Developmental pattern of transcription of the modulo gene indicates that messengers are maternally provided to the embryos and that zygotic transcription is required during subsequent development (Krejci, 1989).
Using a specific monoclonal antibody, the accumulation pattern of Mod protein during embryogenesis was examined. The maternally derived protein is first detected before the blastoderm cellularization in all somatic nuclei, precisely when pericentromeric heterochromatin becomes visible. After the first cell division, Mod protein is expressed in lineages of specific embryonic primordia. Based on its dominant phenotype, expression pattern and DNA-binding activity of its product, it is proposed that mod regulates chromatin structure and activity in specific cell lineages (Garzino, 1992).
During embryogenesis, the pattern of mod expression closely resembles that of dmyc. In stage 10 embryos mod transcripts are visible in the mesoderm, the anterior and posterior midgut precursors, the salivary gland precursors in parasegment 2 and the anal plate. This pattern is indistinguishable from that of pitchoune (pit), a potential dMyc target. In ovaries, transcripts of dmyc, pit and mod, are likewise identically detected throughout egg chamber development, except at stage 2 (Perrin, 2003).
Variegation in Drosophila is a manifest illustration of the important role played by chromatin structure in gene expression. Mutants of modulo (mod) have been isolated and this gene is shown to be a dominant suppressor of variegation. Null mutants are recessive lethal with a melanotic tumour phenotype. The mod protein directly binds DNA, which indicates that it may serve to anchor multimeric complexes promoting chromatin compaction and silencing (Garzino, 1992).
To analyse the consequences of mod loss of function in mitotically active cells of the imaginal disc, clones of cells homozygous for the null allele A4-4L8 were generated by FLP-mediated recombination. In these experiments, clones were identified on the adult epidermis of mosaic animals by the loss of yellow and Stubble bristle markers. mod-deficient clones were found in adult flies on head, thorax, legs and abdomen. They present three main features: (1) they are systematically of reduced size when compared to controls (wild-type clones induced at the same stage); (2) mutant thoracic cells produce short slender bristles, which are best seen when the clone encompasses a macrochaete; (3) the effect of the mod mutation appears to be cell-autonomous, as no alteration in cell or bristle morphology is seen in wild-type cells surrounding the clone. These features are reminiscent of the dominant phenotypes of Minute mutations, which are believed to affect ribosomal protein genes (Perrin, 1998).
Lack of mod results in an extended larval third instar and in a cell-autonomous Minute-like phenotype. modL8 is used to determine the function of mod in controlling growth. Homozygous modL8 larvae reach the third larval stage with a 6-hour delay and do not exhibit significant size differences relative to wild type. modL8 larvae enter pupariation with a 3-day delay (Roman, 1999). Most die as pharate adults. The few escapers were reduced in body weight (26%) and total wing area (6%). Remarkably, the wing cell area was strongly reduced by 22%. Although these defects are consistent with mod being required for growth and cell size regulation, overall larval growth is not impaired in 5 day-old larvae. To gain insight into this striking phenotype, larval tissues were analyzed during the third instar. Interestingly, larval tissues in modL8 animals are normal. The endoreplicative cells of the larval gut were of normal size in modL8 mutants. The results indicate that mod function is not required for growth of endoreplicative cells (Perrin, 2003).
The larval midgut also contains diploid intestinal adult precursor cells (apc) that proliferate during larval stages to form groups of approximately 4-8 cells prior to pupariation in wild type (5 days AEL). In contrast, at the same age, the modL8 midgut have fewer isolated intestinal apc, usually as doublets, or occasionally as groups of 3-4 cells. In addition, mutant apc appear smaller than wild type. Proliferation is, however, not arrested, since apc eventually form groups of 4-8 cells in 7 days AEL mutant larvae. Taken together, these observations suggest that mod is selectively required in diploid cells to sustain growth (Perrin, 2003).
Imaginal discs are composed mainly of proliferative cells. To better understand modL8 related growth defects, imaginal discs were analyzed during larval development. modL8 wing imaginal discs are severely delayed in growth. However, at the extended third instar (7-8 days AEL), the mutant discs attain a size comparable to prepupal wild type discs, suggesting that mod is required for growth of imaginal tissues. The reduced growth rate was not due to an enhancement of cell death, since no increase of apoptotic cells is observed in modL8 mutant imaginal discs stained with acridine orange. Somatic modL8 clones exhibit a growth-related phenotype characterized by short slender bristles and reduced cell number. Within third instar discs, modL8 clones contain very few cells when induced in a wild type background, confirming the cell autonomous nature of mod function. In contrast, clones are larger and comprise many more cells when induced in a Minute background. Since Minute cells are known to be at a growth and proliferative disadvantage, these results indicate that modL8 cells grow and proliferate at a lower rate than wild type cells (Perrin, 2003).
To address the question of a potential effect during cell cycle progression, dissociated cells from modL8 prepupal wing discs were analyzed with a fluorescence-activated cell sorter (FACS). Cell cycle profile analysis did not reveal any significant difference in mutant versus wild type cells. However, the mod mutation is associated with a reduction in size of both G1 and G2 cells. This observation suggests that Mod also acts on cell size control as reported for other growth regulators (Perrin, 2003).
Thus, loss-of-function experiments show that Mod is necessary for cell growth. Whether over-expression suffices to stimulate cell growth was tested. Using the UAS/Gal4 system, Mod was overproduced together with GFP in the entire posterior compartment of wing imaginal discs. This over-expression does not significantly increase posterior compartment size. Using GFP as a landmark, the determination of the posterior/anterior area ratio shows that compartment size is not affected. To examine a possible effect on apoptosis, wing discs over-expressing Mod in their posterior compartments were stained with Acridine Orange. Clearly, apoptotic cells are more abundant in the posterior than in the anterior compartment, indicating that an excess of Mod results in increased cell death. Interestingly, although co-expression of P35 with Mod efficiently suppresses apoptosis, this does not modify the posterior/anterior ratio. Taken together, these results show that Mod-induced apoptosis does not influence compartment size (Perrin, 2003).
Nevertheless, the fact that over-expressed Mod is not able to overcome the limit on compartment size could mask specific effects on individual cells. Therefore, imaginal disc cells either over-expressing GFP alone or Mod+GFP marker within their posterior compartment, were dissociated and analyzed by FACS. mod gain-of-function within the whole posterior compartment leaves the cell cycle profile unaltered. However, both these G1 and G2 cells were slightly bigger, whereas cells from the anterior compartment (GFP minus) were similar in size. This observation suggests that over-expression of Mod can trigger a cell size increase but not compartment growth (Perrin, 2003).
To further investigate the function of Mod in the context of cell competition, the 'flip out'technique was used to generate clones over-expressing Mod. Over-expression of Mod was accompanied by GFP as marker, and P35 to inhibit Mod-induced apoptosis. In control clones, proliferation and growth rates were identical, congruent with the expected coupling of cell growth and proliferation in wild type imaginal discs. Unexpectedly, clonal over-expression of Mod deos not modify the growth rate, but is associated with significantly slower proliferation. While these observations show that Mod is not sufficient to trigger cell growth, they also implicate Mod over-expression in increased cell size. To verify the apparent cell size increase, dissociated cells from discs containing GFP clones were analyzed by FACS. Consistent with the delay in cell doubling, Mod+ cells are indeed slightly larger than wild type, although the effect is more pronounced in G2 than in G1 cells. Moreover, analysis of the cell cycle profile reveals that the population of over-expressing cells is reduced in G1 relative to G2. In contrast, the cell cycle profile is not changed upon mod mis-expression within the entire compartment, either in loss-of-function mutants or in compartment over-expression. A similar discrepancy has been reported in loss-of-function mutants for Drosophila TOR: in mosaic wing discs, mutant cells accumulate in G1, whereas the cell cycle profile is unchanged when the entire wing disc is mutant. The molecular mechanism underlying cell competition might be responsible for this discrepancy, creating interference with cell cycle progression in heterogeneous cell populations (Perrin, 2003).
In Mod+ clones, the cell size increase along with the G2 accumulation raises the possibility that Mod slows down cell division by delaying G2/M transition. To address this question, String (the rate limiting phosphatase for G2/M) was expressed alone or in combination with Mod using the `flip out' technique. Cell division was slightly faster in String+ cells as compare to wild type, and further accelerated upon co-expressing Mod and String together. If Mod functions as a brake of G2/M transition, co-expression of String might counteract this effect. However, expressing Mod and String together accelerates cell division rate more than String alone does. Hence, this cooperative effect rather excludes that Mod could antagonize String at G2/M transition. Strikingly, String over-expression promotes growth of Mod+ cells. No evident hypothesis can be proposed to explain how the overgrowth induced by Mod and String co-expression is accomplished. However, this observation indicates that Mod, while insufficient to direct growth alone, can nonetheless cooperate with Stg to promote growth (Perrin, 2003).
Consistent with mod being a dMyc target, experiments reveal that Mod is required for growth of imaginal cells. However, in contrast to dMyc, over-expression of Mod does not trigger cell growth on its own, indicating that additional target genes are required to mediate the growth enhancement induced by dMyc. Also, that loss-of mod function only affects proliferative cells reveals that different target genes are recruited to accomplish the dMyc-dependent growth in endoreplicative tissues (Perrin, 2003).
Over-expression of Mod alone does not increase cell growth but leads to a slight increase in cell size. In Mod+ clones, the cell size increase is accompanied by an increase in G2 cells, which might be due to a block at G2/M transition leading to a subsequent slow down of cell division rate. Since dMyc over-expression does not affect G2/M transition it is, therefore, unlikely that Mod, a dMyc-target, could repress G2/M transition. Such a Mod-induced negative effect on cell cycle progression has also been ruled out, since over-expressing Mod and String together accelerates cell division more than String alone does. This unambiguously excludes a Mod negative effect on cell cycle regulators, but somewhat suggests an acceleration of G1/S transition in Mod+ clones. Since the decrease of the G1/G2 ratio does not occur when Mod is over-expressed within the entire compartment, it is, however, unlikely that Mod acts directly on cell cycle progression. In contrast, the fact that in adult wings of modL8 escapers cell size is more reduced (22%) than overall wing area (6%), suggests that Mod could directly act on cell size through a not yet characterized mechanism. This hypothesis is further supported by the observation that change in Mod dosage always induces cell size variations, which can be observed in absence of cellular growth effect (Perrin, 2003).
Angelov, D., et al. (2006). Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes. EMBO J. 25(8): 1669-79. Medline abstract: 16601700
Alexandre, E., Graba, Y., Fasano, L., Gallet, A., Perrin, L., De Zulueta, P., Pradel, J., Kerridge, S. and Jacq, B. (1996). The Drosophila Teashirt homeotic protein is a DNA-binding protein and modulo, a HOM-C regulated modifier of variegation, is a likely candidate for being a direct target gene. Mech. Dev. 59(2): 191-204. 8951796
Arn, E. A., Cha, B. J., Theurkauf, W. E. and Macdonald, P. M. (2003). Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins. Dev. Cell 4(1): 41-51. 12530962
Bantignies, F., Goodman, R. H., Smolik, S. M. (2002). The interaction between the coactivator dCBP and Modulo, a chromatin-associated factor, affects segmentation and melanotic tumor formation in Drosophila. Proc. Natl. Acad. Sci. 99(5): 2895-900. 11854460
Finger, L. D., Trantirek, L., Johansson, C. and Feigon, J. (2003). Solution structures of stem-loop RNAs that bind to the two N-terminal RNA-binding domains of nucleolin. Nucleic Acids Res. 31(22): 6461-72. Medline abstract: 14602904
Garzino, A. Pereira, P. Laurenti, Y. Graba, R. W. Levis, Y. Le Parco et al. (1992). Cell lineage-specific expression of modulo, a dose-dependent modifier of variegation in Drosophila. Embo. J. 11: 4471-4479. 1425581
Graba, Y., Laurenti, P., Perrin, L., Aragnol, D. and Pradel, J. (1994). The modifier of variegation modulo gene acts downstream of dorsoventral and HOM-C genes and is required for morphogenesis in Drosophila. Dev. Biol. 166: 704-715. 7813788
Grinstein, E., et al. (2006). Cell cycle-controlled interaction of nucleolin with the retinoblastoma protein and cancerous cell transformation. J. Biol. Chem. 281(31): 22223-35. Medline abstract: 16698799
Kim, K., et al. (2005). Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation. Mol. Cell. Biol. 25(6): 2463-74. Medline abstract: 15743838
Krejci, E., Garzino, V., Mary, C., Bennani, N. and Pradel, J. (1989). Modulo, a new maternally expressed Drosophila gene encodes a DNA binding protein with distinct acidic and basic regions. Nucleic Acids Res. 17: 8101-15. 2510129
Mikhaylova, L. M., Boutanaev, A. M. and Nurminsky, D. I. (2006). Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line. Proc. Natl. Acad. Sci. 103(32): 11975-80. Medline abstract: 16877538
Nurminsky, D. I., Nurminskaya, M. V., De Aguiar, D. and Hartl, D. L. (1998). Selective sweep of a newly evolved sperm-specific gene in Drosophila. Nature 396(6711): 572-5. Medline abstract: 9859991
Perrin, L., et al. (1998). Dynamics of the sub-nuclear distribution of Modulo and the regulation of position-effect variegation by nucleolus in Drosophila. J. Cell Sci. 111. 2753-2761. 9718368
Perrin, L., et al. (1999). The Drosophila modifier of variegation modulo gene product binds specific RNA sequences at the nucleolus and interacts with DNA and chromatin in a phosphorylation-dependent manner. J. Biol. Chem. 274: 6315-6323. 10037720
Perrin, L., et al.(2003). Modulo is a target of Myc selectively required for growth of proliferative cells in Drosophila. Mech. Dev. 120: 645-655. 12834864
Rickards, B., Flint, S. J., Cole, M. D. and LeRoy, G. (2007). Nucleolin is required for RNA polymerase I transcription in vivo. Mol. Cell. Biol. 27(3): 937-48. Medline abstract: 17130237
Roger, B., Moisand, A., Amalric, F. and Bouvet, P. (2002). Repression of RNA polymerase I transcription by nucleolin is independent of the RNA sequence that is transcribed. J. Biol. Chem. 277(12): 10209-19. Medline abstract: 11773064
Roman, G., He, J. and Davis, R. L. (2000). kurtz, a novel nonvisual arrestin, is an essential neural gene in Drosophila. Genetics 155: 1281-1295. 10880488
date revised: 15 April 2007
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