pipsqueak


DEVELOPMENTAL BIOLOGY

Antibody staining of ovaries shows that PsqA is a nuclear protein, expressed in both the somatically derived follicle cells and in the germline. The apparent association of the PsqA protein with chromatin in the nurse cells, and the specific fluorescence in distinct dots in the oocyte nucleus, argue that PsqA protein is associated, directly or indirectly, with DNA. These observations suggest that PsqA plays a role in regulating gene expression in oogenesis (Horowitz, 1996).

Siegel (1993) has indicated that ovaries from psq mutant females have reduced levels of vasa mRNA and protein, implicating psq in the regulation of vasa expression. Expression of vasa is initially observed in the germarium; PsqA is both abundant in the early germarium and required very early in oogenesis, correlating with the presence of Vasa at this time. High levels of PsqA are observed in the posterior polar cells at stages 5-6, a time when these cells are purported to be involved in receiving and responding to a signal from the posteriorly localized Grk. It is possible that PsqA is involved in regulating gene expression as part of this complex signaling process (Horowitz, 1996).

The striking and specific pattern of PsqA localization in the oocyte nucleus at stages 6-10 (seen as two small bright spots of fluorescence) suggests that PsqA may bind, either directly or indirectly, to a specific site on the DNA in the oocyte nucleus. It is intriguing to consider the possibility that psq may be directly affecting the expression of a particular locus in the oocyte nucleus (Horowitz, 1996).

Finally, psq could also play a more general role in regulating gene expression by affecting chromatin structure. PsqA protein is associated with the nurse cell chromatin; Siegel (1993) found that psq mutants fail to undergo the normal decondensation of nurse cell DNA at stage 5. Interestingly, two other BTB-containing proteins, E(var)3-93D and GAGA, have been implicated in the modulation of chromatin structure, as well. It is possible that the BTB-containing fusion protein produced in the psq PZ mutants may disrupt the protein-protein interactions of these or other BTB-containing modulators of chromatin structure, leading to some aspects of the mutant phenotypes observed in these lines (Horowitz, 1996).

Given the copurification and genetic interactions between psq and Pc-G, one might expect Psq to be detected on polytene chromosomes at sites corresponding to ANTP-C (at 84AB) and BX-C (at 89E), where strong Pc-G signals are normally observed. Using antibodies against Psc and Psq for double immunofluorescent staining of polytene chromosomes under standard fixation conditions (i.e., 3.7% formaldehyde), no consistent Psq staining at ANTP-C and BX-C sites was found. It was possible, however, that the Psq staining at these sites might have been masked by other proteins of the complex that are apparently layered upon it. Thus, conditions to alleviate any potential masking effect were tested. Using 2.5% formaldehyde for fixation, it was found that Psq signals can be detected at ANTP-C and BX-C sites, with the Psc staining being consistently weaker than what was observed under standard conditions. The staining of Psc and Psq at these sites was not always coextensive. For example, the Psq signal represents only a part of the broader region of Psc staining at the ANTP-C site. The significance of this observation remains unclear (Huang, 2002).

The staining patterns on polytene chromosomes from transgenic flies containing insertions of small PRE fragments (e.g., PRE-D) were examined. While these fragments showed ectopic Psc staining as expected, most of these fragments appeared to insert at sites that correspond to endogenous Psq sites on wild-type chromosomes. Thus, this approach could not be used to map the exact locations of Psq binding sites within the PRE (Huang, 2002).

The pipsqueak (psq) gene is expressed at high levels in the R3/R4 precursors during eye development. Such expression depends on seven-up. Strong psq alleles are dominant suppressors of mutant svp induced cone cell transformation phenotype. The gene is a member of the maternal posterior group of genes, but the effect of strong semilethal alleles demonstrate an additional specific requirement for psq function downstream of svp for the development of photoreceptors R3/R4. Interestingly, all viable alleles with a maternal posterior group phenotype cluster around one specific 5' exon, while all semilethal alleles have lesions that map to a different alternative 5' exon (Weber, 1995)

Effects of Mutation or Deletion

A new member of the posterior group of genes has been identified and termed pipsqueak. pipsqueak acts after the establishment of the Oskar posterior anchor but before the localization of Vasa protein during oogenesis. Characterization of multiple alleles at the pipsqueak locus shows that pipsqueak, like vasa, is required for early stages of oogenesis, including but not limited to formation of the egg chamber and progression through Stage 6 of oogenesis. Genetic interaction studies suggest that pipsqueak acts at least partially through vasa; molecular studies indicate that pipsqueak affects vasa level in the ovary. vasa and pipsqueak mutant phenotypes have been compared in order to determine whether pipsqueak acts solely through vasa: a model is presented for the role of pipsqueak in posterior pattern formation (Siegel, 1993).

A number of alleles have been isolated and they have been ordered into a hypomorphic series. The weakest alleles (psqP1, psqP4) reduce the level of vasa mRNA, resulting in the absence of Vasa protein from the posterior pole of the oocyte and embryo and, concomitantly, in posterior group defects. Stronger alleles cause more significant reductions in vasa level, resulting in earlier blocks in oogenesis. psq13B6 egg chambers contain no detectable vasa transcript and, like vasa deficiency egg chambers, rarely enter vitellogenesis. psq alleles also exhibit phenotypes not seen in a vasa deficiency, suggesting additional roles for the psq locus. Some alleles seem to disrupt egg chamber formation, leading to a supernumerary germ cell phenotype. Some alleles also alter the morphology of the nurse cell nucleus, apparently blocking the polytene to polyploid transition. Finally, in trans to a deficiency, psq causes a rudimentary ovary phenotype, suggesting that germ cells are either lost or fail to produce cystoblasts during development (Siegel, 1993).

Insertional mutagenesis screens using the P[lacZ, rosy+] (PZ) transposable element have provided thousands of mutant lines for analyzing genes of varied function in the fruitfly. As has been observed with other P elements, many of the PZ-induced mutations result from insertion of the P element into the promoter or 5' untranslated regions of the affected gene. A novel mechanism for mutagenesis by this element has been documented. Sequences present within the element direct aberrant splicing and termination events that produce a mRNA composed of 5' sequences from the mutated gene (in this case, pipsqueak) and 3' sequences from within the P[lacZ, rosy+] element. These truncated RNAs could yield proteins with dominant mutant effects (Horowitz, 1995).

Mutations at the pipsqueak locus affect early patterning in the Drosophila egg and embryo. pipsqueak is a large and complex gene, encoding multiple transcripts and protein isoforms. One protein, PsqA, is absent in all of the mutants examined. PsqA is a nuclear protein present in the germ cells and somatically derived follicle cells throughout oogenesis, and it is required prior to stage one of oogenesis. PsqA contains a BTB (POZ) domain at its amino terminus; additionally, an evolutionarily conserved motif of unknown function present four times in tandem at the C terminus of the protein has been identified. PZ pipsqueak mutants produce a putative fusion protein containing the pipsqueak BTB domain fused to sequences resident on the PZ element (Horowitz , 1995). Expression of this fusion protein in wild-type flies has a dominant effect, resulting in infertility and eggshell defects. These dominant phenotypes are discussed in light of current theories on the role of the BTB domain in protein-protein interactions (Horowitz, 1996).

To learn more about the functional relationship between Trl and Psq, a possible genetic interaction between Trl and psq was analyzed, and their roles in the control of homeotic gene expression was compared. Animals doubly heterozygous for the null allele TrlR85 and the alleles psqRF13, psq0115, or Df(2R)psq-lolaDelta18 showed no readily apparent defects or reduced viability. Thus, a single copy of each gene, together with a substantial maternal contribution of Trl and psq gene products, seems to be sufficient for normal development. Trl has been shown to have properties of a PcG gene. Thus, Trl is a component of some PcG complexes and GAGA-binding sites are required to maintain the silencing activity of the bxd, iab-7, and MCP PREs. Consistent with a role of Trl in PRE function, Trl alleles enhance the extra sex combs phenotype of Pc mutants. To test whether psq shows a similar genetic interaction, animals doubly heterozygous for the allele Pc3 and alleles psqRF13, psq0115, or Df(2R)psq-lolaDelta18 were examined. In all three combinations of psq alleles with Pc3, the frequency of animals with ectopic sex combs was significantly higher than in the presence of the Pc3 allele alone. The same interaction, as well as an interaction between polyhomeotic and psq, has been observed by others (Hodgson, 2001, and S. Sakonju, personal communication to (Schwendemann, 2002). On the basis of its interaction with Ubx and Abd-B, Trl has originally been identified as a member of the trxG of genes, which are required for the maintenance of homeotic gene expression. Animals doubly heterozygous for the alleles TrlR85 and Ubx130 show haltere-to-wing and notal transformations with a penetrance of about 8%. In contrast, the same transformations are observed at a much lower frequency in animals doubly heterozygous for the psq allele Df(2R)psq-lolaDelta18 and Ubx130. It was therefore asked whether replacement of one wild-type copy of psq by a mutant allele would have an influence on the dominant genetic interaction between TrlR85 and Ubx130. Both the psq alleles Df(2R)psq-lolaDelta18 and psqF112 clearly enhance this interaction, leading to a 4- to 5-fold increase in the frequency of haltere and notal transformations. The weak dominant genetic interaction observed between psq and Ubx thus seems to be indeed indicative of a requirement of psq wild-type function for Ubx expression. It is concluded that psq has similar functions as Trl, not only in silencing homeotic genes, but also in their activation. This conclusion is further supported by the finding that both psq and Trl alleles are dominant enhancers [E(var)s] of PEV. The white (w) gene is suppressed in a clonally inherited manner when juxtaposed to centromeric heterochromatin by a chromosomal rearrangement, In(1)wm4h, leading to a variegated eye color. The ability to enhance (or suppress) PEV has been exploited to genetically identify factors that counteract (or promote) chromatin-mediated gene silencing. Like the Trl13C allele, the psq alleles Df(2R)psq-lolaDelta18 and psq0115 clearly enhance the variegated eye phenotype of In(1)wm4h. The similar behavior of psq and Trl in this assay is consistent with a common mechanistic basis for the actions of the products encoded by the two genes (Schwendemann, 2002).

To determine the role of psq in vivo, a dosage-sensitive assay was used to test the genetic interactions between Pc and psq mutations. Two classes of psq mutations have been previously identified, each has been implicated in a distinct developmental function. For example, the 0115, 2403, and 8109 alleles (referred to as class I mutations) that result from P element insertion mutations in the ~40-kb intron appear to primarily affect oogenesis, giving rise to the grandchildless phenotype (Horowitz, 1996, Siegel, 1993), whereas the F112, E34, and E39 alleles (referred to as class II mutations) that are clustered around the first exon of psq-B (or psq-2 by Horowitz (1996) appear to affect eye development (Weber, 1996). Although none of these psq mutations alone have been known to cause homeotic phenotypes in adults, this does not necessarily preclude their role in homeotic gene silencing, since an increasing number of Pc-G interacting genes have been found to cause little, if any, homeotic phenotype by themselves. Thus, females heterozygous for various psq alleles were crossed with male Pc4 heterozygotes and were examined for their effects on homeotic leg transformation, i.e., production of ectopic sex comb teeth on the second and third legs of F1 males. The average number of ectopic sex comb teeth is strikingly enhanced for all alleles of class I mutations when doubly heterozygous with the Pc4 mutation (ranging from 9 to 10 teeth per leg), and the second and third legs were almost completely transformed into the first leg. In contrast, class II alleles showed relatively weak but significant effects on leg transformation, giving rise to 3 to 5 ectopic teeth per leg (Huang, 2002).

Since three major protein complexes containing different combinations of Pc-G proteins have been described, it is interesting to determine whether the function of psq is generally or specifically required for these complexes. Pc-G mutations that are representative of these protein complexes were tested. Preliminary results show that there is a remarkable increase in the number of ectopic sex comb teeth in the progeny carrying both Psc1 and psq2403 mutations compared to those carrying the Psc1 mutation alone. The ScmD1 mutation displays an intermediate level of interaction, giving rise to approximately two- to three-fold increases, whereas no significant enhancement was found for the esc10 or E(z)63 mutations. These results suggest that psq is crucial for proper function of a subset of Pc-G proteins that are constituents of CHRASCH (Huang, 2002).

The effect of psq mutations on Ubx expression in imaginal discs was examined. Ubx proteins are normally expressed at high levels in the haltere- and third-leg discs but at low levels in the peripodial membranes of the wing discs. No significant change was observed in larvae heterozygous for either the Pc4 or psq mutation. However, high levels of Ubx proteins were observed in the medial sections of the wing discs from larvae doubly heterozygous for Pc4 and psq. In addition, substantial amounts of Ubx proteins could be detected in the first- and second-leg discs (Huang, 2002).

Class I psq mutations cause a more-severe reduction in the level of Psq-A than in the level of Psq-B (Horowitz, 1996). This observation raises an interesting possibility that different classes of psq alleles might have differential effects on these two major protein species and that such effects might be correlated with their contribution to the silencing function. The relative abundance of these proteins from adult ovaries of several homozygous mutants was determined. There are indeed some fluctuations in the relative abundance of these two proteins, however the fluctuations between different alleles within one class or between different classes do not seem to be consistent with the possibility that a specific Psq protein may be more critical for gene silencing. For example, while both psq0115 and psq2403 mutants contain much less Psq than psq8109, psq2403 shows consistently weaker interaction with Pc than psq0115 and psq8109. In addition, although psq8109 and psqE34 contain similar amounts of Psq, psq8109 shows substantially stronger interaction with Pc than psqE34. Thus, it seems unlikely that the homeotic phenotype can be attributed to the mere presence of different Psq in a simple manner (Huang, 2002).

In addition to their effects at the level of gene expression, however, class I mutations can also result in the synthesis of a novel BTB-domain-containing fusion product [i.e., psq1-l(3)S12] due to aberrant splicing (Horowitz, 1995). It has further been shown that overexpression of this fusion protein in wild-type flies is sufficient to solicit phenotypes similar to those of class I mutations (Horowitz, 1996). It is believed that this fusion protein may interfere with the function of Psq-A through the shared BTB protein interaction domain (Horowitz, 1996) since this domain is present in Psq-A protein but not in Psq-B protein. Taking this possibility into consideration, the stronger interaction of class I mutations with the Pc mutation might result from a combination of reduced expression and an interference with Psq-A protein, leading to a specific loss of Psq-A function. To substantiate this possibility, whether the loss of Psq-A alone is sufficient to cause a strong interaction with the Pc mutation was tested. psqDelta18 is a homozygous lethal mutation that deletes the 5' exons of psq-1 as well as the intergenic region between psq and a divergently transcribed gene, lola. As expected, while the level of Psq-B remains normal, very little Psq-A is detected in ovaries from psq0115/psqDelta18 transheterozygotes, indicating that Psq-A is specifically affected by the psqDelta18 mutation. When heterozygous psqDelta18 females were crossed with heterozygous Pc4 males, a very strong leg transformation was observed in their male progeny. Indeed, both the penetrance and expressivity caused by the psqDelta18 mutation are comparable to, if not stronger than, the two strongest psq alleles. Therefore, these results strongly support an essential role for Psq-A in homeotic gene silencing (Huang, 2002).

Epigenetic silencers Lola and Pipsqueak collaborate with Notch to promote malignant tumours by Rb silencing

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, two Polycomb group epigenetic silencers, Pipsqueak and Lola, have been identified that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis (Ferres-Marco, 2006).

Correct organ formation depends on the balanced activation of conserved developmental signalling pathways (such as the Wnt, Hedgehog and Notch pathways). If insufficient signals are received, organ growth may be deficient. By contrast, excess signalling leads to an overproduction of progenitor cells and a propensity to develop tumours. When such hyperproliferation is associated with the capacity of cells to invade surrounding tissue and metastasis to distant organs, cancer develops. Indeed, activation of the Wnt, Hedgehog and Notch pathways is a common clinical occurrence in cancers. Curiously, activation of any of these pathways in animal models seems to be insufficient for cancer to develop, indicating that synergism with other genes is required for these pathways to produce cancer (Ferres-Marco, 2006).

Cellular memory or the epigenetic inheritance of transcription patterns has also been implicated in the control of cell proliferation during development, as well as in stem-cell renewal and cancer. Proteins of the Polycomb group (PcG) are part of the memory machinery and maintain transcriptional repression patterns. The upregulation of several PcG proteins has been associated with invasive cancers. Thus, increased amounts of EZH2, the human homologue of the Drosophila histone methyltransferase E(z), is associated with poorer prognoses of breast and prostate cancers (Ferres-Marco, 2006).

Another histone methyltransferase implicated both in gene silencing and in cancer is SUV39H1, a homologue of Drosophila Su(var)3-9. SUV39H1 and Su(var)3-9 methylate histone H3 on lysine 9 (H3K9me), and this epigenetic tag is characteristic of heterochromatin and DNA sequences that are constitutively methylated in normal cells. DNA methylation is another mechanism involved in cellular memory that actively contributes to cancer. Indeed, numerous tumour-suppressor genes, including the retinoblastoma gene RB, are silenced in cancer cells by DNA hypermethylation. Inactivation of the RB tumour-suppressor pathway is considered an important step towards malignancy; thus, it is important to understand how these damaging epigenetic changes are initiated in cells that become precursors of cancer. Moreover, it is equally important to determine the connection between these processes and the developmental pathways controlling proliferation (Ferres-Marco, 2006).

Forward genetic screening in suitable animals is a powerful tool with which to identify tumour-inducing genes and to reveal changes that precede neoplastic events in vivo. The developing eye of Drosophila melanogaster is a good model for such studies because it is a simple and genetically well-defined organ. The growth of the eye depends on Notch activation in the dorsal-ventral organizer by its ligands Delta (human counterparts, DLL-1, -3, -4) and Serrate (human counterparts, JAGGED-1, -2). This study used the 'large eye' phenotype, produced by overexpression of Delta, as a tool to screen for mutations that interact with the Notch pathway and convert tissue overgrowths into tumours. One mutation, eyeful, was isolated that combined with Delta induces metastatic tumours. eyeful forces the transcription of two hitherto unsuspected growth and epigenetic genes, lola and pipsqueak (psq). The identification of eyeful has been a starting point from which to unravel crosstalk between the Notch and epigenetic pathways in growth control and tumorigenesis. The fact that many epigenetic factors are involved in cancer suggests that these processes may be more generally involved in tumorigenesis than at first it might seem (Ferres-Marco, 2006).

To identify genes that interact with the Notch pathway and that influence growth and tumorigenesis, the Gene Search (GS) system was used to screen for genes that provoked tumours when coexpressed with Delta in the proliferating Drosophila eye. The ey-Gal4 line was used for both eye-specific and ubiquitous induction, resulting in the transactivation of UAS-linked genes throughout the proliferating eye discs. It was through such a screen that the GS88A8 line was isolated. Generalized overexpression of Delta by ey-Gal4 (hereafter termed ey-Gal4 > Dl) produces mild eye overgrowth. In most of the flies in which the GS88A8 line was coexpressed with Delta, tumours developed in the eyes. Moreover, in ~30% of the mutant flies, secondary eye growths were observed throughout the body, and in some flies the whole body filled up with eye tissue. These secondary eye growths had ragged borders, indicating invasion of the mutant tissue into the surrounding normal tissue. As a result, the GS88A8 line was named 'eyeful' (Ferres-Marco, 2006).

A developmental analysis of the tumours was undertaken. To facilitate analyses, a triple mutant strain was generated carrying the eyeful, UAS-Dl and ey-Gal4 transgenes all on the same chromosome (ey-Gal4 > eyeful > Dl. In this strain, mutant eye discs showed massive uncontrolled overgrowth (some discs were more than five times their normal size). In most discs, the epithelial cells had lost their apical-basal polarity, and some had a disrupted basement membrane and grew without differentiating (Ferres-Marco, 2006).

These results were extended to the wing disc. (1) dpp-Gal4 was used to direct coexpression of eyeful and Delta along the anterior-posterior boundary of the wing (perpendicular to the endogenous Delta domain along the dorsal-ventral boundary. In a normal wing disc, the dpp-Gal4 driver typically establishes a stripe of green fluorescent protein (GFP) expression with a sharp border at the boundary. Whereas wild-type (or single eyeful) cells expressing GFP conformed with this pattern, some of the eyeful and Delta cells were found outside this stripe, indicating that the mutant cells can disseminate and invade adjacent regions of the disc. (2) The MS1096-Gal4 line was used to direct expression in the dorsal wing disc compartment. Under these conditions, the wing tissue grew massively and aggressively, and the mutant tissue failed to differentiate. These observations suggest that, when coupled with Delta overexpression, an excess of the gene products flanking the eyeful insertion site induces the formation of tumours capable of metastasising (Ferres-Marco, 2006).

The genomic DNA flanking the eyeful P-element was isolated and sequenced. eyeful is inserted in an intron of the gene longitudinals lacking (lola), which is known to be a chief regulator of axon guidance. lola encodes 25 messenger RNAs that are produced by alternative splicing and that generate 19 different transcription factors. All of the different isoforms share four exons that encode a common amino terminus, which contains a BTB or POZ domain. In addition, all but one of these transcription factors are spliced to unique exons encoding one or a pair of zinc-finger motifs (Ferres-Marco, 2006).

The GS P-elements allow Gal4-dependent inducible expression of sequences flanking the insertion site in both directions. The nearest gene in the opposite direction to transcription of lola is the psq gene. This gene encodes nine variants produced by alternative splicing and alternative promoter use, generating four different proteins. Three of the psq isoforms contain a BTB or POZ domain in the N terminus, and a histidine- and glutamine-rich region downstream of this domain. Two of the BTB-containing isoforms and the isoform that lacks this domain contain four tandem copies of an evolutionarily conserved DNA-binding motif, the Psq helix-turn-helix (HTH) motif (Ferres-Marco, 2006).

psq was initially identified for its 'grandchildless' and posterior group defects and was subsequently shown to have a role in retinal cell fate determination. Psq is essential for sequence-specific targeting of a PcG complex that contains histone deacetylase (HDAC) activity. Psq binds to the GAGA sequence, which is present in many Hox genes and in hundreds of other chromosomal sites (Ferres-Marco, 2006).

Both polymerase chain reaction with reverse transcription (RT-PCR) and in situ hybridization experiments confirmed that transcription of lola and psq was influenced by eyeful in response to Gal4 activation (Ferres-Marco, 2006).

To determine whether lola and/or psq was responsible for the tumour phenotype, 11 enhancer promoter (EP) P-elements inserted into the lola and psq region were tested. In contrast to the GS lines, the EP lines allows Gal4-dependent inducible expression of sequences flanking only one end of the P-element. It was found that none of the EP lines induced tumours; thus, it was reasoned that the deregulation of both genes might be required to produce the tumours (Ferres-Marco, 2006).

The complexity of lola and psq loci, which together produce 23 proteins, hampers identification of the transcripts responsible for the eyeful phenotype by gain-of-expression mutants (that is, by expressing individual or combinations of isoforms). Therefore, this issue was resolved by isolating point mutations that reverted the phenotype caused by deregulated expression of lola and psq. In this analysis, the chemical mutagen ethyl-methane sulphonate (EMS) was used to induce preferentially single nucleotide changes (Ferres-Marco, 2006).

The parental eyeful GS line was viable in trans with deficiencies that removed both lola and psq. In contrast, a set of 14 EMS-induced mutations on the eyeful chromosome failed to complement these deficiencies and were found to be alleles of psq or lola. The EMS-induced mutations that best recovered a normal eye size were sequenced. Each individual mutation had a single base change or a small deletion that considerably altered the predicted Psq or Lola proteins (Ferres-Marco, 2006).

All psq- mutations induced on the eyeful chromosome prevented eyeful from producing eye tumours and metastases. Three alleles affected the BTB domain (psqrev2, psqrev7 and psqrev9), and three other alleles contained either a premature stop codon that would produce truncated proteins lacking the Psq HTH repeats (psqrev4 and psqrev14) or a missense mutation that would change a conserved amino acid in the third Psq HTH repeat (psqrev12). All lola- mutations induced on the eyeful chromosome, including the presumptive null allele (lolarev6), reduced eye tumour size but still permitted sporadic secondary growth (Ferres-Marco, 2006).

These data show an unequal contribution of Psq and Lola in this process, whereby Psq is the most important factor in the tumorigenic phenotype. The BTB subfamily of transcriptional repressors includes the human oncogenes BCL6 and PLZF. In these oncogenes, the BTB domain is crucial for oncogenesis through the recruitment of PcG and HDAC complexes. It is therefore speculated that deregulated Psq and Lola could lead to tumorigenesis by epigenetic processes and that Drosophila counterparts of HDACs and PcG proteins might be involved in the progression of these tumours. Indeed, genetic evidence was found that both Lola and Psq function as epigenetic silencers in vivo (Ferres-Marco, 2006).

Attempts were made to determine the specific epigenetic mechanisms through which deregulation of Psq and Lola might induce tumorigenesis in conjunction with Delta overexpression. Methylation of histone on lysine is a central modification in both epigenetic gene control and in large-scale chromatin structural organization. For example, trimethylation of histone H3 on K4 (H3K4me3) is associated with the active transcription of genes and open chromatin structure. By contrast, histone hypoacetylation and H3K9 and H3K27 methylation are characteristic of heterochromatin state and gene silencing. To determine whether any changes in these epigenetic markers might coincide with the induction of tumorigenesis, eye discs were immunolabelled with antibodies against specific histone H3 modifications. Because dorsal eye disc cells are refractory to Delta, the dorsal region of the discs provided an internal control for these studies (Ferres-Marco, 2006).

With the exception of some scattered cells, a prominent loss or strong reduction of H3K4me3 was observed in the ventral region of the mutant discs. Notably, although the loss of Notch in clones does not affect this epigenetic tag, overexpression of Delta caused a significant reduction in staining for H3K4me3. The H3K4me3 depletion was already apparent in discs showing moderate hyperplasia and thus preceded neoplasm formation. Changes in other epigenetic tags (such H3K9me3 and H3K27me2) could not be reproducibly resolved; perhaps more sensitive methods or antibodies might facilitate detection of such changes (Ferres-Marco, 2006).

H3K4 methylation is thought to be permissive for maintaining and propagating activated chromatin and is thought to neutralize repressor tags by precluding binding of the HDAC complex and impairing SUV39H1-mediated H3K9 methylation. Thus, H3K4me3 depletion may contribute to tumour formation by permitting aberrant chromatin silencing. It was found that a 50% reduction in dosage of the HDAC gene Rpd3 or of Su(var)3-9 decreased the tumour phenotype dominantly. In contrast, reducing the activity of the H3K4 histone methyltransferase genes Trx (known as ALL1 or MLL in humans) or Ash1, which would be expected to deplete the H3K4me3 tag further, did not visibly enhance the tumours (Ferres-Marco, 2006).

E(z) when complexed with the Extra sex combs (Esc) protein becomes a histone methyltransferase. The E(z)-Esc complex and its mammalian counterpart Ezh2-Eed show specificity for H3K27 but may also target H3K9. The complex also contains the HDAC Rpd3, and this association with Rpd3 is conserved in mammals. H3K27 methylation facilitates binding of the chromodomain protein Pc (HPC in humans), which then creates a repressive chromatin state that is a stable silencer of genes (Ferres-Marco, 2006).

Although loss of E(z) does not cause proliferation defects within discs, halving the E(z) gene dosage dominantly suppressed tumorigenesis, indicating that histone methylation by the E(z)-Esc complex is also a prerequisite for the excessive proliferation of these tumours. Accordingly, Esc- or Pc- mutations also notably reduced the tumours (Ferres-Marco, 2006).

Together, these findings suggest that the development of these tumours involves, at least in part, changes in the structure of chromatin brought about by covalent modifications of histones. These changes probably switch the target genes from the active H3K4me3 state to a deacetylated H3K9 and H3K27 methylation silent state (Ferres-Marco, 2006).

From this above data, it is considered that the tumours might form as a result of aberrant gene silencing. If so, then the expression of genes involved in cell-cycle control is likely to be altered in the mutant cells. The transcription of 12 tumour-related genes in the mutant and wild-type discs was compared. Transcription of the gene Rbf, a fly homologue of the RB/Rb family of genes, was strongly downregulated in this assay (and even in ey-Gal4 > Dl flies). A second Rb gene, Rbf2, remained unchanged in the different genetic backgrounds, highlighting the specificity of Rbf silencing (Ferres-Marco, 2006).

It was found that Rbf depletion seems to be intricately associated with tumorigenesis: (1) reducing Rbf gene dosage by 50% enhanced tumour growth; (2) re-establishing Rbf expression in the eye (using an UAS-Rbf transgene) consistently prevented eye tumours and occurrence of secondary growths (Ferres-Marco, 2006).

Inactivation of RB1 in retinoblastoma, a form of eye cancer in children, can occur through DNA hypermethylation of the promoter. Unlike in mammals, however, there is little cytosine methylation of the genome in Drosophila during developmental stages, and its potential role during tumorigenesis is unknown. DNA methylation seems to depend on one DNA methyltransferase, Dnmt2, that preferentially methylates cytosine at CpT or CpA sites. The fly genome also encodes one methyl-CpG DNA-binding MBD2/3 protein. Because there are no known Dnmt2 loss-of-function mutations, the role of this gene in tumorigenesis could not be tested (Ferres-Marco, 2006).

Nevertheless, whether the CpG islands that were observed in the Rbf gene were potential targets for repression by DNA methylation was tested by two methods. (1) Methylation-sensitive restriction enzymes analysis was used; this showed that the regions around the promoter and transcription start site of the Rbf gene are susceptible to methylation. This approach showed aberrant DNA hypermethylation of Rbf in eyeful and Delta eye discs and mild hypermethylation in Delta discs; however, at best only very mild methylation was detected in discs from wild-type flies or from flies with the control psq gene (Ferres-Marco, 2006).

(2) Direct bisulphite sequencing of genomic DNA was carried out from mutant discs. This approach confirmed the notable increase in methylated DNA in eyeful and Delta discs when compared with wild-type discs (and a moderate increase in methylated DNA in the Delta discs). Hypermethylation of the Rbf promoter was not simply the result of de novo transcription of Dnmt2 (ey-Gal4 > Dnmt2), indicating that activation of the Notch pathway is a crucial step in this de novo hypermethylation of Rbf (Ferres-Marco, 2006).

This study has used Drosophila genetics to search for genes that collaborate with the Notch pathway during tumorigenesis in vivo. Psq and Lola were identified as decisive factors to foment tumour growth and invasion when coactivated with the Notch pathway. These proteins are presumptive transcription repressors that contain a BTB domain and sequence-specific DNA-binding motifs and behave as epigenetic silencers in vivo (Ferres-Marco, 2006).

In addition, crosstalk between the Notch pathway and different epigenetic regulators was identified. It is likely that alterations in this crosstalk provoke the aberrant epigenetic repression (and perhaps also derepression) of genes that contribute to cellular transformation. The Rbf gene was identified as one target for this epigenetic regulation and Rbf depletion was shown to directly contribute to the tumours (Ferres-Marco, 2006).

It is proposed that the sequence of events that leads to these tumours commences with hyperactivation of the Notch pathway, which initiates gene repression. Subsequently, or at the same time as Notch, Psq-Lola could bind to the silenced genes and enforce silencing by recruitment of HDAC or PcG repressors. Given the conservation of the Psq-like HTH domains in Psq and of BTB domains, it seems likely that other transcriptional repressors containing such domains strongly influence the tumour-inducing capacities of HDACs and PcG repressors in human cancers (Ferres-Marco, 2006).

Finally, the collaboration between PcG-mediated cellular memory and the Notch pathway may have implications in other processes controlled by Notch, including the second mitotic wave in the Drosophila eye, and the organization of eye and wing growth. In these processes, the memory mechanism could ensure that cells keep a record of the Notch signals received at an earlier stage or when the progenitor cells were closer to the Delta source. In this way, they might remain proliferative without having to receive continuous instructions from Notch. Likewise, such a situation could be conceived for tumorigenesis. The oncogenic signals could opportunistically take advantage of the memory mechanism to fix and to maintain their instructions of continuous proliferation in progenitor or stem cells, thereby fostering tumour growth and metastasis (Ferres-Marco, 2006).


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pipsqueak: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 5 August 2011

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