InteractiveFly: GeneBrief

sunspot: Biological Overview | References

The Wingless (Wg)/Wnt signaling pathway is highly conserved throughout many multicellular organisms. It directs the development of diverse tissues and organs by regulating important processes such as proliferation, polarity and the specification of cell fates. Upon activation of the Wg/Wnt signaling pathway, Armadillo (Arm)/beta-catenin is stabilized and interacts with the TCF family of transcription factors, which in turn activate Wnt target genes. This study shows that Arm interacts with a novel BED (BEAF and Dref) finger protein that has been termed Sunspot (Ssp). Ssp transactivates Drosophila E2F-1 (dE2F-1) and PCNA expression, and positively regulates the proliferation of imaginal disc cells and the endoreplication of salivary gland cells. Wg negatively regulates the function of Ssp by changing its subcellular localization in the salivary gland. In addition, Ssp was found not to be involved in the signaling pathway mediated by Arm associated with dTCF. These findings indicate that Arm controls development in part by regulating the function of Ssp (Taniue, 2010).

Arm is composed of 12 imperfect protein interaction repeats (Armadillo repeat domain) flanked by unique N and C termini. In an attempt to identify novel Arm-binding proteins, a yeast two-hybrid screen of a Drosophila embryo cDNA library was performed using the Armadillo repeat domain of Arm as bait. Positive clones containing the same insert of a novel gene (CG17153) were isolated that were named sunspot (ssp; named after the phenotype of mutant flies). Sequence analysis of the full-length cDNA revealed that it encodes a protein of 368 amino acids. A region near its N terminus (amino acids 34 to 98) shows similarity to the BED (BEAF and Dref) finger domain, which is predicted to form a zinc finger and to bind DNA (Taniue, 2010).

To confirm the interaction between Ssp and Arm, whether Ssp produced by in vitro translation could interact with the Armadillo repeat domain of Arm fused to glutathione S-transferase (GST) was tested. Ssp specifically interacted with the Armadillo repeat domain of Arm (amino acids 140 to 713), but failed to interact with Pendulin (Pen), a Drosophila homolog of importin α, which also possesses the Armadillo repeat domain. Pull-down assays with a series of deletion fragments of Ssp showed that a fragment of Ssp containing amino acids 235 to 307 (termed the ABR, the Arm-binding region) binds to Arm in vitro. Also, it was found that Armadillo repeats 2-8 of Arm are responsible for binding to Ssp. Although TCF is known to bind to Armadillo repeats 3-10 of Arm, Ssp did not compete with TCF for binding to Arm (Taniue, 2010).

Next, whether Ssp is associated with Arm in living cells was examined. Drosophila Schneider-2 (S2) cells were transfected with Arm along with GFP-Ssp, GFP-SspδC (amino acids 1 to 217; a mutant lacking the ABR) or GFP-SspABR (amino acids 235 to 342; a fragment containing the ABR). GFP-fusion proteins were immunoprecipitated from S2 cell lysates and subjected to immunoblotting with anti-GFP and anti-Arm antibodies. For immunoprecipitation of GFP-fusion proteins, a 13-kDa GFP-binding fragment was used derived from a llama single chain antibody, which was covalently immobilized to magnetic beads (GFP-Trap-M), as the molecular weight of GFP-SspδC is the same as that of IgG. It was found that Arm is associated with GFP-Ssp and GFP-SspABR. By contrast, Arm barely co-immunoprecipitated with GFP-SspδC. In addition, pull-down assays were also performed with a mixture of lysates of S2 cells transfected with Arm alone and GFP-Ssp alone, respectively. It was found that Ssp and Arm co-precipitate only when both proteins are co-expressed in S2 cells, excluding the possibility that Ssp and Arm associate after cells are lysed. Taken together, these results suggest that Ssp interacts via its ABR with Arm not only in vitro but also in vivo (Taniue, 2010).

One lethal P-element insertion line, l(3)j2D3^j2D3, was found in which a P-element had been inserted into the gene adjacent to ssp, CG6801, which is located about 250 bp upstream of the 5' end of ssp. RT-PCR analysis revealed that the expression level and size of the CG6801 transcript were not changed compared with in wild-type larvae, which is consistent with the P-element being inserted into an intron in CG6801. To generate mutants that have a deletion in ssp but have intact CG6801, a local hop and imprecise excision approach was used. l(3)j2D3^j2D3 was used in a local hop to generate a P-element insertion line, sunspot^P, that completely complemented the lethality of l(3)j2D3^j2D3. Then ssp mutants were generated by imprecise excision of the P-element from sunspot^P. One allele was found that has a deletion of about 600 bp, and this was designated as ssp⁵⁹⁸. Sequence analysis showed that the deletion extends from a position 60 bp downstream of the presumptive ssp transcription start site to the ssp gene locus. Because this deletion removes the start codon and the BED finger domain of ssp, it is presumed that ssp⁵⁹⁸ represents a null allele for ssp. RT-PCR analysis revealed that ssp⁵⁹⁸ generates a truncated transcript. The truncated transcript encodes a peptide consisting of 13 amino acids, which is unrelated to Ssp. By contrast, RT-PCR analysis revealed that the intact CG6801 transcript is expressed in ssp⁵⁹⁸ mutant larvae, and that the expression level of CG6801 is unchanged in ssp⁵⁹⁸ mutant larvae compared with that in wild-type larvae. Furthermore, ssp⁵⁹⁸ fully complemented the phenotype of l(3)j2D3^j2D3, indicating that this mutant contains intact CG6801 (Taniue, 2010).

The imaginal discs, salivary glands and central nervous system of larvae homozygous for ssp⁵⁹⁸ were smaller than those of their normal counterparts. ssp⁵⁹⁸ homozygotes reached the third instar stage, but failed to reach the pupal stage and died between 10 and 20 days after egg laying (AEL). Furthermore, melanotic pseudotumors were formed in ssp⁵⁹⁸ mutant larvae. Melanotic pseudotumors are groups of cells within the larvae that are recognized by the immune system and encapsulated within a melanized cuticle. One or more small melanotic pseudotumors first appeared in the ssp mutants at 6 days AEL, and the number and size of these melanotic pseudotumors increased during the development of the larvae. Similar phenotypes were observed with hemizygotes for ssp⁵⁹⁸ and Df(3L)BK9, which has a deletion larger than that of ssp⁵⁹⁸ and lacks ssp. In situ hybridization analysis of imaginal discs using the coding region of the ssp cDNA as a probe revealed that ssp transcripts are expressed ubiquitously. Therefore whether ubiquitous expression of ssp restores the phenotypes of ssp⁵⁹⁸ homozygous animals was examined. It was found that ubiquitous expression of the full-length ssp cDNA with the Gal4-UAS system rescued the lethality and other phenotypes associated with ssp⁵⁹⁸ homozygous animals. Taken together, these results suggest that the phenotypes of ssp⁵⁹⁸ homozygotes are caused by the loss of ssp function, and that ssp is required for cell proliferation and morphogenesis of the imaginal disc and central nervous system (Taniue, 2010).

Arm is a key transducer of Wg signaling and many of the Arm-binding proteins are known to function as a component of the Wg signal transduction pathway. To explore the possibility that Ssp is related to the Wg signal transduction pathway, the effect of Wg on the distribution of GFP-Ssp was examined. Because imaginal disc cells are too small for detailed study, focus for this analysis was placed on the third instar salivary glands, and whether the subcellular localization of GFP-Ssp is linked to Wg signaling was studied. The larval salivary gland mainly consists of secretory gland cells and imaginal ring cells. Gland cells are large polyploid epithelial cells. Small imaginal ring cells reside at the proximal end of the secretory gland. Immunostaining with anti-Wg antibody revealed that Wg is expressed in imaginal ring cells. Furthermore, Drosophila frizzled 3 (dfz3)-lacZ, a target gene of Wg signaling, was found to be expressed in imaginal ring cells and proximal gland cells, which reside within several cell diameters of the Wg-expressing cells. These results suggest that Wg signaling is active in the proximal region in the third instar salivary gland. When GFP-Ssp was expressed ubiquitously under the control of dpp-Gal4 in the larval salivary gland, GFP-Ssp was found to be localized predominantly at the nuclear envelope in proximal gland cells. In addition, GFP-Ssp was detected as aggregates in the nucleus in the distal region of the salivary gland. To examine whether this region-specific subcellular localization of GFP-Ssp is related to Wg signaling, Wg or Axin, a negative regulator of Wg signaling, was overexpressed in the salivary gland under the control of dpp-Gal4. It was found that expression of Wg along with GFP-Ssp resulted in the accumulation of a certain population of GFP-Ssp at the nuclear envelope in both the distal and proximal regions. Again, a significant amount of GFP-Ssp was localized in nuclear aggregates in both distal and proximal cells, suggesting that ectopic expression of Wg can also change the subnuclear localization of Ssp in proximal cells, from the nuclear periphery to nuclear aggregates. This result also suggests that ectopic expression of Wg in distal cells is not sufficient to change the subnuclear localization of all GFP-Ssp protein, from nuclear foci to the nuclear periphery. By contrast, when Axin was expressed along with GFP-Ssp, GFP-Ssp was detected as nuclear aggregates, not only in the distal region but also in the proximal region, but was no longer detected at the nuclear envelope. These results suggest that the subcellular localization of Ssp is regulated at least in part by Wg signaling in the third instar salivary gland (Taniue, 2010).

To examine whether the effect of Wg signaling on Ssp localization is mediated by the direct interaction between Arm and Ssp, Ssp localization was studied in larvae expressing an RNAi targeting Arm. It was found that Ssp was localized in nuclear aggregates and that Wg overexpression did not alter its localization when the expression of Arm was suppressed by RNAi. Thus, Arm is required for Wg-induced Ssp relocalization. Ssp localization was also examined in cells expressing δArm, a mutant of Arm that localizes at the plasma membrane. It was found that overexpression of δArm under the control of dpp-Gal4 results in the localization of GFP-Ssp at the plasma membrane throughout the salivary gland. Next the subcellular localization of SspδC, a mutant that lacks the ABR and is unable to interact with Arm, was examined. When GFP-SspδC was expressed ubiquitously, it was found to localize homogenously in the nucleus of both distal and proximal cells. This result indicates that the localization of Ssp to nuclear aggregates requires the ABR and suggests that Ssp requires a direct interaction with Arm to localize to its target sites in the nucleus. Furthermore, it was found that the localization of GFP-SspδC was not changed by coexpression with Wg, or δArm. Taken together, these results suggest that the direct interaction between Arm and Ssp is required for the regulation of Ssp localization by Wg signaling (Taniue, 2010).

The N-terminal region of Ssp contains a BED finger domain. This presumptive DNA-binding domain is known to be contained in several Drosophila proteins, such as Dref and BEAF-32. Dref regulates the transcription of genes involved in DNA replication and cell proliferation, including dE2F-1 and PCNA, the promoters of which contain BED finger-binding elements (BBEs). To clarify whether Ssp regulates the transcription of these genes, the expression levels of dE2F-1 and PCNA were examined. For this purpose, the P-element (lacZ) insertion lines E2F⁰⁷¹⁷² and PCNA⁰²²⁴⁸ were used. dE2F-1-lacZ and PCNA-lacZ expression were found to be high in distal cells compared with proximal cells in the larval salivary gland. When ssp was ectopically expressed in the salivary gland, dE2F-1-lacZ expression was markedly elevated in distal cells, whereas it was only slightly elevated in proximal cells. However, PCNA-lacZ expression was markedly elevated throughout the salivary gland. By contrast, dE2F-1-lacZ and PCNA-lacZ expression were not elevated in distal cells of ssp mutant salivary glands compared with in wild-type salivary glands, and dfz3-lacZ expression in ssp mutant and Ssp-overexpressing salivary glands was not changed compared with in wild-type salivary glands, suggesting that Ssp is not involved in Arm-dTCF-mediated transactivation of Wg target genes. In addition, overexpression of Wg resulted in a decrease in the expression levels of dE2F-1-lacZ and PCNA-lacZ in distal cells. Thus, Ssp is active in the distal region where Wg signaling is not active, and Ssp is aggregated in the nucleus. Conversely, Ssp is not very active in the proximal region where Wg signaling is active, and Ssp is accumulated in the nuclear envelope (Taniue, 2010).

The expression of dE2F-1, PCNA and dfz3 was examined in the wing disc. Clones of cells lacking Ssp function were generated by FLP/FRT-mediated somatic recombination. Clones of ssp mutant cells underwent only a few divisions after they were generated in the presumptive wing blade: the mutant cells proliferated slowly and either died or were actively eliminated from the disc epithelium. Therefore, a Minute mutation, M(3)65F, was used to confer a growth advantage upon cells homozygous for ssp. When mitotic recombination was induced in a M(3)65F background using enhancer trap lines, ssp mutant cells exhibited reduced levels of dE2F-1-lacZ and PCNA-lacZ expression but did not show any change in the levels of dfz3-lacZ and Arm expression. These results suggest that ssp regulates the expression of dE2F-1 and PCNA, but is not involved in Arm-dTCF-mediated Wg signaling (Taniue, 2010).

To confirm these results, endogenous expression of dE2F-1 and PCNA was examined by quantitative real-time RT-PCR analysis using RNA from late third instar larvae. Flies carrying heat-shock-inducible Gal4 (hs-Gal4) were crossed with transgenic flies carrying UAS-GFP, UAS-ssp or UAS-wg. Consistent with the above results, overexpression of ssp resulted in elevated steady state levels of dE2F-1 and PCNA transcripts. Furthermore, overexpression of Wg induced decreases in the numbers of dE2F-1 and PCNA transcripts. These results suggest that dE2F-1 and PCNA expression is regulated positively by Ssp and negatively by Wg (Taniue, 2010).

Also whether Ssp regulates the expression of dE2F-1 by binding directly to its promoter region was examined. Electrophoretic mobility-shift assays (EMSA) showed that GST-Ssp, but not GST, bound to a 40-mer oligonucleotide corresponding to a region in the dE2F-1 promoter that contains three BBEs. By contrast, GST-Ssp barely bound to a mutated probe in which CG in each BBE had been replaced with AA. Binding of Ssp to the wild-type probe was inhibited in the presence of an excess amount of unlabeled wild-type probe, whereas the mutated probe did not inhibit the interaction significantly. When anti-Ssp antibody was included in the reaction mixture, the Ssp band was not detected. Furthermore, it was found that GST-SspδBFD, a mutant Ssp lacking the BED finger domain, did not bind to the wild-type probe. These results suggest that Ssp regulates dE2F-1 expression by binding directly to the BBEs in the dE2F-1 promoter region via its BED finger domain (Taniue, 2010).

To further elucidate the function of Ssp and Wg, the third instar salivary glands of ssp and wg mutants were examined. In the third instar salivary gland, the distal region undergoes greater endoreplication than does the proximal region. As a result, the nuclear size of distal gland cells is markedly larger than that of proximal gland cells. However, the nuclear size of ssp mutant distal cells was found to be smaller than that of wild-type distal cells. By contrast, the nuclear size of wg mutant proximal cells was larger than that of wild-type proximal cells. Thus, the difference in nuclear size between proximal and distal cells was also small in the salivary glands of wg mutants (Taniue, 2010).

To confirm these results, the effects were examined of Ssp and/or Wg overexpression on the nuclear size of salivary gland cells. When Ssp was overexpressed, the nuclear size of both proximal and distal cells was heterogenous. Overexpression of Wg decreased the nuclear size of distal cells: the difference in nuclear size between Wg-overexpressing proximal and distal cells was small. However, when Wg was overexpressed along with Ssp, the effect of Ssp was suppressed and the heterogeneity of nuclear size was not observed. Furthermore, to confirm that Ssp and Wg play important roles in the regulation of endoreplication, δArm-expressing clones were generated using the flip-out technique. It was found that the nuclear size of δArm-expressing cells is much smaller than that of surrounding cells. This result suggests that δArm mislocalizes Ssp to the plasma membrane, thereby negatively regulating Ssp activity for endoreplication (Taniue, 2010).

To directly show that ssp mutant cells undergo fewer endoreplications than do wild-type cells, BrdU-labeling experiments were performed. When wild-type salivary glands were labeled with BrdU, distal cells efficiently incorporated BrdU, indicating that they underwent at least one round of DNA replication during the labeling period. By contrast, very few nuclei of ssp mutant cells and Wg-overexpressing cells were labeled with BrdU (Taniue, 2010).

dMyc has also been reported to be required for the endoreplication of salivary gland cells. It is therefore interesting to examine the relationship between dMyc, Wg and Ssp in endoreplication. It was found that dMyc expression was unchanged in both ssp mutant and Ssp-overexpressing salivary glands. Thus, Ssp might not be involved in the regulation of dMyc (Taniue, 2010).

Taken together, these results suggest that Ssp and Wg play important roles in the regulation of endoreplication in the third instar salivary gland, and that Wg might exert its effect by negatively regulating the function of Ssp. It is interesting to speculate that Ssp plays a general role for endoreplication in all larval endocycling tissues (Taniue, 2010).

It is believed that Wg/Wnt target genes are transactivated by Arm/β-catenin associated with TCF. However, expression of some human genes is transactivated by β-catenin that is associated with proteins other than TCF. For example, β-catenin interacts with the androgen receptor in an androgen-dependent manner and enhances androgen-mediated transactivation. In the present study, it was shown that Arm interacts with Ssp and negatively regulates its function. Ssp transactivates dE2F-1 and PCNA expression, and positively regulates the endoreplication of salivary gland cells. Furthermore, the Wg signal represses the function of Ssp by altering the subcellular localization of Ssp in the salivary gland: the Wg signal induces the accumulation of Ssp at the nuclear envelope. Interestingly, recent studies indicate that the nuclear membrane provides a platform for sequestering transcription factors away from their target genes. For example, it has been shown that the tethering of transcription factors such as c-Fos and R-Smads to the nuclear envelope prevents transcription of their target genes. The results appear to be consistent with these findings. Although the precise mechanism remains to be investigated, the interaction between Arm and Ssp appears to be required for the regulation of Ssp localization by Wg signaling. It remains to be investigated whether the mechanisms identified in the salivary gland are applicable to other tissues (Taniue, 2010).

Search PubMed for articles about Drosophila Sunspot

Taniue, K., et al. (2010). Sunspot, a link between Wingless signaling and endoreplication in Drosophila. Development 137(10): 1755-64. PubMed ID: 20430750

date revised: 15 January 2011

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