BIOLOGICAL OVERVIEW

The path taken by Decapentaplegic signals in their travels to the nucleus from outside the recipient cell is only partially understood. Bunched is the first known intracellular component in this signaling path; its role is inhibitory. bunched, also known as shortsighted (shs) is downstream of DPP in the morphogenetic furrow of the eye disc, and acts in the developing brain. Receptors for DPP include Punt, Thickveins and Saxophone. The only known transcription factors activated downstream of DPP are schnurri, an immediate downstream target, and mothers against dpp.

Bunched has no basic DNA-binding domain typical of leucine zipper transcription factors. The leucine zipper in bZIP transcription factors is a dimerization domain. If SHS dimerizes to bZIP transcription factors, inhibition might be achieved by sequestering these factors in the cytoplasm, analogous to NFkB-IkappaB interaction (see dorsal and cactus). Alternatively, SHS may have a cytoplasmic function unrelated to transciption regulation (Treisman, 1995).

A set of dorsal follicle cells is patterned by the oocyte in a cell-cell signaling event occurring at stages 8 and 9 when the germinal vesicle (nucleus) migrates to the dorsal anterior of the oocyte. The anterodorsally positioned oocyte nucleus produces Gurken mRNA, a proposed ligand for the Epidermal growth cell receptor gene present on the overlying follicle cells. Activating Egfr transmits a signal through a Raf-dependent signaling pathway to generate anterior dorsal follicle cell fates, resulting in the respective specializations of the eggshell, including the dorsal appendages. A ventral follicle cell subpopulation that does not experience induction by Gurken produces molecular cues for a different inductive event, directing embryonic dorsal-ventral embryonic axis formation (Dobens, 1997 and references).

A Drosophila sequence homologous to the mammalian growth factor-stimulated TSC-22 gene was isolated in an enhancer trap screen for genes expressed in anterodorsal follicle cells during oogenesis. In situ hybridization reveals that bun transcripts localize to the anterior dorsal follicle cells at stages 10-12 of oogenesis. Additional staining is evident in the border cells at the nurse cell/oocyte border and in a group of posterior polar follicle cells. The centripetally migrating follicle cells, just anterior to the stained columnar cells of the anterodorsal patch do not stain. Changes in bun enhancer trap expression in genetic backgrounds that disrupt the grk/Egfr signaling pathway suggest that bun is regulated by growth factor patterning of dorsal anterior follicle cell fates. In fs(1)K10 mutant egg chambers, dorsal follicle cell fates expand at the expense of ventral follicle cell fates, presumably due to mislocalization of GRK mRNA from the anterodorsal portion of the oocyte to more ventral positions. In fs(1)K10 females, expression of bunched expands ventrally, with two maxima in the anterodorsal anteroventral follicle cells, diminishing laterally. In stage 10 follicles from Egfr mutants expression of bun is lost from the dorsal anterior; reduced bun expression is shifted to more posterior follicle cells. Egg chambers from a gurken mutant completely lack dorsal appendages. No bunched expression is seen in the dorsal anterior follicle cells from stage 10 gurken mutant egg chambers. Clonal analysis shows that bun is required for the proper elaboration of dorsal cell fates leading to the formation of the dorsal appendages. Eggs from bunched mutants are shortened and their dorsal appendages are short and often wide, with branched and split ends (Dobens, 1997).

Preliminary evidence indicates the bunched is sensitive to decapentaplegic levels in the follicle cells. It is therefore thought that normal bunched expression in the dorsal anterior follicle cells is the result of the combined actions of the Egfr receptor for Grk and serine/threonine kinase receptors for Decapentaplegic (Dobens, 1997).

DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Opposing DPP and EGF signals are shown to set the boundary for an ovarian follicle cell (FC) fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. Global increase in DPP levels, using heat-shock-GAL4 to drive UAS-dpp expression throughout all FCs gives rise to eggs that show expanded opercula and reduced dorsal appendages. In other respects, the eggshells are normal. At the extreme anterior, normal micropyles were formed. The mutant opercula generally have a normal organization of large cell imprints surrounded by a raised structure, the collar. Significantly, expansion of the operculum always occurs over the dorsal side of the egg, indicating that dorsal-ventral patterning is unperturbed. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein Mad; ectopic expression of Bunched suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. The bunched expression pattern requires the EGF receptor ligand Gurken. Activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. It is proposed that the juxtaposition of cells with high and low Bunched activity organizes a sharp boundary for the operculum fate (Dobens, 2000).

A role for bunched as an antagonist of operculum patterning correlates well with the strict exclusion of bunched-lacZ from the centripetal migrating follicle cells (CMFC), the anteriormost population of columnar FCs. The dorsal CMFC undergo changes in shape that lead to the unique cell imprints in the operculum. Expression of bunched-lacZ occurs throughout the posterior FCs, and is strongest over the dorsal anterior FCs. This expression pattern reflects the pattern of BUN-1 mRNA accumulation, which encodes the isoform that can block A359 expression. It is concluded from these data that the dorsal anterior boundary of BUN-1 mRNA expression defines the future boundary of the operculum (Dobens, 2000).

Gurken signaling through the Egfr is necessary for normal bunched-lacZ expression in the dorsal anterior FC. Ectopic expression of activated Egfr is sufficient to induce ectopic bunched-lacZ in the centripetal migrating FCs. Conversely, Dpp signaling is both necessary and sufficient to repress bunched-lacZ in columnar FCs. Thus the dorsal anterior boundary of bunched-lacZ expression is set by a balance of positive EGF and negative Dpp signals. Dpp also sets the anterior boundary for Broad-Complex expression; however, the regulation of this gene by EGF signaling is more complex. In summary, a model is proposed where the boundary for the operculum is set by the boundary of Bunched activity, which is positioned by opposing activity of Dpp and EGF signals in the dorsal FCs. Dorsal anterior FC are exposed to high levels of EGF ligands Grk, Spitz and Vein, and thus have elevated bunched expression. High anterior Dpp signaling represses bunched expression. The close apposition of these signals in the dorsal anterior FCs creates a sharp boundary of bunched expression. BUN-1 functions to repress A359 and define the boundary to centripetal migrating FC fates, including the operculum. These data indicate that the ventral operculum boundary is also set by bunched; however, another signal appears to promote ventral bunched expression at late stages. The normal operculum border is defined by the eggshell collar. This structure is lost as bunched activity is lowered, suggesting that the boundary of bunched expression may serve to further organize cell fates at the operculum boundary (Dobens, 2000).

Although the data suggest that EGF signals antagonize operculum patterning, EGF signaling is essential for operculum formation. (1) grk and Egfr mutant eggs have no opercula. (2) Overexpression of activated Egfr can result in operculum expansion, although interpretation of the specific phenotype is not straightforward. Thus, it is expected that Dpp does not prevent all Egfr-induced events in the operculum-forming FCs. It is likely that EGF signaling is active in cells that lack Bunched activity, and that Dpp inactivation of Bunched modifies the response of these cells to EGF signals. In cultured mammalian cells, RTK signaling can directly antagonize BMP signaling by preventing nuclear accumulation of Smad protein, offering a possible molecular mechanism for these interactions (Dobens, 2000).

The data presented here indicate that the A359 locus may be a direct target for negative regulation by Bunched. The sensitive A359 repression assay also shows that the TSC domain is critical to Bunched function: altering these amino acids makes BUN-1 inactive. The role of bunched in other tissues is poorly understood. Embryos homozygous for bunched mutations die with morphological defects in the peripheral neurons and subtle defects in cuticle pattern. bunched maternal effect phenotypes are pleiotropic, ranging from very early defects to segmental defects in the embryonic cuticle. During eye development, bunched promotes photoreceptor differentiation, and shows genetic interactions with dpp, wingless, hedgehog and components of the Egfr signaling pathway. It remains to be determined whether bunched has a similar role throughout development, for example as an RTK target gene or a repressor of Dpp target genes. Bunched antagonizes Dpp function in the follicle cells. This finding is surprising, for mutations in the dpp and bunched genes synergize to severely arrest eye development (Treisman, 1995). A mechanistic interpretation of this genetic interaction awaits better understanding of Dpp functions during eye development. It has been noted that the bunched eye phenotype is rescued by the BUN-2 transcript (Treisman, 1995), whereas BUN-1 is the antagonist of Dpp in the FC. The BUN-2 transcript is expressed in the operculum-forming FC, raising the possibilities that this isoform has a distinct role, or that it is subject to post-transcriptional regulation. Further studies of the functions of the two isoforms will be needed to resolve these differences (Dobens, 2000 and references therein).

It is proposed here that the boundary to a Dpp-induced fate in the follicle cells is set by transcriptional regulation of a downstream transcriptional repressor, Bunched. Recently, a similar role has been proposed for the gene brinker in setting threshold gene expression responses to Dpp in the Drosophila wing. Thus, Dpp induction of gene expression through negative regulation of a negative regulator may be a common theme in development. Regulation of the expression of these key downstream repressors provides a powerful mechanism to modulate responses to Dpp signaling (Dobens, 2000 and references therein).

GENE STRUCTURE

There are two transcripts: a long form coding for a protein of 1212 amino acids, and a short form coding for a protein of 225 amino acids. The two proteins differ in their 5'UTR and N-terminal amino acids, indicating use of two transcriptional start sites. The common 3'UTR has 1182 bases. The 5'UTR of the short form is 907 bases. The UTR of the long form is 914 amino acids (Treisman, 1995).

PROTEIN STRUCTURE

Structural Domains and Evolutionary Homologies

Both the long and short forms have a common C-terminal region containing a leucine zipper domain with homology to mouse gene TSC-22, which is transcriptionally induced in response to TGF-beta. The same gene is a target for FSH in Sartoli cells (Treisman, 1995).

The TSC box, immediately preceding the zipper subregion in the central domain is a highly conserved sequence common to the TSC family. The domain shows no strong matches with other proteins in the database and is also unrelated to the similarly located basic domain of the bZIP class of proteins (Dobens, 1997).

Previous studies on the promoter function of the human C-type natriuretic peptide (CNP) gene have revealed the existence of two GC-rich cis elements essential for gene transcription in rat pituitary-derived GH3 cells. To isolate transcription factors that bind to those GC-rich elements, a lambda ZAP cDNA library derived from GH3 cells was screened. Several positive clones with specific binding abilities were obtained; one is identical as TSC-22, a speculated transcriptional modulator stimulated by transforming growth factor beta (TGF-beta) of unknown function. TSC-22 significantly enhances CNP promoter activity in GH3 cells. In adults, human TSC-22 mRNA is highly expressed in brain, lung and heart. TSC-22 gene expression in GH3 and human aortic endothelial cells is stimulated by cytokines, including TGF-beta, in concert with the CNP mRNA increase. These results suggest that TSC-22 is a transcriptional regulator of the CNP gene and transmits signals from cytokines, such as TGF-beta, to CNP gene expression (Ohta, 1996).

The molecular mechanisms underlying the pleiotropic effects of FSH were investigated by screening a plasmid cDNA library for clones hybridizing to FSH-regulated RNAs from FSH-treated Sertoli cells. One clone encodes the rat homolog of transforming growth factor-beta 1-stimulated clone 22 (TSC-22), which contains a putative leucine zipper region. Regulation of rat TSC-22 mRNA was analyzed in primary Sertoli cell cultures. TSC-22 mRNA transiently increases 4-fold in the presence of FSH, reached maximal levels at 3 h, and returns to prestimulation levels by 12 h. The FSH-stimulated increase is independent of protein synthesis because it occurs in the presence of cycloheximide and FSH. TSC-22 mRNA is detected in all tissues examined in male and female rats; the highest levels in the 16-day animal were observed in the testis, ovary, uterus, and lung. Testicular 1.8-kilobase (kb) TSC-22 mRNA decreases by 50% from 14 to 60 days of age. A 5-kb transcript becomes detectable by 30 days and decreases after 50 days of age. Ovarian 1.8-kb TSC-22 transcript levels increased about 2-fold during the same maturation period (Hamil, 1997).

A gene sequence (TSC-22) that is induced by transforming growth factor (TGF) beta 1 was isolated by differential screening of a cDNA library constructed from poly(A)+ RNA of mouse osteoblastic cells treated with TGF-beta. TSC-22 gene expression is transcriptionally activated by TGF-beta 1. It is also induced by phorbol 12-myristate 13-acetate, serum, cholera toxin, or dexamethasone, but not appreciably by epidermal growth factor. Its induction is rapid and transient, reaching a peak 2 h after TGF-beta 1 treatment, and is resistant to cycloheximide like that of c-jun. The nucleotide sequences of TSC-22 cDNA show no homology with any known gene sequence. The open reading frame and in vitro translation product indicate that the gene encodes a polypeptide of 143 amino acids with a molecular mass of 18 kDa that contains a putative leucine zipper structure. Polyclonal antibody was raised against TSC-22 protein expressed in Escherichia coli cells; the antibody detects a 18-kDa protein in both the cytoplasmic and nuclear fractions of cells. These results indicate that the TSC-22 gene is a new member of the family of early response genes, and encodes a small polypeptide that is a putative transcriptional regulator (Shibanuma, 1992).

Human gastric carcinoma cell line HSC-39 has been shown to undergo apoptotic cell death in response to treatment with transforming growth factor beta1 (TGF-beta1). To understand better the cell death mechanism in this TGF-beta1-mediated apoptosis, the effect of the expression of TGF-beta-stimulated clone 22 (TSC-22) on cell death was investigated. TGF-beta1 induces TSC-22 gene expression in HSC-39 cells only when the cells have previously been adapted to the serum-free culture conditions required to undergo TGF-beta1-mediated apoptosis. HSC-39 cells transfected with a TSC-22 expression vector show a significant decrease in cell viability compared with those transfected with a control vector. The cellular events characteristic of apoptosis, chromatin condensation and DNA fragmentation, are observed only in cells transfected with a TSC-22 expression vector. On immunostaining of the transfected cells, almost every cell that expresses TSC-22 tagged with influenza virus hemagglutinin exhibits the morphology of an apoptotic cell. Partial protection from the cell death effect of TGF-beta1 on HSC-39 cells is observed when cells are treated with acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspart-1-al (Ac-DEVD-CHO, an inhibitor specific for CPP32-type protease). Protection against cell death by the transfection of a TSC-22 expression vector is also offered by Ac-DEVD-CHO addition. These results suggest that TSC-22 elicits the apoptotic cell death of human gastric carcinoma cells through the activation of CPP32-like protease and mediates the TGF-beta1 signaling pathway to apoptosis (Ohta, 1997).

TGF-beta-stimulated clone-22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved during evolution. Two homologs are known that share a similar leucine zipper domain and another conserved domain (designated the TSC box). Only limited data are available on the function of TSC-22 and its homologs. TSC-22 is transcriptionally up-regulated by many different stimuli, including anti-cancer drugs and growth inhibitors; recent data suggest that TSC-22 may play a suppressive role in tumorigenesis. TSC-22 forms homodimers via its conserved leucine zipper domain. Using a yeast two-hybrid screen, a TSC-22 homolog (THG-1) is found to be a heterodimeric partner. The presence of two more mammalian family members with highly conserved leucine zippers and TSC boxes is reported. Interestingly, both TSC-22 and THG-1 have transcriptional repressor activity when fused to a heterologous DNA-binding domain. The repressor activity of TSC-22 appears sensitive for promoter architecture, but not for the histone deacetylase inhibitor trichostatin A. Mutational analysis shows that this repressor activity resides in the non-conserved regions of the protein and is enhanced by the conserved dimerization domain. These results suggest that TSC-22 belongs to a family of leucine zipper-containing transcription factors that can homodimerize and heterodimerize with other family members and that at least two TSC-22 family members may be repressors of transcription (Kester, 1999).

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), were synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicate that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it has been concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein reveals that the left-handed super helical structure of the leucine zipper type sequence is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids, the structure of pDIP is stabilized by the formation of interhelical salt bridges. This result has been confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed (Seidel, 1997)

The leucine zipper transcription factor TSC-22 (TGF-beta1 Stimulated Clone-22) was first isolated from a mouse osteoblast cell line as an immediate-early target gene of TGF-beta1. However, work with other cell lines, as well as with a Drosophila homolog, bunched, suggests that it is an effector gene of various growth factors and potentially involved in the integration of multiple extracellular signals. Throughout mouse embryogenesis TSC-22 is expressed in a dynamic pattern. Although early TSC-22 expression is ubiquitous in 6.5 day embryos, as development proceeds TSC-22 expression is upregulated at sites of epithelial-mesenchymal interactions such as the limb bud, tooth primordiun, hair follicle, kidney, lung, and pancreas. TSC-22 is also expressed in many neural crest-derived tissues including the mesenchyme of the branchial arches, the cranial, dorsal root, and sympathetic ganglia, as well as the facial cartilage and bone. Other areas of expression are the otic and optic vesicles, the heart, and cartilage and bone forming regions throughout the embryo (Dohrmann, 1999).

This study was undertaken to clarify the molecular mechanism of the effect of a new anti-cancer drug, vesnarinone, on a human salivary gland cancer cell line, TYS. TSC-22cDNA was isolated as a vesnarinone-inducible gene from a cDNA library constructed from vesnarinone-treated TYS cells. TSC-22 was originally reported as a transforming growth factor (TGF)-beta-inducible gene. The expression of TSC-22 is up-regulated within a few hours after treatment with vesnarinone and continues for 3 days. The level of TSC-22 mRNA in TYS cells continuously increases until the cells reach confluence. Furthermore, the induction of TSC-22 by vesnarinone is inhibited by treatment with cycloheximide. When cells are treated with an antisense oligonucleotide against TSC-22 mRNA under quiescent conditions, the antisense oligonucleotide stimulates the growth of TYS cells; however, under growing conditions the antisense oligonucleotide does not affect cell growth. Furthermore, the antisense oligonucleotide suppresses the antiproliferative effect of vesnarinone. These results suggest that TSC-22 may be a negative growth regulator and may play an important role in the antiproliferative effect of vesnarinone (Kawamata, 1998).

Transcriptional Regulation

bunched requires hedgehog signal for initiation of transcription in the eye-imaginal disc (Treisman, 1995).

DEVELOPMENTAL BIOLOGY

See the embryonic expression pattern of bun at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site

Larval

bunched is expressed in a hedgehog-dependent stripe in the undifferentiated cells just anterior to the morphological furrow of the eye imaginal disc. It appears to be involved in the transmission of the differentiation-inducing signal; a reduction in bunched function leads to a delay in differentiation and to a loss of photoreceptors in the adult. bunched is also required for a morphogenetic movement in the brain that reorients the second optic lobe relative to the first. Input from the eye is required for this rotation. Additional functions of shs include a role in wing margin development and a requirement for both male and female fertility (Treisman, 1995).

Effects of mutation or deletion

The mutant is lethal, but a few surviving adults have slightly small, rough eyes. The first observable phenotype evinces a delay in neural differentiation. The mutation is enhanced by a single copy of hedgehog or decapentaplegic. The mutant phenotype is suppressed by the loss of one copy of wingless (Treisman, 1995).

REFERENCES

Dobens, L. L., et al. (1997). The Drosophila bunched gene is a homologue of the growth factor stimulated mammalian TSC-22 sequence and is required during oogenesis. Mech. Dev. 65(1-2): 197-208. Medline abstract: 97398458

Dohrmann, C. E., Belaoussoff, M. and Raftery, L. A. (1999). Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development. Mech. Dev. 84(1-2): 147-51. Medline abstract: 99400109

Dobens, L. L. (2000). Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary. Development 127: 745-754. Medline abstract: 20115428

Hamil, K. G. and Hall, S. H. (1994). Cloning of rat Sertoli cell follicle-stimulating hormone primary response complementary deoxyribonucleic acid: regulation of TSC-22 gene expression. Endocrinology 134(3): 1205-1212. Medline abstract: 94164020

Kawamata, H., et al. (1998). Induction of TSC-22 by treatment with a new anti-cancer drug, vesnarinone, in a human salivary gland cancer cell. Br. J. Cancer 77(1): 71-8. Medline abstract: 98119236

Kester, H. A., Blanchetot, C., den Hertog, J., van der Saag, P. T. and van der Burg, B. (1999). Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity. J Biol Chem 274: 27439-27447. Medline abstract: 99419018

Ohta, S., Shimekake, Y. and Nagata, K. (1996). Molecular cloning and characterization of a transcription factor for the C-type natriuretic peptide gene promoter. Eur. J. Biochem. 242(3): 460-466. Medline abstract: 97175009

Ohta, S., Yanagihara, K. and Nagata, K. (1997). Mechanism of apoptotic cell death of human gastric carcinoma cells mediated by transforming growth factor beta. Biochem. J. 324(3): 777-782

Seidel, G., Adermann, K., Schindler, T., Ejchart, A., Jaenicke, R., Forssmann, W.-G. and Rosch, P. (1997). Solution structure of porcine delta sleep-inducing peptide immunoreactive peptide A homolog of the shortsighted gene product. J. Biol. Chem. 272; 30918-30927. Medline abstract: 98049560

Shibanuma, M., Kuroki, T. and Nose, K. (1992). Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors. J. Biol. Chem. 267(15): 10219-10224. Medline abstract: 92268052

Treisman, J. E., Lai., Z. C., and Rubin. G. M. (1995). Shortsighted acts in the decapentaplegic pathway in Drosophila eye development and has homology to a mouse TGF-beta-responsive gene. Development 121: 2835-2845 Medline abstract

date revised: 28 February 2000  

Please e-mail comments/corrections to brodyt@codon.nih.gov