[Research Interests] [Representative Publications] RESEARCH INTERESTSDEVELOPMENTAL GENETICS, IMAGINAL DISC DEVELOPMENT IN DROSOPHILA STUDIED IN LETHAL AND TEMPERATURE SENSITIVE MUTANTS The long-term goal of the research in this laboratory is to understand how the functions of specific genes are integrated into developmental processes. We have been studying the processes of proliferation and determination using the imaginal discs of Drosophila as a model system because of the availability of powerful genetic, developmental and molecular techniques. During the proliferative phase of imaginal disc development, which occurs during larval development, a significant developmental program occurs which includes transmission of the determined state and a regulated pattern of cell proliferation.
I. Genes required for the normal determination of imaginal discs The determined state of Drosophila imaginal discs depends on stable disc-specific patterns of homeotic selector gene expression. The proteins encoded by two groups of conserved genes, the Polycomb and trithorax groups, have been proposed to maintain, at the level of chromatin structure, the expression pattern of these homeotic genes during development. The proteins encoded by the Polycomb group are postulated to repress transcription of homeotic genes outside of their normal expression domain and the proteins encoded by the trithorax group are postulated to activate transcription of homeotic genes within their normal expression domain. This paradigm is based primarily on the analysis of mutant phenotypes but also on some biochemical studies. The ash1 gene is a member of the trithorax group. The symbol ash is an acronym for absent, small or homeotic imaginal discs. In ash1 mutants, expression of homeotic selector genes is disrupted. The primary translation product of the 7.5 kb ash1 transcript is predicted to be a basic protein of 2144 amino acids. ASH1 is a nuclear protein that contains a SET domain and a double zinc finger called a PHD finger. Both of these motifs are also found in the products of other trithorax group and Polycomb group genes. [Work done by former postdoctoral fellow Nick Tripoulas and former graduate student Dennis La Jeunesse. The SET domain, named for its presence in Su(var)3-9, Enhancer of Zeste, and trithorax, has been shown, in some cases, to have histone methyl transferase activity. Methylation on lysine 4 of histone H3 is associated with transcriptional activation, whereas methylation on lysine 9 of histone H3 is associated with transcriptional repression. We have found that an ash1 null mutation causes severely reduced lysine 4 methylation of histone H3 but little effect on lysine 9 methylation of histone H3. A missense mutations of ash1 that affects a conserved residue within the SET domain causes a similar phenotype. Moreover, a fusion of the ASH1 SET domain with GST can pull down histone H3. ASH1 has previously been reported to be a component of a 2Mda multimeric protein complex. These new data suggest that one function of this complex is to methylate lysine 4 residues of histone H3. We infer that this methylation affects chromatin structure and thereby maintains active transcription of specific target genes. [Work done by former graduate student Kristin Nastase.]
Mutations in different trithorax group genes show intergenic non-complementation. We have used this property as the basis for a deficiency screen to identify new members of the trithorax group. Of 133 deficiencies tested, 5 uncover one or more unknown genes that fail to complement an ash1 null allele. For one of these deficiencies, we have tested all of the lethal P-element insertions known to be located in the deleted region, for failure to complement null alleles of ash1. We found two allelic lethal insertions that failed to complement. We have named the gene identified by these insertions little imaginal discs (lid) based on the phenotype of pupal lethal homozygotes. The lid gene product is the Drosophila homolog of Retinoblastoma binding protein 2. It is predicted to be a 203 kDa protein with a number of conserved domains including an ARID (AT-rich interaction domain), three PHD fingers, a leucine zipper and a bi-partite nuclear localization signal. [Work done by former graduate student John Gildea, former graduate student Kristin Nastase and Evelyn Hersperger.] Surprisingly, four of the deficiencies that fail to complement an ash1 null allele, uncover five genes previously classified as Polycomb group genes [E(Pc), Su(z)2, Psc, Asx, and Scm]. Mutations in four of these five genes (all but Scm) also fail to complement brm and trx mutations. This led us to examine mutations in all of the genes previously classified as members of the Polycomb group for failure to complement ash1, brm and trx null alleles. We found that mutations in seven of these genes (Pc, Pcl, ph, pho, mxc, esc, and sxc) complement trithorax group gene mutations and suppress the phenotype of trithorax group double mutants. Mutations in 2 others [Sce, and E(z)] fail to complement trithorax group gene mutations and enhance the phenotype of trithorax group double mutants. Su(z)2, Scm, and E(z) were also identified in screens for suppressors of zeste. We have found that 3 of 4 other suppressors of zeste also fail to complement mutations in trithorax group genes. We previously reported that loss of function mutations in E(z) (also known as pco) behave like mutations in a trithorax group gene. They show intergenic non-complementation with mutations in trithorax group genes and they cause loss of homeotic gene expression in larval imaginal discs as do mutations in trithorax group genes. At the time of that report it was not clear whether the dual nature of E(z) was an anomaly or whether it indicated a new paradigm needed to be developed to account for the maintenance of segment specific homeotic gene expression. We now know that some of the genes previously classified as Polycomb group genes and many of the genes classified as suppressors of zeste are like E(z) ; they show intergenic non-complementation with mutations in both Polycomb and trithorax group genes. To account for the maintenance of segment specific homeotic gene expression, we postulate that genes previously classified as Polycomb group genes represent two functional groups: the Polycomb group and a new group that we propose be called the ETP group (Enhancers of trithorax and Polycomb group mutations). Loss of function mutations in Polycomb group genes show intergenic non-complementation with each other and suppress the phenotype of mutations in the trithorax group; loss of function mutations in the ETP group genes show intergenic non-complementation with mutations in both trithorax group genes and Polycomb group genes. This implies that the products of this latter group of genes act as both positive and negative trans-regulatory factors. [Work done by John Gildea, Rocio Lopez, and Evelyn Hersperger.] We also identified the missing imaginal precursors (mip) gene (CG4539, AKA Bekka) in the screen for mutations that exhibit intergenic non-complementation with ash1 mutations. However, we find that it also exhibits intergenic non-complementation with Pc mutations. So we classify it as a gene in the ETP group along with genes such as E(z) and Asx. The MIP protein is similar in sequence to DNA-binding helix turn helix proteins. Since mip mutations affect Ubx transcription, we tested whether MIP binds to Ubx regulatory sequences. We found that in vitro translated MIP causes a shift in the electrophoretic mobility of a 400 bp PRE/TRE fragment of the Ubx upstream regulatory element. Mutant mip homozygotes die as second instar larvae with no detectable imaginal discs. The requirement for mip function is disc-autonomous (assayed by embryo transplantation) but is not cell-autonomous (assayed by clonal analysis). This suggested the hypothesis that MIP is required for the transcription of a signaling molecule such as dpp. We found that mip homozygotes have reduced accumulation of Spalt and Vestigial both of which are upregulated by dpp signaling. [Work done by former graduate student Kristin Nastase.] The ash2 gene is also a member of the trithorax group. The primary translation product of the 2.0 kb ash2 transcript is predicted to be a protein of 554 amino acids. ASH2 has previously been reported to be a component of a 0.5 Mda multimeric protein complex. We performed a yeast two hybrid screen to identify components of this complex that directly interact with ASH2. One of the components identified was the product of the skittles gene, SKTL. The biological significance of this observation is supported by intergenic non-complementation between ash2 mutations and sktl mutations. SKTL is similar in sequence to phosphatidylinositol-4-phosphate 5-kinases (PIP kinases) but it has a nuclear localization signal. Apparently Drosophila, as well as other organisms, has several different genes encoding proteins with this activity and SKTL is a version that acts in nuclei. We have detected FLAG-tagged SKTL in nuclei and on polytene chromosomes where it co-localizes with ASH2. [Work done by former graduate student Mimi Cheng.]
II. Genes required for the normal growth of imaginal discs The hyperplastic discs (hyd) gene was originally identified by a temperature-sensitive mutation that causes imaginal disc overgrowth in mutant larvae raised at a restrictive temperature. Sequence analysis of HYD revealed similarity to a portion of the C-terminus of human E6-AP, a ubiquitin ligase. We have shown that ubiquitin can indeed be transferred to the C-terminus of HYD in vitro and that a different fragment of HYD can bind to the bendless gene product, a ubiquitin E2 protein. In a yeast two-hybrid screen, we identified 14-3-3, the leonardo gene product and a truncated actin binding protein, D-TABP, as HYD partners. The binding of HYD to D14-3-3 and to D-TABP was confirmed in vitro. The biological significance of these physical interactions has been confirmed by showing that mutations in bendless, and leonardo enhance the hyd phenotype. The 14-3-3 protein is believed to be involved in signal transduction because of its association with RAF. TABP is also involved in signal transduction; it negatively regulates filamin which tethers cell surface receptors to the actin cytoskeleton. [Work done by former postdoctoral fellow Kazuhito Amanai and Evelyn Hersperger.] Mutations in the minidiscs (mnd) gene cause all of the imaginal discs and the brain lobes to be small. Although the product of the mnd gene is required for the growth of all discs, its requirement is neither disc nor cell autonomous. The mnd gene is located in the 70F/71A region distal to Bearded. We have identified mnd within a genomic clone and have isolated numerous cDNA clones. A transgene containing the largest cDNA with a heat shock promoter can rescue the lethal phenotype. The mnd gene encodes a 54 kDa protein which is predicted to have 12 transmembrane domains and is similar in sequence to the catalytic subunit of an amino acid transporter. Many years ago, we showed that co-culture with fat body is required for proliferation of imaginal discs in vitro. Dietary amino acids are required for the fat body to secrete a mitogen that stimulates proliferation of larval neuroblasts. We believe, in response to uptake of dietary amino acids by the MND transporter, the fat body secretes a factor required for imaginal disc proliferation. [Work done by former graduate student Jennifer Martin.]
The abnormal wing discs (awd) gene encodes the subunit of a hexameric nucleoside diphosphate kinase (NDP kinase). Killer of prune (Kpn) is a mutation in the awd gene which substitutes Ser for Pro at position 97. The awdKpn mutation causes no phenotype by itself, but causes dominant lethality in individuals that do not have a functional prune (pn) gene. In such lethal individuals there is adequate AWD/NDP kinase activity for viability and KPN mutant subunits form a normal distribution of hexamers with non-mutant subunits. In order to understand the biochemical basis for the conditional lethality of the awdKpn mutation, we performed a screen for dominant suppressors of pn/awdKpn lethality. We mutagenized pn mutant males, mated them to attached X females homozygous for awdKpn and screened for male survivors. No males, but 243,000 females were recovered. Work with awd transgenes revealed that relatively low levels of AWD accumulation (~5% of wild-type) is sufficient to rescue the awd null lethal phenotype. This suggested that the failure to recover suppressors was due to the high level of KPN mutant protein that accumulates even in awdKpn heterozygotes. Based on this idea, we devised a modified screen for suppressors using transgenic awdKpn as a source of KPN mutant protein. In this modified screen, we mutagenized pn mutant males and mated them to attached X females that were homozygous for transgenic awdKpn. All of the male progeny of such a cross would be heterozygous for transgenic awdKpn and hemizygous for pn. In control experiments, without mutagenizing the males, we established that this genotype causes lethality. In this screen, we have recovered 120,000 female progeny and 3 males. Subsequent analysis revealed that all three of these males had suppressor mutations on the third chromosome. These mutations are homozygous lethal and allelic! While all three of these suppressors can, to varying degrees, rescue the lethal interaction in the presence of transgenic levels of KPN, none of the three can rescue the lethal interaction in the presence of endogenous levels of KPN at all. These data support our interpretation that the previous failure to recover suppressors was due to the high level of KPN mutant protein that accumulates even in awdKpn heterozygotes. Indeed while individuals that are homozygous or hemizygous for pn and heterozygous for any one of the suppressor alleles can survive if they are heterozygous for transgenic awdKpn, they cannot survive if they are homozygous for transgenic awdKpn. In other words they are very sensitive to the level of KPN protein accumulation. [Work done by Grafton Hersperger and Allen Shearn.]
III. Growth, metastasis, and invasiveness of Drosophila tumors Several tumor suppressor genes have been identified in Drosophila by loss of function mutations that lead to overgrowth of specific tissues. This overgrowth is called neoplastic when it is accompanied by the loss of capacity of these tissues to differentiate. Mutations in either of two genes lethal giant larvae(lgl) or brain tumor(brat) cause such neoplastic overgrowth of the brain and imaginal discs. As heterozygotes, brain tumor mutations have a low penetrance of wing defects. Three regions of the second chromosome were identified in a screen for deficiencies that exhibited intergenic non-complementation for such wing defects. For one of these three regions, loss of thick veins function is responsible for the intergenic non-complementation. Since thick veins encodes a receptor for DPP signaling, this suggested that brain tumor acts positively to regulate DPP signaling. This hypothesis was confirmed by showing that brain tumor mutants have reduced accumulation of spalt and optomotor blind, gene products that are up-regulated by DPP signaling. lethal giant larvae mutants have reduced accumulation of both of these gene products and also of the vestigial gene product. [Work done by former graduate student Christopher Gee.] Upon transplantation into wild type female adults, cells from fragments of either lgl or brat tumorous brains proliferate to fill the body cavities of the hosts. Using accumulation of a reporter protein to unambiguously identify donor tumor cells, Woodhouse et al (1998) detected tumors cells associated with the head, thorax, and ovaries of the hosts. Because Drosophila has an open circulatory system, the spread of tumor cells throughout the body does not by itself prove that these cells are metastatic. Careful examination of dissected ovaries from adult hosts that had received transplanted tumorous brains revealed in many cases that tumor cells were attached to the ovaries or were between ovarioles but had not actually invaded through any of the basement membranes or cell layers that surround individual ovarioles. To more precisely assay metastatic potential of Drosophila tumor cells we have refined the previous assay to focus on the ability of tumor cells to invade individual ovarioles and form secondary tumors. The formation of such secondary tumors requires tumor cells to invade a structure that is surrounded by a basement membrane and a muscle cell layer. The advantage conferred by this assay results from the stringency of its test for invasion; it provides a means to compare the invasiveness of genetically different tumors. Using this assay, we report that lgl mutant brain cells invade ovarioles at a much higher rate than do brat mutant brain cells. [Work done by former graduate student Julie Goodliffe and Evelyn Hersperger.] Woodhouse et al. (1994) identified a 49 kDa Drosophila protein with gelatinolytic activity that cross-reacts with an antibody to a human matrix metalloprotease. They also reported that lgl homozygous brain tissue contains more of this gelatinase than do wild type brains (Woodhouse et al., 1994). We call the gene that encodes this protein machete. The predicted amino acid sequence of the Machete protein suggests that it is a serine protease. Aside from three other predicted Drosophila serine proteases (60%, 56% and 53% identical respectively), the most similar protein we found in a database search (47% identical) is a chymotrypsinogen from Lucilia cuprina, the green bottle fly. A serine protease that has collagenolytic activity from Celuca pugilator, the fiddler crab, is 40% identical. Interestingly, Machete is 32% identical to human Prostate Specific Antigen, a serine protease whose levels are elevated in the blood of prostate cancer patients. Targeted expression of machete within the ovaries of adult hosts facilitates invasion by brat tumor cells. This is the first evidence that expression of a specific protease promotes the invasiveness of Drosophila tumor cells. [Work done by former graduate student Julie Goodliffe.] Drosophila has two genes predicted to encode matrix metalloproteases. MMP1, the product of one of these genes, CG4859, has been shown to have metalloprotese activity. It is predicted to be a secreted protein. MMP2, the product of the other of these genes, CG1794, is predicted to be a membrane-bound protein. The catalytic domain of this protein has also been shown to have metalloprotease activity. An enhancer trap insertion into CG1794 causes ß-galactosidase accumulation in the optic neuroblasts of larval brain lobes. In an lgl mutant, accumulation of ß-galactosidase from this enhancer trap is found distributed throughout larval brain lobes. We interpret this altered distribution as the consequence of mutant neuroblasts invading other regions of larval brain lobes. [Work done by Michelle Beaucher.] Representative PublicationsBeaucher, M., Hersperger, E., Page-McCaw, A., Shearn, A. 2007. Metastatic ability of Drosophila tumors depends on MMP activity. Developmental Biology. 303:625-634. Beaucher, M., Goodliffe, J., Hersperger. E,, Trunova. S., Frydman, H., Shearn, A. 2007. Drosophila brain tumor metastases express both neuronal and glial cell type markers. Developmental Biology. 301 :287-297.
Provost E, Shearn A. (2006). The Suppressor of Killer of prune, a unique glutathione S-transferase. J Bioenerg Biomembr. Timmons, L. and Shearn, A. 2000. Role of AWD/Nucleoside Diphosphate Kinase in Drosophila Development. Journal of Biochemistry and Biophysics 32: 293-300. Arama, E., Dickman. D., Kimchie, Z., Shearn, A., and Lev, Z. 2000. Mutations in the ß-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasms in the larval brain. Oncogene 19: 3706-3716. |
