucleosomes, protecting them from modification, and in doing so maintain a repressive state that is both stable and readily reversible (Korenjak, 2004).

One might predict that dREAM would act synergistically with histone deacetylases. Indeed, the C. elegans synMuv B class includes an ortholog of HDAC1, and the dRPD3 histone deacetylase coimmunoprecipitates with RBF from extracts of cell lines. However, dRPD3 is not a stoichiometric component of dREAM complexes. Furthermore, inhibition of histone deacetylases in SL2 cells, either by the depletion of dRPD3 or treatment with deacetylase inhibitors, does not derepress group E genes (Taylor-Harding, 2004). Thus, while histone deacetylation may be a prerequisite for histone binding by dREAM, deacetylases are not required to maintain repression of E group genes (Korenjak, 2004).

The discovery that E2F/RBF complexes contain five subunits of a recently described dMyb complex is particularly intriguing. This complex binds the ACE3 element of a chorion gene locus and has been suggested to regulate chorion gene amplification in ovary follicle cells. Amplification involves both cessation of general genomic replication and relicensing and firing of origins in a temporally and spatially restricted manner. Remarkably, dE2F2, dDP, RBF1, and Mip130/TWIT are all needed to shut off genomic replication in vivo. The discovery of dREAM complexes offers a mechanistic explanation for these genetic results and implies that dREAM complexes function to shut off genomic replication in this cell type. Interestingly, dRPD3 and histone deacetylation have been shown to counteract chorion origin firing (Aggarwal, 2004), lending further support to the idea that 'transcriptional' regulators can also influence DNA replication events (Korenjak, 2004).

Recent genetic studies have suggested that the effects of Mip130/TWIT are reversed at rereplicating sequences by dMyb, possibly following an activating modification of dMyb itself (Beall, 2004). Interestingly, dE2F1 and dMyb colocalize to amplifying foci, and dE2F1, like dMyb, is needed to promote rereplication. Since dE2F1 works at least in part by overriding dE2F2-mediated repression and dMyb has been proposed to selectively counteract Mip130/TWIT activity, the discovery that dE2F2 and Mip130/TWIT reside in the same complex suggests that dE2F1 and activated dMyb may collaborate at the ACE3 locus to reverse repressive effects of dREAM complexes. In this setting, dMyb and E2F appear to share a similar mechanism of action, relying on an activator (dE2F1) or an activating event (modification of dMyb) to relieve the effects of a common repressor (Korenjak, 2004).

It should be noted that mutant alleles of de2f2 and mip130/twit are not lethal but do suffer from reduced viability and fertility. Hence, dREAM complexes are not essential. Amplification of chorion loci in follicle cells represents a highly specialized case of DNA replication. The general patterns of DNA replication are unaffected by mutation in de2f2 and mip130/twit, arguing against a strict requirement for replication per se. Nevertheless, several studies of mammalian cells have linked pRb and E2F proteins to various aspects of DNA replication, but their precise roles in replication remain to be established (Korenjak, 2004).

dREAM complexes are the first native RBF repressor complexes to be purified, but it is noted that additional complexes likely exist. Fractionation reveals an additional complex containing RBF1, dE2F1, and dDP that might act at other E2F targets that were unaffected by the depletion of dREAM components. The results show that specific RBF-containing complexes are important at specific subsets of dE2F-regulated promoters. It is becoming clear that pRb/RBF tumor suppressors assemble distinct molecular machines to exert distinct functions. More work is needed to determine which complexes are needed for each of their ascribed functions. The striking parallels between studies of pRb and E2F orthologs in C. elegans and Drosophila indicate that their basic mechanisms of action are well conserved. Perhaps the most definitive picture will emerge by integrating information from each of the available model organisms (Korenjak, 2004).

The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. Drosophila Myb has been shown to form a stable complex with four additional proteins, Mip130, Mip120, Mip40, and Caf1/p55. This five-subunit complex was originally identified as an activity present in Drosophila extracts that specifically recognizes two critical control elements (ACE-3 and ori-beta) required for chorion gene DNA replication-mediated amplification in the follicle cells surrounding the developing oocyte. To further understand biochemically the functions of the Myb complex, Drosophila embryo extracts were fractionated, relying upon affinity chromatography. E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, both associate with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription (Lewis, 2004).

The discovery that the Myb complex proteins are needed to repress developmentally regulated genes raises the possibility that some of the phenotypes observed in mip130 mutant animals may be due in part to the inappropriate expression of differentiation factors. The number of such target genes regulated by the repressive Myb-MuvB complex identified here is likely quite large. Multiple site-specific DNA-binding proteins contained together in one complex (such as Myb, Mip120, and E2F2:DP), increase the potential diversity for DNA sites that may be bound. Thus, at certain enhancers, the E2F2 site in combination with Mip120 may target the assembly, while at other sites the Myb DNA-binding activity may be important. Furthermore, although the majority of the Myb complex subunits in embryo extracts are present in the Myb-MuvB complex, it seems likely that the Myb and E2F2 proteins function independently at some chromosomal positions. It is also posited that the Myb complex may be modified in such a way as to provide a signal for activation rather than repression. In work to be presented elsewhere, by using microarray analysis and genomic localization of the Myb complex, it has been found that a family of transcripts is indeed dependent upon the Myb complex for expression. Thus a network of chromosomal domains may be independently regulated by these factors. In the context of such a network, it is intriguing that as an activator of DNA amplification, it appears as if Myb plays an active role in targeting the Mips and associated activities to the ACE-3 site. In contrast to the transcriptional repression studied in cell culture, Myb does not seem important for such targeting. Clearly understanding the DNA sequence context and associated factors in activation may shed some light on this difference (Lewis, 2004).

The Drosophila repressor characterized in this work has been called the Myb-Muv B complex because of the striking resemblance of its protein composition to that encoded by the synMuv class B genes of C. elegans. Elegant genetic screens have defined a regulatory pathway essential for vulval development that is entirely consistent with a model in which these synMuv proteins are individual members of a large complex. Together with the known expression patterns of the proteins associated with the Drosophila Myb-MuvB complex and phenotypes of the mutants for many of the factors of the complex, the biochemical data argue for a general role for the repressor in many tissue types. Phenotypic differences between the putative nematode complex and the Drosophila counterpart may ultimately be ascribed to subunit composition or perhaps other more complex differences in the actual developmental programs between the two organisms. To highlight these in vivo differences, it is worthwhile to briefly review the SynMuv mutant phenotypes (Lewis, 2004).

Wild-type C. elegans hermaphrodites contain a single vulva organ, while synMuv mutants may posses multiple vulva. In the wild-type organism, the activity of the synMuv genes antagonize the effects of the basal activity of the RTK/Ras pathway by repressing transcription of vulval genes. The class B synMuv genes likely inhibit vulval induction by a conserved mechanism whereby the class B synMuv proteins form a repressive complex with the sequence-specific transcription factor EFL-1/E2F protein, and recruit corepressor proteins to inhibit the transcription of vulval specification genes via EFL-1/E2F-binding sites. As a result, those cells adopt the nonvulval fate. However, in the key vulval precursor cells, the antagonistic action of the synMuv genes is inactivated or can be overcome by the activated RTK/Ras pathway, thereby permitting downstream activation and transcription of keys genes for vulval fate. Some of the findings from studies on the biochemical properties of the components of the Drosophila Myb-MuvB complex may be relevant to a putative nematode complex. The Mip120 protein binds specifically to the ACE-3 and ori- and is probably involved in sequence-specific interactions for the Myb-MuvB complex. It is therefore possible that the C. elegans Mip120 homolog, LIN-54, is also a sequence-specific DNA-binding protein that helps direct the class B gene complex to specific promoters for repression of vulval genes (Lewis, 2004).

L(3)MBT, a homolog of LIN-61, is similar to the Drosophila polycomb group protein Sex Combs on Midleg (SCM), which is a member of the PRC1 complex. PRC1 is thought to primarily repress gene expression through blocking the nucleosome remodeling activity of SWI/SNF. As shown in this study, RNAi directed against L(3)MBT indicates that it is required for transcriptional repression at many of the sites coordinately repressed by E2F2 and the Myb-associated proteins. The L(3)MBT protein appears substoichiometric relative to core Myb-MuvB complex subunits. Like L(3)MBT in the Myb-MuvB complex, the SCM protein is present in substoichiometric quantities relative to the other subunits of the human and Drosophila hPRC-H and PRC1 complexes. Both L(3)MBT and LIN-61 contain multiple MBT repeats that are evolutionarily conserved domains found throughout metazoa. The X-ray crystal structure of the MBT repeats provide some hints as to how both LIN-61 and the Drosophila L(3)MBT protein may function. The MBT repeat consists of a five-stranded beta-barrel core domain that shares structural similarity to the Tudor and chromodomains. The Tudor domain interacts with methylated arginine residues, and chromodomains interact with methylated lysine residues of histone H3. Consistent with the speculation that this domain is critical for function of the MBT family and that the proteins bind modified histones, several hypomorphic mutations in Drosophila SCM map to residues within the putative ligand-binding pocket. The MBT domains in LIN-61 and L(3)MBT may maintain a repressed chromatin domain through interaction with histone tails methylated at specific lysine residues on neighboring nucleosomes, thus hindering the nucleosome mobility by chromatin remodeling factors. For maximum repression, genes regulated by the Myb-MuvB or the putative nematode complex may require additional mechanisms of repression such as histone modification and thus the association of the deacetylase Rpd3 (Lewis, 2004).

Like the Myb-MuvB complex the putative C. elegans complex may also play a wide role in repression in different tissues. For example, several C. elegans class B synMuv genes, including the homologs of Mip130, E2F2, DP, and RBF, have been shown to function independently of synMuv A genes for regulation of the G1/S transition (Lewis, 2004).

The particular genes regulated by the Myb-MuvB complex are likely determined in a tissue-specific and cell-type-specific manner. In Drosophila tissue culture cells, E2F2 appears to function primarily for repression of developmentally regulated genes, while E2F1/RBF1 complexes are involved in regulating genes involved in cell cycle progression. However, microarray studies performed in e2f2 and rbf1 mutant follicle cells indicate that both E2F2 and RBF1 are involved in the repression of several S-phase genes, including CDT1 and the ORC and MCM complex subunits. Therefore, it is likely that the set of genes regulated by the Myb-MuvB complex may change depending on the developmental context (Lewis, 2004).

In the embryo extracts that were fractionated, the repressive form of the Myb complex seems to predominate. However, other much less abundant complexes between the previously identified Myb complex and activators should also be found. Certainly at ACE3, E2F1 and the Myb complex cooperate for amplification and proper ORC localization, and it has been proposed that a switch between E2F2 and E2F1 at ACE3 may be epistatic to activation. Other RNAi studies using Drosophila cell lines indicate that E2F1 and E2F2 primarily occupy and regulate the expression of a non-overlapping set of genes, and the work presented here implies that this non-overlapping control may be dictated by other proteins associated with the well-studied E2F proteins. The Myb complex might assemble with either activators or repressors of the E2F family to regulate either transcription or DNA replication in response to appropriate developmental cues. In future work it will be important to understand how in a given cell type, the cis-acting DNA sites and chromosomal context determine a region for either repression or activation (Lewis, 2004).

tMAC, a Drosophila testis-specific meiotic arrest complex paralogous to Myb-Muv B, contains Myb, E2F2, Caf1/p55 and Aly, and can activate or repress gene transcription

The Drosophila Myb-Muv B (MMB)/dREAM complex regulates gene expression and DNA replication site-specifically, but its activities in vivo have not been thoroughly explored. In ovarian amplification-stage follicle cell nuclei, the largest subunit, Mip130, is a negative regulator of replication, whereas another subunit, Myb, is a positive regulator. A mutation has been identified in mip40, and a mutation has been generated in mip120, two additional MMB subunits. Both mutants were viable, but mip120 mutants had many complex phenotypes including shortened longevity and severe eye defects. mip40 mutant females had severely reduced fertility, whereas mip120 mutant females were sterile, substantiating ovarian regulatory role(s) for MMB. Myb accumulation and binding to polytene chromosomes was dependent on the core factors of the MMB complex. In contrast to the documented mip130 mutant phenotypes, both mip40 and mip120 mutant males were sterile. Mip40-containing complexes were purified from testis nuclear extracts and tMAC, a new testis-specific meiotic arrest complex was identified that contains Mip40, Caf1/p55, the Mip130 family member, Always early (Aly), and a Mip120 family member, Tombola (Tomb). Together, these data demonstrate that MMB serves diverse roles in different developmental pathways, and members of MMB can be found in alternative, noninteracting complexes in different cell types (Beall, 2007).

Coordinating developmentally regulated transcription and replication patterns in metazoans is critical for differentiation of tissue-specific cell types. Central to these processes is the modification and/or remodeling of chromatin by multisubunit complexes through association of site-specific DNA-binding proteins. A multisubunit complex in Drosophila, the Myb-Muv B (MMB) or dREAM complex contains the previously identified five-subunit Myb complex (containing Myb, Caf1/p55, Mip40, Mip120, and Mip130), in addition to E2f2, Rbf1 or Rbf2, DP, and Lin-52. Curiously, the complex contains both activator (Myb) and repressor (Mip130, Rbf1, Rbf2, and E2f2/DP) proteins. That MMB is widely expressed in different tissues has led to the idea that MMB may function both as an activator or repressor at specific chromosomal locations. Depending on the developmental pathways in particular tissues or cell types, different signals might regulate switching of function at a particular site. It has been suggested that at sites known to be repressed by MMB, Myb is a silent member not participating in the transcriptional repression, even though Myb itself is present at the cis-acting site, and that activation of MMB at a subsequent time would depend on Myb function (Beall, 2007).

The finding that Lethal (3) Malignant Brain Tumor [L(3)MBT], NURF, and the histone deacetylase Rpd3 associate with MMB suggests that histone binding and/or modification are possible mechanisms by which MMB acts to repress transcription and/or replication. Thus, MMB and individual subunits are poised to change their measured role by switching off repression (or activation) in a given cell lineage by post-transcriptional modifications or association of coactivators (or repressors) (Beall, 2007).

The genes regulated by MMB in Drosophila tissue culture cells are primarily differentiation and development-specific genes, and most often, MMB is a transcriptional repressor. Recent genomic profiling in Kc cells of five MMB members (Mip130, Mip120, Myb, E2F2, and Lin-52) showed that these proteins were bound together at thousands of chromosome sites, and RNA interference (RNAi) experiments revealed that MMB participated in either transcriptional repression or activation for many genes. In cell culture and in vivo, the accumulation of Myb and E2F2 proteins, but not mRNAs, depends on the integrity of MMB: Loss of Mip130 dramatically affects the levels of both proteins. These data, together with the biochemical finding that essentially all of Myb is found in complex with MMB, led to the proposal that most if not all of the phenotypes previously identified as Myb-specific (or E2F2-specific) must be evaluated in terms of loss of MMB function in either myb or e2f2 mutants (Beall, 2007).

myb is an essential gene in Drosophila. However, mutations in the largest subunit of MMB, mip130, are viable and suppress myb lethality. Furthermore, homozygous mip130 mutant females have drastically reduced fecundity. Cytological and developmental studies of egg chambers from several MMB subunit mutants were critical in building a heuristic model for MMB function. Normally in the ovary, a developmentally controlled replication program occurs in the somatic follicle cell nuclei surrounding the developing oocyte. In these nuclei, overall genomic replication ceases at stage 9 during egg chamber development and is followed at stage 10 by specific DNA replication at four loci that results in amplification of the genes critical for egg shell formation. Myb binds to the well-defined enhancer for one such amplicon in vivo. When myb is removed by genetic manipulation, replication no longer occurs at the four foci, demonstrating a direct and positive role for Myb in replication at these sites. In contrast, mip130 mutant ovaries display global genomic replication in amplification-stage follicle cell nuclei, indicating a negative role for mip130 in replication at sites other than at the chorion origins. Based on these observations, it has been suggested that MMB functions as either an activator or repressor of chromosomal functions depending on the chromosomal and developmental context. Critically in this model, the essential function of Myb is to activate a repressive complex to which it belongs. In its absence, this unchecked repressive activity by the partial MMB complex is lethal. The presumption was that animals lacking MMB (as in mip130 mutants) maintained expression (or repression) of normal target genes through less robust or redundant mechanisms, resulting in viability of these mutants. Furthermore, a critical, but previously untested prediction of this model is that an MMB complex devoid of Myb could still be targeted to chromosomes (Beall, 2007).

In order to gain further insight into the role(s) for MMB in vivo, a mutation was generated in the second largest subunit, mip120. In addition, a P-element-induced allele was identified in another subunit, mip40. As with mip130, it was found that mip40 and mip120 mutants were viable and displayed either sterility (mip120) or reduced fecundity (mip40) of mutant females. Moreover, mip40 and mip120 suppressed myb lethality, again suggesting that the essential function of Myb in vivo is to counter a repressive activity of MMB. Immunostaining of polytene chromosomes revealed that the association of Myb with specific chromosomal sites was dependent on Mip120, reinforcing the idea that Myb needs MMB for chromatin binding. Conversely, Mip120 and Mip130 did not require Myb for polytene chromosome binding (Beall, 2007).

Unanticipated and in contrast to mip130 mutants, it was found that mip40 and mip120 mutant males were sterile, thus defining a new role for these proteins in male fertility. Given that Mip130 has a paralog in the testis called Always early (Aly), the possibility was investigated that Mip40 and/or Mip120 might either function in a testis-specific Aly complex or that one or both might have testis-specific paralogs (Beall, 2007).

A combination of affinity, ion-exchange, and gel filtration chromatography was used to isolate Mip40-containing complexes from testis nuclear extracts. In addition to MMB, tMAC, a new testis-specific meiotic arrest complex in which the only MMB subunits found were Mip40 and Caf1/p55, was identified, in addition to the testis-specific proteins Aly, Cookie monster (Comr), Matotopetli (Topi), and Tomb. It is suggested is that MMB functions as a cell-type- and developmental-stage-specific regulator of transcription and replication with various subunits contributing to a 'Swiss Army Knife' type of versatility: the ability to interact specifically with numerous cis-elements, and to interact with numerous coactivators or corepressors as determined by context. This versatility extends beyond MMB itself, as some subunits are part of other tissue-specific complexes involved in gene expression (Beall, 2007).

These genetic findings that clearly separate developmental functions for Mip40 and Mip120 do not provide mechanistic insights into how the pleiotropic effects are manifested. For example, partial MMB complexes, resulting from loss-of-function alleles, may assume neomorphic activity. It has been argued that myb lethality in Drosophila is a consequence of such effects. The model that rationalizes 'silent subunits' present at a given location to promote switching from repression to activation (or vice versa) adds genetic complexity to the timing of critical execution functions for different MMB factors. This model anticipates that loss-of-function alleles of genes for different MMB subunits would manifest different arrest points. The possibility was considered that MMB subunits do not always function as a unit. The finding that mip40 mutant males displayed a staining pattern for Mip40 protein in testes quite distinct from Mip130 or Mip120 was curious. Epitope masking of one or another protein in MMB, due to changing coactivator or corepressor association, might explain such staining patterns. However, further work led to the search for a putative testis-specific complex that contained Mip40, where it reasonably would act in a distinct way from its functions in MMB (Beall, 2007).

A complex was purified from testis nuclear extracts that contains MMB members Mip40 and Caf1/p55, in addition to the testis-specific meiotic arrest proteins Aly, Comr, Topi, and Tomb. This complex was named tMAC, because aly, comr, topi, and mip40 mutants all display the same testis phenotype: an arrest at the primary spermatocyte stage of development, consistent with the notion that they are all acting together in a complex to promote differentiation and meiotic cell cycle progression. It seems likely that other proteins might interact with tMAC to aid in the regulation of testis-specific transcripts. This idea stems from what is already known about MMB, where proteins such as Rpd3 and L(3)MBT physically associate with MMB only during early steps in the biochemical purification process and are critical for function at different DNA sites. To date, no other alternative or subcomplexes containing members of MMB have been isolated from either embryo or tissue culture nuclear extracts. Furthermore, genomic profiling in KC cells of key MMB members (Myb, E2F2, Lin-52, Mip120, and Mip130) substantiates the hypothesis that these proteins work together as a group rather than as isolated factors on DNA. Had testis-enriched starting material had not been examined, it would have been impossible to identify tMAC. Thus, despite the present data showing that the MMB core factors always work as an ensemble, it is possible that some of the pleiotropic phenotypes observed for different MMB subunit mutants could reflect the activity of a subunit functioning outside of MMB (Beall, 2007).

It is striking that both tMAC and MMB contain proteins, other than Mip40 and Caf/p55, that are similar to each other in domain architecture: Aly (tMAC) or Mip130 (MMB) and Tomb (tMAC) or Mip120 (MMB). Given that MMB and tMAC contain multiple site-specific DNA-binding proteins (Myb, E2F2/DP, Mip120, Mip130 in MMB; and Tomb and Topi in tMAC), a potentially large number of genes may be regulated by tMAC as is now know is true for MMB (Beall, 2007).

Antisera against the MMB subunit, Lin-52, failed to coimmunoprecipitate Comr or Aly; therefore, it is not likely a tMAC subunit. However, it is interesting to note that there is another Lin-52 family member in Drosophila (CG12442). The adult Drosophila gene expression database indicates that this gene is, indeed, highly expressed in testis relative to other tissues. Given that this protein is extremely small (predicted molecular weight of 16 kDa), it is possible that it was not present in sufficient quantities to be detected in the mass spectrometry analysis and may in fact be part of tMAC. If so, that would be the third MMB subunit to have an alternative 'testis-specific' form present in tMAC (Beall, 2007).

Gonad-specific forms of proteins that are ubiquitously expressed and generally found in complexes that regulate transcription may, indeed, be a common theme. For example, gonad-specific components of the basal RNA polymerase II transcription machinery are crucial for developmentally regulated gene expression programs in these tissues. Five testis-specific TATA-binding protein-associated factors (TAFs) have been identified in Drosophila (encoded by the can, sa, mia, nht, and rye genes). All are required in spermatocytes for the normal transcription of target genes involved in post-meiotic spermatid differentiation (the so-called can class of genes). It is thought that these testis-specific TAFs may associate with some of the general TAF subunits to create a testis-specific TFIID (tTFIID) that carries out the developmentally regulated transcriptional program in spermatocytes. The mip40-null allele is also in the can class, suggesting that tMAC may interact with tTFIID at can class gene promoters. It will be interesting to explore the possibility that tMAC is a testis-specific coactivator with tTFIID. Other tMAC subunits fall into the aly class and might be what is expected for a large complex paralogous to MMB, where one or another subunit may be silent and subsequently required at a later stage or developmental pathway (Beall, 2007).

Mip130 family members, such as Aly, share a domain that is called a 'DIRP' (domain in Rb-related pathway) domain that is thought to be responsible for interaction with Rb. The DIRP domain of human-Lin-9 (Mip130) is necessary for association with Rb; however, the interaction between hLin-9 and Rb may be indirect as hLin-9 may exist in a complex with other proteins that directly touch Rb. Neither of the two Drosophila Rb proteins was in tMAC-containing fractions. When alignments were made with Mip130 family members, a region was noticed within the DIRP domain that was conserved between all family members except Aly. It is possible that this divergent region within the DIRP domain is critical for Rb interaction in other family members and has been lost in Aly. Although no direct understanding is available of how Aly works for transcriptional activation, it is possible that tMAC contains both activating and repressing components similar to MMB and that repression at particular loci does not require E2F/Rb (Beall, 2007).

When examined for replication profiles in mutant ovaries, an absence of amplification-stage egg chambers was found in mip120 mutants, and widespread BrdU incorporation and Orc2 staining in mip40 mutant amplification-stage follicle cell nuclei. The mip40 egg chamber phenotype is similar to that of mip130 and is consistent with a negative regulatory role for these proteins in genome-wide replication at these stages. It is suggested that both Mip40 and Mip120 are functioning in complex with MMB in ovaries and that both are required for normal patterns of replication in amplification-stage follicle cell nuclei. It is speculated, based in part on unpublished studies of the intricate regulatory network of MMB in Kc cells, that the different mutant phenotypes may simply reflect differences in gene expression profiles that result when individual MMB complex members are missing. A key role for Mip120 in the stability of chromatin-bound MMB might, therefore, explain the more severe phenotype of mip120 mutants. More specifically, MMB may regulate the expression of genes critical for amplification-stage egg chamber development either directly or indirectly, and Mip120 is required for targeting MMB to these gene promoters at a particular developmental stage prior to amplification stages. In contrast to Mip120, Myb, and E2F2/DP, Mip40 has no direct DNA-binding ability. Mip40 may be required for repression or activation only after MMB is targeted to chromosomal sites (Beall, 2007).

As with myb; mip130 mutants, it was found that myb; mip40 and myb; mip120 double mutants were viable, further demonstrating that function(s) of MMB without Myb are responsible for myb lethality. Myb protein was no longer associated with chromatin in mip120 and mip130 mutant polytene spreads. However, staining of polytene chromosomes demonstrated that Mip120 and Mip130 proteins were still bound to chromatin in myb mutants in such a way that may prove lethal in the absence of myb. Together, these data support a model in which Myb is critically dependent on members of the MMB complex for both stability and association with chromatin (Beall, 2007).

It is suggested that the presence of MMB at the replication enhancer ACE3 in stage 7-9 egg chambers may actively repress DNA replication here and at other sites in the genome. MMB at ACE3 at these early stages seems poised to await signals for initiation of amplification. The conversion of a repressive MMB complex into an activating complex may require cyclin E/Cdk2 activity, which is required for amplification. In this context it is likely that Rbf association with MMB will persist during amplification as Rbf association with MMB remains unchanged even after saturating hyperphosphorylation by Cdk:cyclin E in vitro. In the future, determining the cell-type-specific signals that target MMB at well-defined cis-regulatory elements at both the follicle cell amplicons and in other tissues will help unravel how MMB functions in vivo (Beall, 2007).



Analysis of polyadenylylated RNA extracted from whole Drosophila shows that Myb is expressed as a single-sized mRNA of 3.2 kb throughout development. To examine the spatial distribution of the Drosophila MYB message, in situ hybridization was performed using RNA from frozen sections of developing embryos, larvae, pupae, and adults. Drosophila MYB transcripts are abundant and evenly distributed in preblastoderm embryos, where they are presumably of maternal origin. Levels of message continue to be relatively uniform during cellular blastoderm formation and germ band extension. Differential expression becomes evident in germ-band-shortened embryos, where message levels are relatively low in developing salivary glands and undetectable in the amnioserosa; in later stages of embryogenesis, MYB message continues to be present at high levels only in the central nervous system (Katzen, 1996).


In climbing stage third instar larvae, Drosophila MYB transcripts are abundant in all tissues that contribute to the adult organism: imaginal discs, brain, ventral ganglia, testes, ovaries, and nests of imaginal cells within the gut. MYB transcripts are not detectable in any nonproliferative polyploid larval tissues. These expression patterns are maintained in prepupae. Transcripts are also present (albeit at lower levels than in imaginal disc cells) in the abdominal histoblasts, which undergo a period of rapid division immediately after puparium formation and in epithelial cells of the developing imaginal gut. After cephalic eversion, levels of MYB message begin to decline in the everted imaginal discs and transcripts are not detectable during the latter half of pupal development. In contrast, abundant levels of message are observed throughout pupal development in neural tissue, ovaries, and testes (Katzen, 1996).


In both newly emerged and mature adults (3-5 days after eclosion), Drosophila Myb is expressed at relatively low, but detectable levels in the brain and thoracic ganglia. Abundant and comparable levels of Drosophila MYB message are detected in ovaries and testes. In ovaries, the message is localized to nurse cells and developing oocytes, confirming that the transcripts present in preblastoderm embryos are of maternal origin. In testes, specific cell types have not been identified, but expression extends well beyond the germinal proliferation center (located at the distal tip) (Katzen, 1996).

Requirements for DNA replication origin function

The developmentally regulated amplification of the Drosophila third chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-ß is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the ~1.2 kb S18 chorion gene and the 840 bp ori-ß. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-ß, thereby confirming the sequence specificity of ori-ß. Deletion mapping of ori-ß identified two required components: a 140 bp 5' element and a 226 bp A/T-rich 3' element called the ß-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2) recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satin eliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like Chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminate foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-ß nucleate the formation of an ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon Chiffon (Zhang, 2004).

The use of insulator elements, the suppressor of Hairy-wing protein binding sites [su(Hw)BSs], protects transgenic chorion gene constructs from chromosomal position effects and allows for detailed analysis of sequence requirements for amplification. The ACE3 replicator and ori-ß origin elements are necessary for efficient amplification. A construct containing only the 320 bp ACE3 and the 840 bp ori-ß ('Small Parent' or SP) demonstrates that these elements are also sufficient for amplification; however, the levels of amplification are moderate and are subject to significant chromosomal position effects even in the presence of the flanking insulator elements. In the BP construct, the 320 bp ACE3 and the 840 bp ori-ß are in their normal context, i.e., spaced by the ~1.2 kb S18 chorion gene, and these sequences supported efficient amplification (~20 fold) with minimal position effects. For this reason, the BP construct was chosen for detailed analysis of ACE3 and ori-ß sequence requirements. Evolutionarily conserved core sequences were found to be sufficient for the majority of ACE3 activity. Deletion of the less conserved 5' and 3' flanking sequences within ACE3 had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. These results and conclusions are analogous to those from a previous study of ACE3 sequence requirements done in the context of a larger, unbuffered construct. No deletion of a subset of ACE3 sequences reduced amplification to the extent of a deletion of all of ACE3. The sequence requirements for ACE3 function in amplification defined in this study correlate well with the sequence requirements previously defined for ORC binding in vitro. The central region of ACE3, corresponding to the evolutionarily conserved sequences, is most crucial for ORC binding, while the 5' and 3' flanking regions within ACE3 stimulates ORC binding. Taken together, the data suggest that the multiple, partially redundant elements that comprise ACE3 are ORC binding sites, and that one crucial function of ACE3 in amplification is to bind ORC (Zhang, 2004).

Recently a protein complex containing Drosophila Myb, p120 and three other proteins was found to bind to both ACE3 and ori-ß sequences, and Myb was found to be required in trans for amplification. Both Myb and p120 are capable of DNA binding on their own, and have binding sites that overlap with the essential core region of ACE3. There are two Myb consensus binding sites (positions 121 to 127 and 137 to 142) and three p120 binding regions (27 to 56, 89 to 105 and 184 to 216) in ACE3 element. Small (30-40 bp) deletions that removed one of Myb consensus binding sites or one of the p120-binding sites in the core region of ACE3 had negative effects on amplification in the context of the BP construct. Taken together, these data suggest that another function of the conserved core region sequences of ACE3 is to bind the Myb complex (Zhang, 2004).

Two-dimensional gel analyses of the endogenous third chromosome chorion gene locus demonstrate that the majority (70%-80%) of initiations occur in a region containing the ori-ß element. In 2D gel analysis of the BP construct, abundant initiation events, as indicated by bubble structures, were associated with the ori-ß element while no initiations could be detected for ACE3. To begin to examine the sequence requirements for ori-ß function, ori-ß was substituted by either the entire 193 bp S. cerevisiae ARS1 origin sequence, or the 20 bp B2 element from ARS1, which is a putative DNA unwinding element. No activity in supporting amplification was detected for either fragment, indicating that ori-ß is not simply an A/T-rich or easily unwound sequence. Deletion mapping suggests two sub-components of ori-ß: an essential 5' 140 bp region that is not particularly A/T-rich, and the 226 bp A/T-rich ß region. The 366 bp fragment containing both regions is sufficient for the majority of ori-ß activity. In addition the 3' most 140 bp of the starting 840 bp ori-ß fragment may have a small stimulatory effect. The portion of the alpha region in ACE3 and the ß region in ori-ß are each A/T-rich and internally repetitive, and have some sequence homology with each other. A large fragment containing the ß-region can bind ORC in vitro. Therefore, it is hypothesized that, like the sequences in ACE3, one required function of the ß region in ori-ß is to bind ORC (Zhang, 2004).

A similar organization has been identified for a developmentally regulated origin in another dipteran fly, Sciara coprophila. In Sciara larvae, the salivary gland cells amplify several loci containing putative pupal case genes, resulting in chromosomal DNA 'puffs'. The ori II/9A DNA replication initiation site has been mapped to the nucleotide level and has similarities to the yeast ARS. Drosophila ORC has been shown (Bielinsky, 2001) to bind to an 80 bp region adjacent to this replication start site (Zhang, 2004).

Analysis of trans-acting gene mutations confirms the intimate association between amplification initiation and the formation of a large focus of ORC2 localization at amplifying chromosomal loci. Mutations in k43 (Orc2) itself, or the newly identified trans-regulatory gene satin, eliminated ORC2 antibody staining and focus formation. Null mutations of chiffon, a dbf4-like gene, completely eliminate amplification. In chiffon-null mutant follicle cells, diffuse ORC2 staining was still present in the nucleus, but it failed to localize into foci at stage 10A. A similar phenotype had previously been observed for mutations in the amplification trans-regulators dDP (a subunit of E2F) and Rbf. A role for chiffon in ORC localization was surprising given the well-characterized order of events known for other organisms. In S. cerevisiae and Xenopus in vitro systems, ORC is bound at origins and is required for the subsequent binding of Dbf4 and its catalytic subunit CDC7, which is one of the last events before origin firing. The data suggest two possible models for the role of chiffon in ORC2 focus formation during amplification. In the first model, Chiffon protein would bind first to the chorion gene sequences, either directly or more likely via an interaction with another DNA-binding protein, since the Chiffon sequence suggests no obvious DNA-binding motifs. Chiffon would then recruit Drosophila ORC2 to the DNA. This model seems unlikely given the opposite order of events observed in yeast and in Xenopus in vitro systems. In the second and favored model, a relatively small amount of ORC binds first to the chorion gene loci, most probably to the conserved core sequences in ACE3 and the ß region in ori-ß. Chiffon protein would then interact with the ORC complex(es) and catalyze the further binding of large amounts of ORC to generate the dramatic foci observed upon staining with ORC2 antibody. A mechanism is envisioned in which the alpha and ß regions nucleate ORC binding, and then through a process dependent upon Chiffon, an ORC-containing chromatin structure spreads along the chromosome to form the dramatic foci. This model is appealing in that it provides a way for ACE3 and ori-ß to interact and form a chromosomal domain activated for DNA initiation events. Previous data have indicated that ACE3 and ori-ß interact during amplification in a way that can be blocked by an intervening insulator element. Moreover, analysis of the endogenous locus indicates that ACE3 is required for the activation of multiple origins spread throughout a chromosomal domain containing the chorion gene cluster. This model is testable in that it predicts that the insulators would form a boundary for this ORC-containing chromatin structure (Zhang, 2004).

The possibility cannot be ruled out that chiffon is not the true Dbf4 homolog in Drosophila, but this appears unlikely. Chiffon shows conservation with Dbf4 homologs from all other species in the key ORC-binding domain (called CDDN2) and the CDC7-binding domain (called CDDN1). Moreover, there is no other gene in the Drosophila genome with detectable homology to Dbf4. However, Chiffon contains an additional large C-terminal protein domain present only in Dbf4 homologs from closely related species, such as Medfly and mosquito. It is speculated that this C-terminal domain may play a specific role in chorion gene amplification. Further experiments will be required to determine if a role in ORC localization is a characteristic of all Dbf4 family members, or whether this represents a function unique to the large chiffon protein (Zhang, 2004).

Consistent with the correlation between ORC2 focus formation and amplification initiation, dramatic ORC2 foci can form at the sites of amplifying transgenic chorion gene constructs. It was therefore surprising that in no cases were foci observed at the sites of actively amplifying BP constructs. This is despite the fact that amplification was readily observed at these sites by BrdU incorporation. One possible explanation might be the moderate amplification level of BP (18- to 20-fold). However, the YES-3.8S construct amplifies to similar levels as BP, and an extra ORC2 focus was observed for every line. In addition multimers of ACE3 with very low amplification levels are capable of creating additional ORC2 foci. Therefore, the lack of focus formation with BP is not simply due to its moderate amplification level, but must reflect the specific sequence content or arrangement in BP. The lack of focus formation in BP is also not simply due to the presence of flanking insulator elements; the YES-3.8S construct contains the same flanking insulator elements. The data suggest two non-exclusive possibilities. The first is that the difference is due to the fact that BP contains less extensive chorion gene sequences than YES-3.8S. Although deletion of these sequences has no significant effect on amplification level, it may be that redundant ORC binding sites have been deleted, thereby dramatically reducing visible focus formation. The second possibility is that the relevant difference is the amount of sequence present inside the insulators. BP contains only 2.4 kb between the insulators, whereas Yes-3.8 contains 9 kb. If the insulators limit the size of the domain in which an ORC containing chromatin structure can spread from ACE3 and/or ori-ß, then the small size of this domain in BP may not create a visible focus. In this model, the insulators would have two significant effects on amplification: they would prevent the spread of negative chromatin structures into the bounded region and thereby prevent negative chromosomal position effects; and they would limit the ORC containing chromatin structure and initiation activity to the bounded region. These models should be testable in the future by CHIP analysis of chromatin structures associated with chorion gene sequences and transgenic constructs. It will be of interest in the future to determine if su(Hw)BS insulators or other types of insulators are involved in organizing the endogenous chorion gene locus and the rest of the genome into domains of DNA replication activity (Zhang, 2004).

Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. It was found that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC; Drosophila RB, E2F and Myb), a tissue-specific version of the mammalian MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).

The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes. In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells in this study did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).

The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).

The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).

Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).

The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor or by heat shock. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).

Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).

The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression, and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011 and references therein).

In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).

Effects of Mutation and Ectopic Expression

Drosophila Myb-1 and Myb-2 are temperature sensitive for lethality (Katzen, 1996). In addition, the lethal phenotype is tighter when either mutation is carried over a deficiency chromosome that deletes the Myb gene than when either of the mutations is homozygous, indicating that both alleles are hypomorphic rather than null. Myb-1 and Myb-2 mutants raised at temperatures permissive for viability are not grossly malformed, but closer inspection of adults reveals several defects, the most obvious of which occur in the wings (Katzen, 1996). These findings indicate that although temperatures of 18°C and 25°C are permissive for viability of Myb-1 and Myb-2 mutants, respectively, they are not completely permissive for myb function. These temperatures are therefore referred to as semi-permissive. When Myb-1 mutant flies are raised at 18°C, wings are approximately the same size and shape as parental wings, but appear to be considerably cruder. In particular, wing veins are thicker and differ slightly in their relative positions. The mutant wings have approximately half the number of hairs as wild-type wings, and mutant hairs are considerably larger than normal. Hairs are less regularly spaced, less uniform in orientation, and occasionally group in small clusters, indicating a disturbance of tissue polarity. Bristles on mutant wings are not obviously reduced in number, but do appear to be larger and less uniform in orientation (Katzen, 1998).

Wings dissected from Myb-2 mutants raised at 18°C are much less severely affected, with respect to both quantity and orientation of hairs, than are Myb-1 wings. However, when wings are dissected from Myb-2/deficiency females, which occasionally survive at 18°C, or from Myb-2 mutants raised at 25°C, the defects are more extreme, closely resembling the Myb-1 phenotype. These results reinforce the evidence from viability studies that Myb-2 is a hypomorphic allele (retains subpar activity), since two copies are better than one (Myb-2 homozygote vs. Myb-2/deficiency) and the severity of wing phenotype is temperature sensitive (Katzen, 1998).

Drosophila Myb is required for the G2/M transition of the cell cycle and for suppression of endoreduplication in pupal wing cells. Studies of the abdominal phenotype in loss-of-function Myb mutants reveal additional roles for Myb in the cell cycle, specifically in mitosis. Abdominal epidermal cells that are mutant for Myb proliferate more slowly than wild-type controls throughout pupation, with particularly sluggish progression through the early stages of mitosis. Abnormal mitoses associated with multiple functional centrosomes, unequal chromosome segregation, formation of micronuclei, and/or failure to complete cell division are common in the later cell cycles of mutant cells. Resulting nuclei are often aneuploid and/or polyploid. Similar defects have also been observed in loss-of-function mutations of the mammalian tumor suppressor genes p53, Brca1 and Brca2. These data demonstrate that in abdominal epidermal cells, Drosophila Myb is required to sustain the appropriate rate of proliferation, to suppress formation of supernumerary centrosomes, and to maintain genomic integrity (Fung, 2002).

In previous analysis of the mutant Myb wing phenotype, wing cells were found to be arrested in G2 of their final cell cycle during pupal development, and some of the arrested cells had entered into endoreduplication, indicating that Myb is required for both promotion of the G2/M transition and suppression of endoreduplication. This study shows that Myb function is required not only to enter mitosis, but to proceed through mitosis. New aspects of Myb function may be apparent in abdominal histoblasts because of the demands of their developmental program. Histoblasts proliferate more rapidly than wing cells (doubling times of less than 3 hours versus 10-12 hours for wing cells), and the levels of Myb mRNA are lower in histoblasts than in wing discs. Comparison of developmental delays indicates that abdominal histoblasts are indeed more sensitive to reductions in Myb function. In Myb mutants, wing development lags behind wild type by only ~1.5 hours, whereas abdominal development lags by 10 to 12 hours. It is also possible that additional mitotic functions for Myb are revealed in abdominal cells because regulation of the G2/M transition is not as restrictive as it is in wing cells (Fung, 2002).

Defects in esg and cdc2 mutants have been attributed to a failure to suppress endoreduplication in abdominal histoblasts during larval development, when they are normally arrested in G2. Although the abdominal phenotype in Myb mutants resembles that of esg and cdc2, mutant Myb histoblast nests contain appropriate numbers of cells with no apparent abnormalities. This indicates that although Myb function appears to be required to suppress endoreduplication during an aberrant G2 arrest, it is not essential for keeping abdominal histoblasts in the normal extended G2 phase that persists throughout larval development (Fung, 2002).

In previous analyses of the mutant myb phenotype, the myb1 allele inevitably produces a stronger phenotype than myb2 at equivalent temperatures. By contrast, the abdominal phenotype is as strong or stronger in myb2 than in myb1 at the same temperatures, indicating that the relationship between the two temperature-sensitive alleles is more complex than previously thought. Although each mutation results in the change of an amino acid perfectly conserved between Myb and its vertebrate counterparts, different regions of the protein are affected: for myb2, the DNA-binding domain; and for myb1, a conserved domain (region IV) near the C terminus for which no specific biochemical activity has yet been ascribed. The finding that the myb1 phenotype is stronger than myb2 in wing cells, but weaker in abdominal cells indicates that Myb activity is differentially regulated in these two tissues (Fung, 2002).

Although several abnormalities are observed in Myb mutant abdominal histoblasts, the earliest developmental defects are likely to represent the primary cellular defect. Abdominal histoblast cells in Myb mutants appear normal during larval development, a period during which they are arrested in G2, but their rate of proliferation after puparium formation is considerably slower than for wild-type cells. The sluggish proliferation does not correlate with a commensurate decrease in mitosis. For example, although histoblasts in myb2/Df(1)sd72a animals lag behind wild-type cells by two to three cell division cycles, the mitotic index in the mutants is considerably higher (5.7% versus 2.7% at 24 hours APF), indicating that at least part of the reduced rate of proliferation in mutants can be attributed to abnormally slow progression through mitosis (Fung, 2002).

Examination of the mitotic cells reveals an enrichment of cells in the early stages of mitosis (pre-metaphase). Even at 24 hours APF, there is an increased ratio of prophase to metaphase cells, and as development proceeds, an increasing number of cells stall in pre-prophase. Whether the majority of pre-prophase cells ever proceed further into mitosis is unclear. It is also possible that some of the cells weakly stained with antiserum against the mitotic-specific phospho-epitope on histone H3 (PH3) were not in pre-prophase, but were instead undergoing chromosome decondensation after having failed to complete mitosis or cytokinesis. These are likely to be in the minority for several reasons: an increased percentage of pre-prophase cells can be seen as early as 24 hours APF, before other mitotic or ploidy abnormalities are evident. Up until 30 hours APF, double staining with rhodamine-labeled phalloidin and DAPI reveals an absolute correspondence of one nucleus per cell. In addition, weakly staining PH3 cells do not have abnormally shaped nuclei, even at later developmental timepoints, as did most cells that failed to complete mitosis; instead, the morphology and PH3 staining of the pre-prophase nuclei closely resembled the occasional pre-prophase nuclei that are observed in wild-type cells. One further possibility, which cannot be ruled out at present, is that the regulation of histone H3 phosphorylation is somewhat abnormal in these cells, and therefore, the weak staining may not always reflect pre-prophase. The scoring of mitotic cells does not distinguish between prometaphase and metaphase, but Bub1 staining patterns suggest that in the mutants, the 'metaphase' population is likely to be enriched for prometaphase cells. Therefore, it is concluded that Myb mutant histoblasts appear to suffer delays or partial blocks in chromatin condensation, in the process of kinetochore attachment to the mitotic spindle and in chromosome alignment on the metaphase plate (Fung, 2002).

The percentage of Myb mutant histoblasts with visible mitotic abnormalities increases dramatically as pupal development proceeds, implying that initial defects are compounded in subsequent divisions. These mitotic defects, which include aberrant numbers of centrosomes, grossly abnormal DNA morphology, aneuploidy and polyploidy, are characteristic of situations in which the coordination of centrosome and nuclear cycles has been disturbed. Failure to precisely coordinate centrosome doubling with the nuclear cell cycle produces mitotic cells with less than or greater than two centrosomes, a situation that generally results in the formation of abnormal spindles. Examples of such abnormalities, which inevitably lead to genomic instability, are represented among Myb mutant abdominal cells. Mitotic cells with only one centrosome forming a monopolar spindle are occasionally seen. These cells cannot divide and will inevitably produce polyploid cells. More commonly observed are mitotic cells with more than two centrosomes forming multipolar spindles. In such a situation, chromosomes will be randomly distributed into multiple daughter cells which will be aneuploid if cell division is successfully completed. Alternatively, if the mitotic cell fails to complete division and returns to an interphase state, the resulting cell would either be multinucleate or contain a single polyploid nucleus. The results indicate that all of these possible outcomes occur in mutant Myb cells. Therefore, the defects observed during later stages of abdominal epidermal development are thought to be the consequence of disturbing the coordination between centrosome reproduction and the nuclear cell cycle (Fung, 2002).

In a recent study that compared gene expression in actively proliferating fibroblasts isolated from people of different ages or from individuals with the premature aging disease progeria, Mybl2 expression was significantly downregulated in both progeria fibroblasts and in 'normal' fibroblasts isolated from old-aged (~90 years) people (Ly, 2000). A significant proportion of these 'aged' fibroblasts exhibit abnormalities, including multilobed nuclei and multiple nuclei within a cell. These nuclear defects are reminiscent of those observed in the abdominal epidermal cells of Myb mutants, suggesting the possibility that reduced levels of Mybl2 might lead to centrosomal abnormalities, which in turn cause aneuploidy and polyploidy (Fung, 2002).

Is the disturbance in centrosome regulation a primary defect of the mutation in Myb or a secondary consequence of the slow rate of proliferation and/or delays in early mitotic stages, which occur earlier in development? The centrosome defect may be primary, because the first abnormalities in centrosome numbers precede the appearance of multilobed and binucleated cells, allowing exclusion of the possibility that a failure in mitosis or cytokinesis represents the primary defect. Once present, however, the extra centrosomes do lead to defective mitoses and failed cell divisions (Fung, 2002).

Many links have been established between the regulation of the cell division cycle and centrosome duplication. Artificial prolongation of mitosis leads to premature splitting and separation of centrosomes, while prolongation of S-phase allows multiple rounds of centrosome duplication to occur. The mildest centrosome defect in Myb mutants appears to be a premature separation of centriole pairs during late anaphase/early telophase, which could be the result of delayed progression through mitosis. Premature separation might explain why the percentage of centrosome abnormalities is always higher in anaphase/telophase cells than in metaphase cells. This early separation may therefore represent the first step in a breakdown of the coordination between nuclear and centrosome cell cycles (Fung, 2002).

Another indication that splitting and/or duplication of centrosomes is not occurring in a coordinated fashion is the frequent occurrence of odd numbers of centrosomes within the mutant cells. Four centrosomes in mutant cells could result from failure to complete the previous cell division, followed by re-entry into the cell cycle. A subsequent mitosis could then easily lead to an assortment of an odd number of centrosomes in each daughter cell (e.g. three in one and one in the other), but they should be duplicated during the subsequent cycle, thereby generating an even number by the following mitosis (six and two, respectively). Intriguingly, odd numbers of centrosomes are also a common feature in vertebrate cells that have suffered a mutation in a tumor suppressor gene such as p53, Brca1 or Brca2 (Fung, 2002).

The normal function of many proto-oncogenes is to participate in signal transduction pathways that regulate cellular proliferation. When proto-oncogenes suffer mutations that convert them to activated oncogenes, they promote uncontrolled cell growth. Specific aspects of the phenotypic defects observed in the Myb mutants, such as sluggish proliferation and stalling or arresting at the G2/M transition, match expectations for loss-of-function mutations in a proto-oncogene. Centrosome amplification and genomic instability, however, are frequently associated with loss-of-function mutations in tumor suppressor genes, not proto-oncogenes. Since genomic instability is associated with oncogenic progression and aggressive tumors, the disturbance in the regulation of centrosome reproduction may be the primary mechanism by which tumorigenesis is promoted when some tumor suppressor genes are mutated. Therefore, the Myb gene shares some properties with proto-oncogenes and others with tumor suppressor genes, raising the possibility that mutations which decrease the activity of one of the vertebrate Myb genes could contribute to genomic instability and subsequent oncogenesis or aging (Fung, 2002).

Drosophila possesses a single gene, Myb, that is closely related to the vertebrate family of Myb genes, which encode transcription factors that are involved in regulatory decisions affecting cell proliferation, differentiation and apoptosis. The vertebrate Myb genes have been specifically implicated in regulating the G1/S transition of the cell cycle. Drosophila Myb is expressed in all proliferating tissues, but not at detectable levels in endoreduplicating cells. Analysis of loss-of-function mutations in Myb has revealed a block at the G2/M transition and mitotic defects, but has not directly implicated Myb function in the G1/S transition. The Gal4-UAS binary system of ectopic expression was used to further investigate the function of Myb. Depending upon the type of cell cycle, ectopic Myb activity can exert opposing effects on S phase: driving DNA replication and promoting proliferation in diploid cells, even when developmental signals normally dictate cell cycle arrest, but suppressing endoreduplication in endocycling cells, an effect that can be overcome by induction of E2F. A C-terminally truncated Myb protein, which is similar to an oncogenic form of vertebrate Myb, has more potent effects than the full-length protein, especially in endoreduplicating tissues. This finding indicates that the C terminus acts as a negative regulatory domain, which can be differentially regulated in a tissue-specific manner. These studies help to resolve previous discrepancies regarding Myb gene function in Drosophila and vertebrates. It is concluded that in proliferating cells, Myb has the dual function of promoting S phase and M phase, while preserving diploidy by suppressing endoreduplication (Fitzpatrick, 2002).

The results reported here reveal apparently contradictory roles for Myb: continuous expression of the Myb protein promotes S phase in diploid cells, while inhibiting DNA synthesis in endoreplicating cells. Similar results have been obtained with continuous ectopic expression of the Cyclin E protein, a paradox that has been at least partially explained. To maintain genomic integrity in proliferating diploid cells, it is necessary to prevent cells from undergoing more than a single round of DNA replication during each cell cycle. To ensure this, initiation of replication requires the assembly of prereplication complexes, an event that occurs in early G1 and is dependent on the low level of Cyclin dependent kinase (Cdk) after the mitotic destruction of cyclins. Although cells that undergo endoreduplication do not undergo mitosis (or at least do not complete mitosis), all endocycles exhibit distinct gap phases between each round of DNA replication. Experimental evidence indicates that endocycling nuclei, like proliferating cells, can only regain the competence to re-enter each S phase after a low point in Cdk activity, which appears to be dependent on decreases in Cyclin E levels. However, it remains unclear why DNA replication in proliferating cells is not inhibited by the continuously high levels of Cyclin E/Cdk2 activity that occur normally during the early cell cycles of Drosophila embryogenesis or that can be driven ectopically in imaginal disc cells (Fitzpatrick, 2002).

The similarities between the responses of both proliferating and endocycling cells to ectopic expression of Myb and Cyclin E suggest the possibility that the effects of ectopic Myb activity might be due to induction (either directly or indirectly) of high levels of Cyclin E, and preliminary results indicate that Cyclin E levels are increased in salivary glands expressing DeltaMyb. However, it was also found that periodic expression of E2F/DP could overcome Myb-induced inhibition of endoreduplication, whereas it has been shown that E2F/DP expression can not override the replication block induced by Cyclin E. The finding that E2F induction can override the inhibition of DNA endoreduplication caused by ectopic Myb activity, suggests that Myb induced inhibition may be upstream of E2F or that E2F can circumvent the Myb-induced block. In addition, it is unlikely that endogenous Myb plays a role in regulating the levels of Cyclin E in endocycling larval cells, since Myb transcripts have not been detected in these cells and no deleterious effects on larval tissues have been observed in loss-of-function mutant alleles of Myb. For these reasons, and because no defects were detected in salivary glands when another Myb construct, which contains the Myb DNA-binding domain fused to an engrailed repressor domain, was ectopically expressed, it is not thought that Myb or DeltaMyb are acting to repress, rather than activate expression of target genes in salivary glands, a phenomena that has been observed with other transcriptional activators when they are overexpressed. Therefore, the results from ectopic expression of Myb reinforce the conclusions from studies of loss-of-function alleles, that one of the functions of Myb is to suppress endoreduplication and maintain genomic stability in proliferating diploid cells (Fitzpatrick, 2002).

The transgenic experiments reported here demonstrate that DeltaMyb is a much more potent inhibitor of endoreduplication than Myb. The C-terminal region of the vertebrate A-Myb and c-Myb proteins has been shown to contain negative regulatory domains that downregulate the DNA-binding and transcriptional activation abilities of the proteins; the equivalent portion of the B-Myb protein contains both negative and positive regulatory sequences. The findings indicate that the C-terminal sequences that were deleted in the Myb protein (by analogy to c-Myb) act to strongly downregulate Myb activity in salivary glands. By contrast, DeltaMyb appeared to be only slightly more active than Myb at promoting proliferation in imaginal disc cells, even those in the zone of non-proliferating cells (ZNC), which should be specifically arrested in either G1 or G2. This finding is in agreement with a growing body of evidence from studies with the vertebrate Myb proteins, that their ability to activate transcription is strongly dependent on the presence and/or abundance of other cellular factors. Therefore, one rationale for the difference between the behavior of the Myb proteins in imaginal discs and salivary glands is that imaginal disc cells may contain an 'activating factor', absent in salivary glands, that interacts with full-length Myb to relieve the repression of its transcriptional activating potential that is mediated via the C-terminal domain. Another possibility is that salivary gland cells contain a factor that specifically interacts with full-length Myb to repress its activity, but this seems less likely since endogenous Myb expression has not been detected in these cells (Fitzpatrick, 2002).

E2F/DP and DREF (DNA replication-related element binding factor) are transcription factors that have been shown to be crucial for cell cycle regulation in Drosophila. These factors promote, and are required for DNA replication in both mitotic and endocycling cells. Like these factors, Myb promotes DNA replication in mitotic cells. However, the situation differs in endocycling cells. Previously reported results have shown that Myb is not required for DNA replication in endocycling cells, and the data presented here demonstrate that Myb can actively inhibit endoreduplication. The ability of Myb to have directly opposing effects on DNA replication, depending upon cell cycle context, makes Myb unique among the transcription factors in Drosophila that have been implicated in cell cycle regulation. Further investigation should elucidate how these transcription factors interact to coordinate cell cycle progression (Fitzpatrick, 2002).

The finding that Myb activity can induce proliferation throughout the ZNC of the wing disc indicates that it can either override or circumnavigate the G1 and G2 blocks established by the Notch and Wingless signaling pathways at the dorsoventral compartment boundary. Preliminary evidence indicates that the levels of protein encoded by some of the genes involved in, or targeted by, these signaling pathways are decreased, but the mechanisms by which this is accomplished are presently obscure. However, disturbances in these pathways may account for the observed increases in apoptosis. Elucidation of these mechanisms should help to further understanding of how cell proliferation and patterning are coordinately regulated in developing organs (Fitzpatrick, 2002).

There is a substantial amount of data indicating that vertebrate Myb genes function to promote the G1/S transition. By contrast, loss-of-function mutations in Drosophila, cause either a block at the G2/M transition followed by endoreduplication or mitotic defects, which have implicated Myb in several aspects of cell cycle regulation, but not directly in the initiation of S phase. These discrepancies have prompted the question of whether the functions of the insect and vertebrate Myb genes are really equivalent? However, mitotic defects (chromosome breakage and cells arrested in metaphase) have recently been observed with mutations in several other genes that are known to be required for DNA replication, including MCM4 (dpa – FlyBase), PCNA (mus209 – FlyBase), and three genes encoding proteins crucial for assembly of the pre-initiation complex: Orc2, Orc5 and dup (also known as cdt1). These findings indicate that the mitotic defects observed in Myb mutants could be secondary consequences of replication defects. By contrast, the findings that Myb is an activator of cyclin B expression in the imaginal eye disc and that Myb activity can induce mitosis in cells within the ZNC that are normally blocked in G2, provide support for the conclusion that Myb has a direct involvement in promoting mitosis. Additionally, three experimental observations provide strong circumstantial evidence that Myb function is not an absolute requirement for DNA replication per se: Myb expression is not detected in larval endoreplicating tissues; endoreduplication in larval tissues appears to occur normally in loss-of-function mutant alleles of Myb; and de novo endoreduplication is observed in mutant wing cells during pupal development (Fitzpatrick, 2002).

Evidence has been presented that, in addition to inducing increased levels of mitosis, Myb, like its vertebrate counterparts, can promote the G1/S transition. These studies also demonstrate that the C termini of the vertebrate and Drosophila Myb proteins share the function of downregulating their activities. Finally, the finding that ectopic Myb can actively inhibit endoreduplication reinforces conclusions from analyses of loss-of-function alleles, that Myb normally acts in proliferating cells to maintain diploidy by suppressing reinitiation of S phase prior to mitosis. The demonstration that at least one aspect of myb function is conserved between the Drosophila and vertebrate Myb proteins, raises the issue of whether one or more of the vertebrate Myb proteins may also act to inhibit endoreduplication and/or to promote mitosis. In conclusion, these studies demonstrate that Myb functions in multiple aspects of the cell division cycle to promote proliferation and maintain the integrity of the genome (Fitzpatrick, 2002).

Mutation of the Drosophila Myb causes genomic instability

Vertebrates have three related Myb genes. The c-Myb protooncogene is required for definitive hematopoiesis in mice and when mutated causes leukemias and lymphomas in birds and mammals. The A-Myb gene is required for spermatogenesis and mammary gland proliferation in mice. The ubiquitously expressed B-Myb gene is essential for early embryonic development in mice and is directly regulated by the p16/cyclin D/Rb family/E2F pathway along with many critical S-phase genes. Drosophila has a single Myb gene most closely related to B-Myb. Two late-larval lethal alleles of Drosophila Myb were isolated. Mutant imaginal discs show an increased number of cells arrested in M phase. Mutant mitotic cells display a variety of abnormalities including spindle defects and increased polyploidy and aneuploidy. Remarkably, some mutant cells have an aberrant S- to M-phase transition in which replicating chromosomes undergo premature histone phosphorylation and chromosomal condensation. These results suggest that the absence of Drosophila Myb causes a defect in S phase that may result in M-phase abnormalities. Consistent with a role for Drosophila Myb during S phase, Dm-Myb protein is detected in S-phase nuclei of wild-type mitotic cells as well as endocycling cells, which lack both an M phase and cyclin B expression. Moreover, it was found that the Dm-Myb protein is concentrated in regions of S-phase nuclei that are actively undergoing DNA replication. Together these findings imply that Dm-Myb provides an essential nontranscriptional function during chromosomal replication (Manak, 2002).

Analysis of two temperature-sensitive alleles of Dm-Myb has suggested that this gene is required for progression from G2 into M phase. To directly test whether a G2/M delay or arrest occurs in the absence of Dm-Myb protein, flow cytometry was used to determine the DNA content of imaginal wing disc cells from late third instar wild-type and MH107 Dm-Myb mutant larvae. Wing imaginal discs contain a largely unsynchronized population of cells that cycle in small clusters, increasingly accumulating in G2 as third instar larval development proceeds. Therefore, if the primary defect in the Dm-Myb mutant occurs during G2/M progression as reported, one would expect to see an increase in the fraction of mutant cells with G2 DNA content. Instead, a decrease in this fraction was observed (Manak, 2002).

The inversion of the G1:G2 ratio of DNA content in mutant vs. wild-type imaginal wing disc cells may be the result of a delay in G1/S. It is also possible that the observed decrease in mutant cells with a G2 DNA content is the result of a developmental delay despite the nearly wild-type size of the discs in late third instar mutant larvae. However, this seems unlikely for two reasons: (1) wandering mutant larvae like those used for DNA content analysis begin to pupariate within a day, implying that they are indeed late third instar; (2) never in mid to late third instar wild-type larvae does the fraction of cells with G2 DNA content exceed the fraction of cells with G1 DNA content (Manak, 2002).

Because only a very small fraction of cells with G2/M DNA content is generally in M phase (1%-2%), mitotic cells in wild-type and mutant imaginal discs were directly enumerated with antibodies directed against condensed chromatin (PH3). A 2-3-fold increase in the percentage of cells with condensed chromosomes was found in mutant imaginal discs, rather than a decrease as would be expected for cells with a primary defect in the G2/M transition. Intriguingly, many of the mutant nuclei appeared to contain hypercondensed chromosomes as judged by the increased intensity of their PH3 staining. Not surprisingly, a substantial increase in apoptosis was also seen in the imaginal discs of the Dm-Myb mutant (Manak, 2002).

Because of the increased number of mitoses in the Dm-Myb mutant, it was of interest to investigate whether M phase proceeded normally. Third instar wild-type and mutant larval brains and imaginal discs were examined at high power with antibodies directed against γ-tubulin and PH3. Wild-type cells with highly condensed chromosomes contained two symmetrically arrayed centrosomes at the spindle poles, whereas mutant cells with highly condensed chromosomes often contained only one or no centrosomes. Many of the PH3-positive mutant cells contained small, sausage-shaped masses of DNA, reminiscent of the hypercondensed chromosomes seen in mutants of morula (Manak, 2002).

To determine whether centromeres were properly assembled in mutant cells, cells were costained with anti-CID (CENP-A) and anti-ß-tubulin Abs. Brightly staining centromeres were clearly visible in both wild-type and mutant chromosomes. However, the mutant cells displayed a variety of abnormalities, including monopolar spindle attachment and massive polyploidy. To examine chromosomal segregation, cells were costained with anti-PH3 and anti-ß-tubulin. Again a variety of mitotic abnormalities were observed in mutant cells, including monopolar and multipolar spindles. Occasionally, condensed chromatin was found untethered to the spindle during metaphase. This configuration could represent either a failure of congression at the metaphase plate or precocious segregation. These data are certainly consistent with defects in chromosomal segregation and/or cytokinesis. For example, if chromatids fail to segregate and the cell undergoes an additional mitotic cycle without cytokinesis, alterations in centrosome number and ploidy could result (Manak, 2002).

Myb and CBP/Nejire work together to promote mitosis

Drosophila melanogaster possesses a single gene, Myb, that is closely related to the vertebrate family of Myb genes, which encodes transcription factors involved in regulatory decisions affecting cell proliferation, differentiation and apoptosis. In proliferating cells, Myb promotes both S-phase and M-phase, and acts to preserve diploidy by suppressing endoreduplication. The CBP and p300 proteins are transcriptional co-activators that interact with a multitude of transcription factors, including Myb. In transient transfection assays, transcriptional activation by Myb is enhanced by co-expression of the Drosophila CBP protein, dCBP/Nejire. Genetic interaction analysis reveals that these genes work together to promote mitosis, thereby demonstrating the physiological relevance of the biochemical interaction between the Myb and CBP proteins within a developing organism (Fung, 2003).

The cellular basis of the cuticular defects is observed in the wings and abdomens of myb mutants. In the wings, the lower hair density reflects a reduction in cell number, a consequence of the majority of mutant myb cells being arrested in the G2 phase of their final cell cycle. A fraction of the arrested cells also lose the ability to suppress endoreduplication, resulting in DNA contents of higher than 4C. In the abdomens, epidermal cells that are mutant for Myb proliferate much more slowly than wild type cells and display a variety of mitotic defects, including abnormal numbers of centrosomes, resulting in aneuploidy and polyploidy. Therefore, Myb function appears to be required for multiple aspects of the cell cycle (Fung, 2003).

Do the enhanced cuticular phenotypes observed in myb mutants with reduced CBP levels reflect qualitative or quantitative defects at the cellular level? To address this question, pupal wing and abdominal tissue samples were prepared from females that were: wild type for Myb but heterozygous for a mutation in CBP (w,nej3/w,+); homozygous for a mutation in Myb but wild type for CBP (w,myb1/w,myb1), and simultaneously homozygous for a mutation in Myb and heterozygous for a mutation in CBP (w,nej3,myb1/w,+,myb1). During embryonic and larval development, the animals were incubated at 18°C, but to maximize differences in cellular morphology between the various genotypes, they were shifted to 24°C at puparium formation, thereby reducing Myb function during pupal development (Fung, 2003).

Postmitotic wing tissues were double stained with DAPI to visualize nuclei and rhodamine-labeled phalloidin, an F-actin specific stain, to visualize either cell boundaries (28-30 h after puparium formation, APF) or 'prehairs' (developing hairs at 34-36 h APF). In control samples (heterozygous for a mutation in CBP), each cell boundary encircled a single nucleus of relatively uniform size. After initiation of hair formation, the pattern and morphology of nuclei and prehairs was highly ordered and uniform, with each cell producing a single distally protruding hair. In myb1 mutants, cell and nuclear sizes and shapes were more variable and generally larger than in controls. A few cells with bi-lobed nuclei (appearing like two fused nuclei) were also observed, an abnormality not detected in previous experiments, presumably because of its rarity. The size, shape and orientation of prehairs were also less uniform, and two prehairs extending from a single cell were occasionally observed. The variability in nuclear and cellular sizes and shapes became more pronounced when CBP levels were reduced within the context of a myb1 mutant, with enlarged, misshapen or multi-lobed nuclei and/or or multiple separated nuclei within a single cell being commonly observed. The enhanced cellular defects associated with decreased CBP levels generated more extreme variability in the number, size and orientation of prehairs. Most notably, single cells producing two or more prehairs were common in these samples, correlating with the adult phenotype (Fung, 2003).

To quantitate the visual observations the areas of pupal wing cells and nuclei from the relevant genotypes were measured at their largest photographic cross-section. These results confirm that on average, myb1 nuclei and cells are larger than controls, but also more variable. Both properties are substantially enhanced by decreased levels of CBP as shown by increased averages, ranges, and standard deviations. Six microscopic fields of wing cells (measuring 0.055 mm×0.07 mm) for each genotype were also examined for the presence of cells with either multi-lobed or more than one nucleus, and for cells with more than one protruding hair. No examples of these abnormalities were detected in control samples. For myb1, a total of six cells with bi-lobed nuclei and 16 cells with multiple wing hairs were observed. These defects were greatly enhanced in nej3,myb1/myb1 samples, where a total of 43 cells with multi-lobed or 2 or more separated nuclei and 83 cells with multiple wing hairs were observed (Fung, 2003).

The presence of multi-lobed or multiple separated nuclei in cells that were homozygous for myb1 and heterozygous for nej3, indicates that the cells enter, but do not complete mitosis or cytokinesis, a phenotype that is qualitatively different from most of the myb1 mutant wing cells, which appear to have been arrested before entering into their final mitosis. On the surface, this result is counter-intuitive since it indicates that the myb1 mutant cells with reduced CBP levels appear to be progressing further in the cell cycle than the myb1 mutant cells with normal levels of CBP. However, the presence of fewer, larger wing cells when CBP levels are reduced, indicates that at least a portion of these cells are failing to complete cell division in the previous (second to last) cell cycle, thereby accounting for the enhanced phenotype (Fung, 2003).

The cells that form the wings and other adult thoracic and head structures proliferate throughout larval development, completing their final one or two cell divisions during early pupation. In contrast, the cells that form the adult abdominal epidermis do not divide during larval development, but undergo rapid proliferation after puparium formation. The first detectable defect in abdominal cells that are mutant for Myb is that they proliferate considerably more slowly than wild type cells. Therefore, the rate of abdominal epidermal development was compared between the same three genotypes used for the wing analysis. By 27 h APF, replacement of the larval abdominal epidermal cells by adult cells is already well underway in controls. This process is clearly retarded in myb1 mutants, but the delay is much more dramatic when CBP levels are reduced within the context of a myb1 mutant. In controls, the majority of polyploid larval cells have already been replaced with adult cells by 32 h APF, even though cell proliferation continues until about 40 h APF. In contrast, small regions of larval cells at the segment boundaries (which are the last to be replaced) were still present in myb1 mutants at 42 h APF in, and much larger regions of larval cells remain when CBP levels are reduced, even at 45 h APF. Since the animals were shifted to a non-permissive temperature at puparium formation (24°C), neither the myb1/myb1 nor the nej3,myb1/+,myb1 females survived to adulthood. Therefore, the cellular defects observed in these experiments are expected to be more extreme than those represented by the cuticular defects in adults that were raised entirely at permissive temperature. However, the dramatic delay in replacement of larval cells by adult epidermal cells is likely to account for the undifferentiated cuticle observed between segments and along the dorsal midline in nej3,myb1/+, myb1 adults (Fung, 2003).

Although mutant myb cells proliferate slowly, the mitotic index is actually higher in the mutant cells than in controls throughout pupal development, indicating a specific delay in progression through mitosis. Using an antibody for a mitotic-specific phospho-epitope on histone H3 (PH3) to identify mitotic abdominal histoblasts, it was found that at 32 h APF, the average mitotic index was 4.5±1.1% for w,nej3/w,+; 10.3±0.9% for w,myb1/w,myb1; and 17.4±1.1% for w,nej3,myb1/w,+,myb1. Similar results were also observed at other timepoints. This data demonstrate that delayed progression through mitosis is dramatically enhanced when CBP levels are reduced within the context of a myb1 mutant. The sluggish mitotic progression could account for most, if not all, of the associated reduction in the rate of cell proliferation (Fung, 2003).

In the later cell cycles of abdominal epidermal cells, abnormal mitoses associated with multiple functional centrosomes, unequal chromosome segregation, formation of micronuclei, and/or failure to complete cell division are common in cells that are mutant for Myb. It seemed likely that the mitotic abnormalities and slowed rates of cellular proliferation in myb mutants are directly related to each other, and it was therefore anticipated that the occurrence of mitotic abnormalities would also be enhanced by reduced levels of CBP. However, the data do not support this expectation. No changes were detected in the timing or rate of centrosomal and chromosomal abnormalities between w,myb1/w,myb1 and w,nej3,myb1/w,+,myb1 samples, suggesting that these defects may be at least partially independent of the reduced rate of proliferation. The size and morphology of the cells and nuclei from the two genotypes were also very similar, an observation that is consistent with the rate of mitotic defects not being increased in these samples. These findings are also consistent with the observation in adults that in regions where differentiated cuticle has formed, the phenotype is not appreciably different between myb1 mutants that are wild type or heterozygous for nej3 (Fung, 2003).

Although there are some discrepancies, these results confirm the conclusions of (Hou, 1997) that co-expression of CBP with Myb enhances the ability of Myb to activate transcription of a reporter construct in transient transfection assays. Taken together with their demonstration of direct binding between CBP and Myb in vitro, it is concluded that like their vertebrate counterparts, the Drosophila CBP and Myb proteins physically interact and that CBP acts as a transcriptional co-activator of Myb (Fung, 2003).

CBP-related protein, p300, can acetylate lysines within a highly conserved region of the human c-Myb protein, and the acetylation enhances the DNA-binding and transactivation capabilities of c-Myb. Of the three lysine residues within the conserved region (region III) that may be acetylated (K471, K480 and K485), the first two are conserved at equivalent positions in the Drosophila Myb protein (K450 and K459). The evolutionary conservation of these lysine residues suggests that they may be targets for acetylation by Drosophila CBP and that the mechanism of activating the Drosophila Myb protein via acetylation may also be conserved (Fung, 2003).

Reducing the levels of CBP in animals mutated for Drosophila Myb enhances virtually all aspects of the mutant phenotype: viability is reduced and cuticular and cellular defects are increased. The genetic interaction between Myb and CBP provides direct evidence that the biochemical interaction between CBP and Myb proteins (demonstrated in mammalian and Drosophila systems) is physiologically relevant within the context of a developing animal. Previous studies have shown that Drosophila CBP functions during multiple stages of development and that mutations in CBP/nej produce pleitropic phenotypes, indicating that CBP may be required for multiple developmental processes. Indeed, CBP/nej has been implicated in several signal transduction pathways that regulate developmental patterning, including the Decapentaplegic, Hedgehog, and Wingless pathways. However, the analysis presented here provides the first explicit evidence that Drosophila CBP is directly involved in regulating cell proliferation (Fung, 2003).

A paradox of CBP/p300 function is that these proteins appear to be capable of having opposing effects on cell proliferation. Mice or humans with mutations that led to reduced levels or activity of CBP display markedly increased susceptibility to tumorigenesis, indicating that they function as tumor suppressors. However, a plethora of biochemical and cell culture studies have shown that CBP/p300 physically interacts with, and activates a number of transcription factors known to promote cellular proliferation, including E2F1 and oncoproteins such as JUN, FOS and MYB. Still, direct evidence for CBP/p300 being able to cooperate with any of these transcription factors to drive proliferation within an animal has been lacking. Therefore, the finding that Drosophila CBP is required in concert with Myb for positive regulation of the cell cycle during Drosophila development validates a physiological role for CBP/p300 in promoting cell proliferation in vivo, and supports the proposal that the pro-or anti-proliferative effects of CBP/p300 are dependent on cellular context (Fung, 2003).

Functional Evolution of the Vertebrate Myb Gene Family: B-Myb, but neither A-Myb nor c-Myb, complements Drosophila Myb in Hemocytes

The duplication of genes and genomes is believed to be a major force in the evolution of eukaryotic organisms. However, different models have been presented about how duplicated genes are preserved from elimination by purifying selection. Preservation of one of the gene copies due to rare mutational events that result in a new gene function (neo-functionalization) necessitates that the other gene copy retain its ancestral function. Alternatively, preservation of both gene copies due to rapid divergence of coding and non-coding regions such that neither retains the complete function of the ancestral gene (sub-functionalization) may result in a requirement for both gene copies for organismal survival. The duplication and divergence of the tandemly arrayed homeotic clusters have been studied in considerable detail and have provided evidence in support of the sub-functionalization model. However, the vast majority of duplicated genes are not clustered tandemly, but instead are dispersed in syntenic regions on different chromosomes, most likely as a result of genome-wide duplications and rearrangements. The Myb oncogene family provides an interesting opportunity to study a dispersed multigene family because invertebrates possess a single Myb gene, whereas all vertebrate genomes examined thus far contain three different Myb genes (A-Myb, B-Myb and c-Myb). A-Myb and c-Myb appear to have arisen by a second round of gene duplication, which was preceded by the acquisition of a transcriptional activation domain in the ancestral A-Myb/c-Myb gene generated from the initial duplication of an ancestral B-Myb-like gene. B-Myb appears to be essential in all dividing cells, whereas A-Myb and c-Myb display tissue-specific requirements during spermatogenesis and hematopoiesis, respectively. The absence of Drosophila Myb (Dm-Myb) causes a failure of larval hemocyte proliferation and lymph gland development, while Dm-Myb(-/-) hemocytes from mosaic larvae reveal a phagocytosis defect. Vertebrate B-Myb, but neither vertebrate A-Myb nor c-Myb, can complement these hemocyte proliferation defects in Drosophila. Indeed, vertebrate A-Myb and c-Myb cause lethality in the presence or absence of endogenous Dm-Myb. These results are consistent with a neomorphic origin of an ancestral A-Myb/c-Myb gene from a duplicated B-Myb-like gene. In addition, these results suggest that B-Myb and Dm-Myb share essential conserved functions that are required for cell proliferation. Finally, these experiments demonstrate the utility of genetic complementation in Drosophila to explore the functional evolution of duplicated genes in vertebrates (Davidson, 2005).

Dm-Myb-/- larvae are completely devoid of visible crystal cells. Crystal cells were detected by heating the larvae to 70°C for 10 minutes resulting in the premature melanization of the cells and making them easily visible through the cuticle of control larvae. Visible crystal cells were also absent in Dm-Myb, Black Cell double mutant larvae, of interest because the dominant Black Cell mutation causes aberrant melanization of crystal cells. The crystal cell lineage development requires the transcription factor Lz; consistent with this requirement, lz null (lzR15) larvae are similarly devoid of visible crystal cells. Sectioning through the Dm-Myb-/- larvae confirmed the complete absence of crystal cells and also revealed reduced plasmatocyte numbers compared to controls. Thus, Dm-Myb is required for the proliferation of larval hemocytes and differentiation of the crystal cell lineage (Davidson, 2005).

Due to the severe proliferation defect of hemocytes both in the circulation and lymph gland of Dm-Myb-/- larvae, mosaic animals were generated using the flp/FRT system to further characterize the role of Dm-Myb in larval hematopoiesis. This system utilizes the flp recombinase to induce site-specific mitotic recombination at the FRT site during the G2 phase of the cell cycle leading to the generation of homozygous mutant cells (-/-) in an otherwise heterozygous animal (+/-). Application of this technique should facilitate the study of the Dm-Myb proliferation defect in the context of wild-type neighboring hematopoietic cells and might allow determination of whether Dm-Myb-/- hemocytes adopt a limited repertoire of cell fates (i.e., a block in hemocyte differentiation) in the presence of a normal lymph gland environment. The Dm-Myb-/- hemocytes were positively labeled with the fluorescent marker GFP via the Mosaic Analysis with a Repressible Cell Marker (MARCM) system, since this technique would facilitate analysis of mutant hemocytes by flow cytometry. This system has in trans to the mutant gene of interest a dominant repressor (GAL80) of GAL4-driven expression of a cell marker (UAS-mCD8a::GFP). Heat-shock induced expression of the flp recombinase results in mitotic recombination events at FRT sites generating homozygous mutant cells that are GFP+ due to loss of the GAL80 repressor of GAL4 activation. Through use of the MARCM system it was possible to generate GFP-positive, Dm-Myb-/- hemocytes both in the lymph gland and in the hemolymph. An analysis of Dm-Myb mosaic lymph glands with antibodies against the previously characterized Drosophila hemocyte markers including Croquemort, a CD36-like protein involved in plasmatocyte phagocytosis, peroxidasin, PDGF/VEGF receptor and Notch did not reveal any difference between Dm-Myb+ and Dm-Myb-/- hemocytes. Using a flow cytometry staining strategy that facilitates the discrimination of Drosophila larval hemocytes from contaminating events acquired during data collection, it was possible to measure phagocytosis in living hemocytes. Phagocytosis was tested by the injection of controlled volumes of fluorescently-labeled, heat-killed E. coli into Dm-Myb-/- mosaic larvae generated using the MARCM system. The flow cytometry staining strategy combined propidium iodide (PI), a cell-impermeable DNA dye that selectively labels dead cells, and monochlorobimane (MCB), a cell-permeable probe for the tripeptide antioxidant glutathione (GSH). Proper intracellular GSH levels are required for cell survival; thus, dead cells and debris lack GSH whereas live hemocytes are highly positive for MCB (GSHhi) and negative for PI (PI-). After the application of flow cytometry, the two populations of hemocytes [Dm- Myb+ (GFP-) and Dm-Myb- (GFP+)] from MARCM mosaic larvae were compared in their ability to take up heat-killed E. coli labeled with the Alexa Fluor594 dye. Calculating the ratio of hemocytes positive for uptake of E. coli to negative hemocytes between the two populations (GFP+ versus GFP-) revealed a phagocytosis defect in the Dm-Myb-/- hemocytes. A comparison of these ratios reveals that Dm-Myb-/- hemocytes demonstrate a 57%-68% decrease in phagocytosis compared to Dm-Myb+ cells (Davidson, 2005).

Consistent with the low number of plasmatocytes observed in whole larval sections, the lymph glands of Dm-Myb-/- animals are undersized relative to those of control animals in similarly staged third instar larvae, the stage of greatest lymph gland hemocyte proliferation. Furthermore, the reduced number of hemocytes contained within the lymph glands of Dm-Myb-/- larvae have aberrant nuclei as revealed by DNA staining. Interestingly, use of an antibody directed against condensed chromatin, phosphohistone H3 (PH3), revealed a substantial increase of Dm-Myb-/- hemocytes positive for this marker of mitosis. Elevated PH3 staining is consistent with previous experiments in which loss of Dm-Myb results in abnormal mitoses and increased PH3 staining in larval brain and imaginal discs. The undersized lymph glands of Dm-Myb mutant larvae in combination with elevated levels of PH3 staining in Dm-Myb-/- hemocytes suggest a cell cycle block and indicates that Dm-Myb is required for larval lymph gland hemocyte proliferation (Davidson, 2005).

To determine the functional redundancy between the vertebrate Myb genes and Dm-Myb, rescue of Dm-Myb-/- larval lymph glands was performed via GAL4-mediated expression of a transgenic cDNA placed under the control of a UAS element. Expression of a transgene was accomplished using a previously characterized lymph gland and pericardiocyte enhancer trap-generated GAL4 driver (GAL4-e33c), and expression of Dm-Myb resulted in the rescue of circulating crystal cells in Dm-Myb-/- larvae. Expression of vertebrate B-Myb using the GAL4-e33c driver also restored circulating crystal cells in Dm-Myb-/- larvae. Furthermore, lymph gland crystal cells were restored in Dm-Myb-/- larvae as detected by Lz immunolocalization in UAS-Dm-Myb rescued and in UAS-B-Myb rescued lymph glands. However, expression of vertebrate A-Myb or c-Myb via the GAL4-e33c driver failed to rescue circulating crystal cells. Indeed, expression of these transgenes with the same driver was not compatible with viability. This is consistent with expression of vertebrate Myb transgenes in the eye of Drosophila; A-Myb and c-Myb expression leads to an aberrant eye phenotype whereas expression of B-Myb and Dm-Myb shows no visible phenotype (Davidson, 2005).

Directed expression of UAS-Dm-Myb and UAS-B-Myb with the GAL4-e33c driver in Dm-Myb-/- lymph glands restored normal levels of PH3 staining in all examined lymph glands. In addition, cell proliferation was restored as measured by the presence of BrdU incorporating cells in UAS-Dm-Myb rescued lymph glands and UAS-B-Myb rescued lymph glands. Furthermore, colocalization of Dm-Myb and B-Myb proteins with BrdU was observed in mitotically cycling hemocytes. In addition, Dm-Myb protein and BrdU also colocalize in pericardiocytes, large polyploid cells whose nuclei undergo endoreduplication without mitosis. These cells flank the dorsal vessel and are interspersed among the lymph gland lobes. Together, these results implicate Dm-Myb in a functional role during or shortly after S-phase in both mitotic and endocycling cells, consistent with the known roles in larval brain cells and endocycling larval fat body cells. In addition, B-Myb can restore normal proliferation of hemocytes and differentiation of the crystal cell lineage in Dm-Myb-/- larvae. Thus, the differentiation of crystal cell lineage in the larval lymph gland and in circulation requires normal proliferation, which is Dm-Myb dependent, and vertebrate B-Myb can complement this function of Dm-Myb. The lethality associated with the expression of A-Myb and c-Myb indicates there is likely to be little functional overlap between these genes and B-Myb and Dm-Myb and supports the neo-functionalization of an ancestral A-Myb/c-Myb gene following the first duplication (Davidson, 2005).

To establish whether Dm-Myb is generally required for proliferation of hemocytes, epistasis experiments were conducted using a Dm-Myb null mutation and dominant substitution mutations of the Toll receptor (Tl10b) and the Jak kinase, hopscotch (hopTuml); dominant gain-of-function mutations in these genes result in hyperactivation of their respective pathways leading to hemocyte overproliferation and abnormal lamellocyte differentiation. To determine whether Dm-Myb is required for the dysregulated overproliferation and differentiation phenotypes of Tl10b and hopTuml mutants, double mutant larvae lacking Dm-Myb in conjunction with these dominant alleles of Toll and hopscotch were generated. It was found that, in addition to an overproliferation of plasmatocytes in the primary lymph gland lobes, the secondary lymph gland lobe hemocytes aberrantly differentiate into lamellocytes in hopTuml mutants. It is thought that the normally smaller secondary lymph gland lobes serve as a reservoir of undifferentiated prohemocytes, however, in hopTuml larvae the secondary lobes enlarge with concomitant abnormal differentiation of lamellocytes. While hemocytes in the secondary lymph gland lobes of hopTuml, Dm-Myb-/- double mutants show an increased expression of the lamellocyte enhancer-trap marker, these ß-gal positive cells fail to overproliferate and do not adopt the flattened shape characteristic of differentiated lamellocytes. In summary, an activated JAK/STAT pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb. In addition, an activated Toll pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb (Davidson, 2005).


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Myb oncogene-like: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 23 August 2020 

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