bunched: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - bunched

Synonyms - shortsighted

Cytological map position - 33E7-8

Function - transcription factor

Keyword(s) - segment polarity, dpp pathway, oogenesis

Symbol - bun

FlyBase ID:FBgn0259176

Genetic map position -

Classification - leucine-zipper, TSC-22 family

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Nie, Y., Li, Q., Amcheslavsky, A., Duhart, J. C., Veraksa, A., Stocker, H., Raftery, L. A. and Ip, Y. T. (2015). Bunched and Madm function downstream of Tuberous sclerosis complex to regulate the growth of intestinal stem cells in Drosophila. Stem Cell Rev. PubMed ID: 26323255
The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. This study shows that the leucine zipper protein Bunched and the adaptor protein Madm are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, these data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells.

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).

Bunched sets a boundary for Notch signaling to pattern anterior eggshell structures during Drosophila oogenesis

Organized boundaries between different cell fates are critical in patterning and organogenesis. In some tissues, long-range signals position a boundary, and local Notch signaling maintains it. How Notch activity is restricted to boundary regions is not well understood. During Drosophila oogenesis, the long-range signals EGF and Dpp regulate expression of bunched (bun), which encodes a homolog of mammalian transcription factors TSC-22 and GILZ. This study shows that bun establishes a boundary for Notch signaling in the follicle cell epithelium. Notch signaling is active in anterior follicle cells and is required for concurrent follicle cell reorganizations including centripetal migration and operculum formation. bun is required in posterior columnar follicle cells to repress the centripetal migration fate, including gene expression, cell shape changes and accumulation of cytoskeletal components. bun mutant clones adjacent to the centripetally migrating follicle cells showed ectopic Notch responses. bun is necessary, but not sufficient, to down-regulate Serrate protein levels throughout the follicular epithelium. These data indicate that Notch signaling is necessary, but not sufficient, for centripetal migration and that bun regulates the level of Notch stimulation to position the boundary between centripetally migrating and stationary columnar follicle cells (Dobens, 2005).

Previous work demonstrated that opposing gradients of EGF and BMP signals established a sharp boundary for expression of one of the Bun transcripts, BunB, and that the resultant protein Bun1 can repress the operculum fate (Dobens, 2000). The data presented in this study indicate that bun is required around the circumference of the FC to prevent changes in cellular architecture that are associated with centripetal migration. These include changes in α-spectrin subcellular localization, non-muscle myosin accumulation and cell shape. Consistent with the model that bun regulates centripetal migration, rare, small, anterior clones of bun4230 have been observed where the mutant cells elongate and pinch into the oocyte. Taken together, these data indicate that bun maintains the columnar FC fate and prevents both centripetal migration and operculum fate determination. However, the data from bun clonal analysis indicate that bun function is position-dependent, suggesting that it regulates competence to respond to additional signals. Strong mutant phenotypes occurred only in columnar bun FC clones that contacted the centripetal migrating FC (Dobens, 2005).

The data indicate that concurrent Notch signaling is required for centripetal migration and that bun blocks this signaling in columnar FC at the time of centripetal migration. Altogether, these data suggest that bun organizes a boundary for morphogenesis, in part by regulating the level of Notch signaling. Thus, fine patterning of the anterior eggshell fates involves the action of three independent signals, Dpp, EGF and Notch, coordinated through expression and activity of bun (Dobens, 2005).

Previously, the role of Notch signaling in anterior FC patterning has been attributed to the early requirement for Notch in patterning the egg chamber termini. Notch signaling is required for differentiation and/or survival of the polar FC, which are necessary for anterior/posterior patterning of both follicle cells and the underlying oocyte. Building on this model, it was recently proposed that centripetal migrating FC are one of several fates that are specified by graded levels of JAK/STAT signaling prior to stage 6, stimulated by Upd produced from anterior polar cells. While JAK/STAT signaling appears necessary for gene expression in centripetally migrating FC, other aspects of the Upd morphogen gradient model are controversial (Dobens, 2005).

Notch signaling is repeatedly required during FC development. Notch is important for initial formation of the two FC lineages, the stalk/polar cell lineage and the epithelial FC lineage. Subsequently, during stage 6, Notch is required throughout the FC epithelium to switch from mitotic cell divisions to endoreplication. It has been proposed that Notch signaling at stage 6 initiates differentiation of the epithelial FC and that all subsequent phenotypes in Nts egg chambers are due to failed differentiation. Indeed, Notch protein levels are high throughout the epithelial FC prior to stage 7 and then decrease substantially by stage 8. By this model, defective anterior morphogenesis would be a deferred consequence of failed Notch signaling during stages 6 and 7 (Dobens, 2005).

However, this study shows that Notch signaling is active in anterior FC during centripetal migration and subsequent morphogenesis of the operculum and dorsal appendages. Multiple Notch target reporters -- Dl-lacZ, 12XSu(H)BS-lacZ and E(spl)mβ7-CD2 -- are expressed in anterior FC at stages 10-14, where Notch, Delta and Serrate are detected during stages 9-14. Furthermore, a temperature-sensitive allele of Notch could be used to disrupt anterior FC morphogenesis without significant perturbation of endoreplication. These observations indicate that late Notch signaling is required for centripetal migration (Dobens, 2005).

An unusual feature of this late Notch activity in anterior FC is the activation of responses in cells that co-express both ligand and receptors. Over-expression studies suggest that high levels of Delta or Serrate can block signaling by Notch from the same cells, which is thought to be important for spatial localization of Notch signaling during wing margin development. This inhibitory effect can be observed in the FC with gratuitous expression of high levels of Serrate. In contrast, low levels of ligand appear to stimulate target gene expression in the same cell, during ectopic expression or under endogenous conditions in proneural clusters and pairs of photoreceptors in the developing eye. Although two ligands are present during anterior FC morphogenesis, the levels for each protein were near the limit of detection for immunofluorescence in wild type FC. Target gene expression levels were also low (Dobens, 2005).

The spatial and temporal restrictions observed for Notch responses suggest that endogenous levels of either Delta or Serrate alone are insufficient to stimulate Notch signaling for anterior morphogenesis. Manipulation of bun activity has strong effects on Serrate accumulation but only weak effects on Delta. Delta gene expression responds to experimental manipulation of Notch activity. It is proposed that stimulation of Notch signaling is localized to centripetally migrating cells through this dual regulation. Serrate accumulates at higher levels in FC with low bun activity, and Delta expression is increased by Notch signaling through a positive feedback loop. Notch signaling in anterior FC may be initiated by Delta signaling from the nurse cells. However, during eggshell deposition, FC expression of ligands is needed to maintain Notch signaling in FC that overlie the oocyte. Detailed analysis of the requirements for Delta and Serrate will be needed to validate the model that both ligands are needed for activation of Notch signaling in centripetally migrating FC. The data presented in this study demonstrate that bun prevents the Notch stimulus from spreading inappropriately into the columnar FC (Dobens, 2005).

The molecular mechanism for bun regulation of Notch signaling is unknown. Two types of non-autonomous effects were observed, suggesting that regulation of Notch stimulation is indirect. First, mutant cells that contact bun+ cells at the edge of the clone often had wild type phenotype. Thus, contact with a wild type cell can rescue these phenotypes in bun mutant cells. Consistent with this, in wild type egg chambers, a single-cell gap between high Notch levels and bun-lacZ expression was observed in ventro-lateral FC during late oogenesis. Second, it appears that high levels of Serrate accumulation can be stimulated by the presence of neighboring cells with higher levels of the Bun1 transcription factor. This non-autonomous effect suggests that even regulation of Serrate levels is indirect. Although numerous genes can modulate Notch signaling, few have been tested for cell-autonomous function. Additional bun target genes are being sought that may modulate Notch signaling (Dobens, 2005).

At present, it is not known if bun regulates Notch activity in other tissues. bun mutant phenotypes occur in the peripheral nervous system, eye, wing margin and denticle belt patterning, all regions that require Notch signaling. In chickens, the bun-related TSC-22 is expressed in developing feather buds at a time coincident with Notch signal activity. Further work will be required to determine whether this is a general function for TSC-22/DIP/BUN family members (Dobens, 2005).

Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila

The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. This study shows that the leucine zipper protein Bunched and the adaptor protein MLF1-adaptor molecule (Madm) are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, these data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells (Nie, 2015).

Homeostasis and regeneration of an adult tissue is normally supported by resident stem cells. Elucidation of the mechanisms that regulate stem cell-mediated homeostasis is important for the development of therapeutics for various diseases. The intestine with fast cell turnover rate supported by actively proliferating stem cells is a robust system to study tissue homeostasis. In the mouse intestine, two interconverting intestinal stem cell (ISC) populations marked by Bmi1 and Lgr5 located near the crypt base can replenish cells of various lineages along the crypt-villus axis Furthermore, recent data suggest that Lgr5+ cells are the main stem cell population and that immediate progeny destined for the secretory lineage can revert to Lgr5+ stem cells under certain conditions [6, 7]. Together, the results suggest previously unexpected plasticity in stem cell maintenance and differentiation in the adult mammalian intestine (Nie, 2015).

In the adult Drosophila midgut, which is equivalent to the mammalian stomach and small intestine, ISCs are distributed evenly along the basal side of the monolayered epithelium to support repair. The maintenance and regulation of Drosophila midgut ISCs depend on both intrinsic and extrinsic factors. When a midgut ISC divides, it generates a renewed ISC and an enteroblast (EB) that ceases to divide and starts to differentiate. The ISC-EB asymmetry is established by the Delta-Notch signaling, with Delta in the renewed ISC activating Notch signaling in the newly formed neighboring EB . Growth factors such as Wingless/ Wnt, insulin-like peptides, Decapentaplegic/BMP, Hedgehog and ligands for the EGF receptor and JAK-STAT pathways are secreted from surrounding cells and constitute the niche signals that regulate both ISC division and EB differentiation. ISC-intrinsic factors including Myc, Target of Rapamycin (TOR) and Tuberous Sclerosis Complex act to coordinate the growth and division of ISCs. Furthermore, chromatin modifiers such as Osa, Brahma and Scrawny function within ISCs to regulate Delta expression or ISC proliferation (Nie, 2015).

This study reports the identification of the leucine zipper protein Bunched (Bun) and the adaptor protein myeloid leukemia factor 1 adaptor molecule (Madm) as intrinsic factors for ISC proliferation. A single bun genomic locus generates multiple predicted transcripts that encode 4 long isoforms, BunA, F, G and P, and 5 short isoforms, BunB, C, D, E, H and O. The first identified mammalian homolog of Bun is TGF-β1 stimulated clone-22 (TSC-22). In the mouse genome four different TSC- 22 domain genes also encode multiple short and long isoforms. All isoforms of Bun and TSC-22 contain an approximately 200 amino acids C-terminal domain where the conserved TSC-box and leucine zippers are located. The originally identified TSC-22 is a short isoform and various assays suggest that it suppresses cancer cell proliferation and may function as a transcriptional regulator. Meanwhile, in Drosophila, the long Bun isoforms positively regulate growth, while the short isoforms may antagonize the function of long isoforms. Transgenic fly assays also demonstrate that the long TSC-22 can rescue the bun mutant phenotypes, whereas short isoforms cannot. These results suggest an alternative model that the long Bun isoforms positively regulate proliferation, while the short isoforms may dimerize with and inhibit the functions of long isoforms (Nie, 2015).

Madm also can promote growth. The long isoform BunA binds to Madm via a conserved motif located in the N- terminus that is not present in the short Bun isoforms. The molecular function of this novel BunA- Madm complex, nonetheless, remains to be elucidated. The results in this report demonstrate that Bun and Madm modulate the Tuberous Sclerosis Complex-target of Rapamycin (TOR)-eIF4E binding protein (4EBP) pathway to regulate the growth and division of ISCs in the adult midgut (Nie, 2015).

This report shows that Bun and Madm are intrinsically required for ISC growth and division. The results suggest a model that Bun and Madm form a complex in the cytoplasm to promote cellular growth and proliferation. The evidence that support this model includes the observation that transgenic expressed Bun localizes in the cytoplasm of midgut precursor cells, similar to the results from transfection in S2 cells and immune-staining in eye discs. Bun physically and functionally interacts with Madm, which has also been proposed as a cytoplasmic adaptor protein. Adding a nuclear localization signal to Bun reduced the growth promoting ability of Bun. Although there is a possibility this signal peptide changes the functionality in an unpredicted way, the interpretation is favored that Bun normally acts in the cytoplasm and with Madm to regulate the proliferation of ISCs. This is in contrast to mammalian TSC-22, which was reported to function in the nucleus (Nie, 2015).

The results seem to contradict a previous publication reporting that TSC-22 arrests proliferation during human colon epithelial cell differentiation. However, this apparent contradiction is resolved when the growing evidence for distinct functions for large and small Bun/ TSC-22 isoforms is considered. The Bun/TSC-22 proteins have short and long isoforms that contain the conserved TSC-box and leucine zippers in the C-terminal domain. The prototypical TSC-22 protein, TSC22D1-001, may act as a transcriptional regulator and repress cancer cell proliferation, particularly for blood lineages. Another recent model suggests that in Drosophila the long Bun isoforms interact with Madm and have a growth promoting activity, which is inhibited by the short Bun isoforms. Similarly, the long isoform, TSC22D1-002, enhances proliferation in mouse mammary glands, whereas the short isoform promotes apoptosis. Unpublished result that transgenic expression of BunB also has lower function than BunA in fly intestinal progenitor cells is consistent with this model where large isoforms have a distinct function, namely in growth promotion (Nie, 2015).

Loss of either Bun or Madm can potently suppress all the growth stimulation by multiple pathways in the midgut as shown in this report. These results are intrepeted to indicate that Bun and Madm do not act specifically in one of the signaling pathways tested but instead function in a fundamental process required for cell growth, such as protein synthesis or protein turnover. It is therefore speculated that Bun and Madm may regulate the TOR pathway. In support of this idea, it was shown that bunRNAi or MadmRNAi efficiently suppresses the Tuberous Sclerosis Complex 2RNAi-induced cell growth and p4EBP phenotypes. A recent study of genetic suppression of TOR complex 1-S6K function in S2 cells also suggests that Bun and Madm can interact with this pathway. Furthermore, proteomic analyses of Bun and Madm interacting proteins in S2 cells have shown interactions with ribosomal proteins and translation initiation factors. Therefore, a model is proposed that Bun and Madm function in the Tuberous Sclerosis Complex-TOR- 4EBP pathway to regulate protein synthesis in ISCs for their growth, which is a prerequisite for ISC proliferation. Suppression of Tuberous Sclerosis Complex mutant cell growth phenotype by bun or Madm RNAi was substantial but not complete. Earlier papers demonstrated that Bun also interacts with Notch and EGF pathway in ovary follicle cells. Therefore by definition Bun and Madm are neither 100% essential nor restricted to the TOR pathway. The genetic data suggest that Bun and Madm work downstream of Tuberous Sclerosis Complex and upstream of 4EBP, but they could also work in parallel to the TOR pathway components (Nie, 2015).

ISCs with loss of Tuberous Sclerosis Complex function have substantial cell size increase. Meanwhile, the Bun/ Madm overexpression caused increased ISC division but not cell hypertrophy. Both loss of Tuberous Sclerosis Complex and overexpression of Bun/Madm should promote cell growth but the phenotypes at the end are different. It is speculated that the reason is the Bun/Madm overexpressing ISCs are still capable of mitosis, while the Tuberous Sclerosis Complex mutant ISCs do not divide anymore thereby resulting in the very big cells. In Bun and Madm overexpressing mid- guts, the p-H3+ and GFP+ cell count showed a significant increase, indicating increased mitosis. Therefore, an explanation is that Bun and Madm overexpression may increase cell size/cell growth, but when they grow to certain size they divide, resulting in rather normal cell size (Nie, 2015). The knockout of the Madm mammalian homolog, NRBP1, can cause accumulation of the short isoform TSC22D2. Up-regulation of Madm/NRBP1 has been associated with poor clinical outcome and increased growth of prostate cancer. Further analysis based on this model may reveal whether high ratio of long Bun/TSC22 isoforms over short isoforms may associate with high Madm activity and poor clinical outcomes (Nie, 2015).


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).


Amino Acids -

Structural Domains

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).

bunched: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 January 2016

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