Myc interacts genetically with Tip48/Reptin and Tip49/Pontin to control growth and proliferation during Drosophila development

Abstract

The transcription factor dMyc is the sole Drosophila ortholog of the vertebrate c-myc protooncogenes and a central regulator of growth and cell-cycle progression during normal development. We have investigated the molecular basis of dMyc function by analyzing its interaction with the putative transcriptional cofactors Tip48͞Reptin (Rept) and Tip49͞Pontin (Pont). We demonstrate that Rept and Pont have conserved their ability to bind to Myc during evolution. All three proteins are required for tissue growth in vivo, because mitotic clones mutant for either dmyc, pont, or rept suffer from cell competition. Most importantly, pont shows a strong dominant genetic interaction with dmyc that is manifested in the duration of development, rates of survival and size of the adult animal and, in particular, of the eye. The molecular basis for these effects may be found in the repression of certain target genes, such as mfas, by dMyc:Pont complexes. These findings indicate that dMyc:Pont complexes play an essential role in the control of cellular growth and proliferation during normal development.repression ͉ transcription M yc proteins are essential regulators of growth, proliferation, and apoptosis in metazoans (1-3). These proteins act as transcription factors to control the expression of numerous target genes involved in growth, metabolism, and other processes (4-7). Less is known about the molecular mechanism that allows Myc to control the expression of these targets. In recent years, different modes of gene activation by Myc have been proposed, notably recruitment of chromatin remodelers (8), histone acetylases (e.g., ref. 9), or RNA pol II kinases (10), but the physiological relevance of these different factors for Myc-dependent biological functions needs to be demonstrated. We therefore set out to study the mechanisms of Myc-controlled growth and proliferation during normal development by using Drosophila as a model system. Initially, we focused on the interaction of Myc with two specific components of cofactor complexes, Tip48 and Tip49, because of the availability of null mutations in the corresponding genes [called reptin (rept) and pontin (pont) in flies, respectively].Tip48 and Tip49 are closely related proteins that show a high similarity to the bacterial ATP-dependent AAAϩ super family DNA helicase RuvB. Orthologs of Tip48 and Tip49 have been identified in plants, yeast, and animals (e.g., refs. 11 and 12). Different observations strongly suggest that one major function of the Tip proteins resides in the control of transcription. Initially, vertebrate Tip49 was found to be a Tata-binding protein-interacting protein (13-16); later Tip48 and Tip49 were also shown to interact physically with the different transcription factors ␤-catenin (11, 14), c-Myc (12), E2F1 (only Tip49) (17), and ATF2 (only Tip48) (18), raising the possibility that the Tip proteins could bridge basic transcription machinery and sequence-specific activators. Both proteins were also purified as part of several multiprotein complexes involved i...

Control of transcription by Pontin and Reptin

“…These data indicate that the inhibition of Miz1 (and hence repression of p21 CIP1 ) is mediated by a complex containing Myc, Pontin and Reptin (55). A similar conclusion was drawn from a separate study in Drosophila (33). In this organism, Myc also shows a strong genetic interaction with Pontin (and a weaker one with Reptin), although a comparison of the transcriptional targets of Myc on one side and Pontin or Reptin on the other side revealed a very limited overlap.…”

“…However, a small number of genes require Myc and Pontin (and possibly also Reptin) for their repression in cultured cells and in vivo, and the promoter of one of these genes, mfas, is indeed bound by both Pontin and Myc. These data also suggest that a complex of Myc and Pontin (probably also containing Reptin) represses genes like mfas, although in this case it is not known whether the repression involves the inhibition of an activator such as Miz1 (33). Neither the Xenopus nor the Drosophila study addresses the enzymatic basis of the repression mechanism, nor the possible involvement of other Tip60 complex components.…”

“…Ino80 is recruited to at least two yeast promoters, and either activates or represses a large number of genes, presumably by mobilizing nucleosomes and altering the accessibility of the underlying DNA to the transcription machinery (19,20). As expected, Reptin is required for the correct expression of many of the same genes (20); other studies showed a significant overlap between the genes controlled by Pontin and those controlled by Reptin (18,(31)(32)(33). This demonstrates that Reptin and Pontin play a role in activation and repression and suggests that they function in the same complex (but see also the section on gene repression below).…”

The conserved Myc box 2 and Myc box 3 regions are important, but not essential, for Myc function in vivo

“…By site-directed mutagenesis the following mutant derivatives were created (numbering relative to untagged Myc): MycΔN: lacking amino acids 1 -65; MycMB2A: containing "AAAA" instead of "DCMW"; MycΔMB2: "GP" substituted for residues 68-84; MycΔMB3: "F" substituted for residues 405-422. The same cDNAs were also cloned directly under the control of the α-tubulin84B promoter (see Bellosta et al, 2005) into an analogous pBSvector lacking UAS-and hsp70-sequences. Full sequences are available upon request.…”

“…As a first step to characterize the biological activities of these mutants, we monitored their ability to activate established endogenous Myc targets in a Myc-null mutant background. For this purpose, the transgenes were first combined with the Myc null allele dm 4 , a "tub-FRT-Myc-FRT-GAL4" transgene (which drives ubiquitous expression of a MycWT cDNA and thereby rescues the lethality of the dm 4 allele; Bellosta et al, 2005) and hs-FLP. Following a massive heat-shock of 2 hours at 37°, virtually all cells have eliminated the rescuing Myc cDNA and express GAL4 instead , which in turn drives the expression of the UAS-controlled transgenes.…”