The Drosophila RYBP gene functions as a Polycomb-dependent transcriptional repressor

Abstract

The Polycomb and trithorax groups of genes control the maintenance of homeotic gene expression in a variety of organisms. A putative participant in the regulation of this process is the murine RYBP (Ring and YY1 Binding Protein) gene. Sequence comparison between different species has identified the homologous gene in Drosophila, the dRYBP gene. We have investigated whether dRYBP participates in the mechanisms of silencing of homeotic genes expression. We first studied its expression by RNA in situ hybridisation and detected dRYBP expression ubiquitously and throughout development. Moreover, we generated a polyclonal anti-dRYBP antibody that recognises the dRYBP protein. dRYBP protein is nuclear and expressed maternally and ubiquitously throughout development. To study the transcriptional activity of dRYBP, we generated a fusion protein containing the entire dRYBP protein and the GAL4 DNA binding domain. This fusion protein functions, in vivo, as a transcriptional repressor throughout development. Importantly, this repression is dependent on the function of the Polycomb group genes. Furthermore, using the GAL4/UAS system, we have over expressed dRYBP in the haltere and the wing imaginal discs. In the haltere discs, high levels of dRYBP repress the expression of the homeotic Ultrabithorax gene. This repression is Polycomb dependent. In the wing discs, dRYBP over expression produces a variety of phenotypes suggesting the overall miss regulation of the many putative genes affected by high levels of dRYBP. Taking together, our results indicate that dRYBP is able to interact with PcG proteins to repress transcription suggesting that the dRYBP gene might belong to the Polycomb group of genes in Drosophila.

High levels of dRYBP induce apoptosis in Drosophila imaginal cells through the activation of reaper and the requirement of trithorax, dredd and dFADD

“…Nub-G4>UAS-dRYBP We have shown that overexpression of dRYBP in wing discs causes the homeotic Ultrabithorax (UBX) protein [24] to be expressed in a limited number of wing disc cells [17,18]. This UBX expression is not dependent on the presence of the NZF domain of the dRYBP protein as overexpression of the dRYBP-DZF and fulllength proteins ( Figure 1L) produced the same pattern of UBX expression in wing discs ( Figure 1O).…”

“…Using the GAL4/UAS system, we observed that overexpression of the dRYBP protein in wing imaginal discs produces small adult wings [17,18]. We asked whether the reduction of wing size was due to the induction of apoptosis.…”

“…Several lines of evidence indicate that dRYBP interacts with the Polycomb group (PcG) and trithorax group (trxG) of proteins [17,18]. We used PcG and trxG mutant backgrounds to investigate whether apoptosis induced by dRYBP is dependent on these proteins.…”

“…For overexpression experiments, the GAL4/UAS system was used [37] at 25 ºC and 29 ºC with the following lines (described in Calleja et al [38]): scalloped-Gal4 (sd-Gal4), nubbin-Gal4 (nub-Gal4), apterus-Gal4 (ap-Gal4), distal less-Gal4 (dll-Gal4, MD23 and EM212) and 248-Gal4. Finally, transgenic lines containing UAS-RNAi dRYBP [18], UAS-RNAi FADD M8 [39], UAS-DIAP1 [40], UAS-EIGER [10], UAS-p35 [23], UAS-Dmp53 [34], UAS-TRX (P-EP-TRX, gift of Isabel Guerrero), UAS-SCE [41], UAS-RYBP murine and UAS-PC (both constructed in this work), UAS-dRYBP [18] and UASdRYBP-DZF [17] were used. For imaginal disc irradiation, eggs were collected during a 24-h laying period, allowed to develop until the formation of second instar larvae and then irradiated.…”

“…We have previously shown that loss of dRYBP function produces phenotypes that are highly variable in both penetrance and expressivity [17]. Moreover, we showed that dRYBP interacts genetically and molecularly with Polycomb (PcG) and trithorax (trxG) proteins and that its overexpression generates a small wing phenotype [17,18].…”