Here we show that bunA functions in growth control. BunA positively regulates growth by adjusting cell number and cell size during Drosophila development. Additionally, we found that the short Bun isoforms can act in a dominant negative way on BunA function.
The bun genomic locus gives rise to six different transcripts. Since each transcript has at least one distinct 5' exon, the expression of the six mRNAs is likely to be controlled by separate promoters. The distinct 5' exons result in Bun proteins with individual N-termini, except for BunF that is almost identical to BunA. All Bun isoforms have a common C-terminus comprising a conserved DNA-binding domain (TSC-box) and a leucine zipper for homo- and heterodimerization [27, 51]. It is conceivable that the Bun isoforms exert different functions, since BunA, but not BunB and BunC, is involved in growth control. BunB and BunC might be (partially) redundant to other proteins, for example BunD and BunE, and hence they would only exhibit their mutant phenotypes in double mutant situations. However, our data allow us to conclude that BunA is the major Bun isoform involved in growth control because restoring bunA function suffices to rescue the lethality and the growth deficit associated with a deletion allele that removes the TSC-box and the leucine zipper and thus presumably represents a complete loss-of-function for all Bun isoforms.
Flies with impaired bunA function are small due to fewer and smaller cells. Consistently, clones of cells lacking bunA function remain smaller than their sister clones, and the reduction in clone area is also caused by a diminution of both cell size and cell number. Since apoptosis is not obviously enhanced in clones of bunA mutant cells, we conclude that bunA is required to adjust cellular growth. In line with our results, Wu and colleagues (manuscript submitted) found that BunA exerts similar growth effects in follicle cells and in cultured Drosophila S2 cells.
The bunA growth phenotypes are reminiscent of the phenotypes caused by an impairment of insulin signaling [52–56]. Furthermore, bunA also affects lipid metabolism, as has been shown for insulin signaling [53, 57]. Therefore, we tested whether bunA would genetically interact with insulin signaling components (data not shown). However, we concluded that BunA is probably not a core component of the insulin signal transduction cascade because we did not detect a clear epistatic relationship with the lipid phosphatase PTEN. It is also unlikely that BunA acts directly in the TOR signaling branch because bunA mutant larvae do not display the pronounced growth deficit of the endoreplicative tissues (salivary glands, fat body) that has been observed in dTOR and Rheb mutant larvae [58, 59].
BunA is clearly distinct from insulin signaling components in that it also affects pattern formation. Flies with lowered bunA function display various eye phenotypes reminiscent of defects associated with reduced Notch signaling activity [45–48]. Dobens and colleagues  have proposed a model whereby bun modulates Notch signaling by indirectly adjusting the amount of the Notch ligand Serrate during eggshell development. A similar relationship between bun and Notch signaling may account for the function of bun in patterning processes such as photoreceptor cell differentiation. bun genetically interacts with the EGF receptor and Dpp (BMP-2/-4 ortholog) signaling cascades during eye development  as well as during oogenesis . Presently, it is unclear whether bunA has distinct patterning and growth functions or whether it operates at the interface between pattern formation and growth regulation by integrating various patterning signals to adjust cellular growth.
BunA influences cellular growth and proliferation yet the mechanism remains unknown. In light of the putative transcriptional regulator function of BunA, it is conceivable that bunA induces the expression of growth-promoting genes or it represses the expression of growth inhibitors. However, Treisman and colleagues  have reported that BunA predominantly localizes to the cytoplasm in the larval eye disc. In addition, we could not detect any nuclear signal upon expression of an N- or C-terminally GFP-tagged BunA in Drosophila S2 or Kc cells (data not shown). Thus, BunA might shuttle between the cytoplasm and the nucleus, and its translocation to the nucleus might be tightly regulated. Alternatively, BunA could function in the cytoplasm in a process distinct from transcriptional regulation. The identification of BunA binding partners should shed light on the subcellular environment in which BunA exerts its function.
Our study on the growth-promoting function of bunA in Drosophila may influence the perspective on the mammalian homologs of Bun, especially on TSC-22 (TSC22D1). Whereas the longer isoform of TSC-22 (TSC22D1.1) is similar to BunA (and BunF), the shorter isoform (TSC22D1.2) resembles BunB, BunD, and BunE. Data from numerous studies suggest that TSC22D1.2 acts as a tumor suppressor [4–7, 10–12], which is at odds with the fact that only bunA is involved in growth regulation in Drosophila, and that BunA behaves rather opposite to a tumor suppressor. The results from our in vivo analysis may be of special interest in this context, since the relative balance of bun transcripts is important (allelic series) and overexpression of bunC (and also bunB) interferes with bunA function in a dominant negative manner. If this interaction is conserved in mammals, we can envision the following scenario for how the TSC-22 locus may be involved in tumor suppression. Whereas the long TSC-22 isoform, TSC22D1.1, positively regulates cellular growth (as does BunA), the short isoform, TSC22D1.2, inhibits growth by competing with TSC22D1.1. The antagonism between the long and the short isoforms can be achieved at several levels. An excess of the short isoform could lead to the formation of non-functional heterodimers, or the two isoforms could compete for another dimerization partner. Provided that TSC-22 functions in transcriptional regulation, the two isoforms might also contribute to differential regulation of target genes. In either case, the long TSC-22 isoform could be hyperactivated as a consequence of the loss of the short isoform. Thus, the short isoform could act as a tumor suppressor by keeping the long isoform in check. Our findings should encourage further studies in mammals that distinguish between the TSC-22 isoforms and that primarily focus on the function of the long TSC-22 protein, TSC22D1.1.