The tumor suppressor p14 ARF gene is induced by ectopically expressed E2F, a positive regulator of the cell cycle. The gene is expressed at low levels in normally growing cells in contrast to high levels in varieties of tumors. How p14 ARF gene is regulated by E2F in normally growing cells and tumor cells remains obscure. Here we show that regulation of p14 ARF gene by E2F is distinct from that of classical E2F targets. It is directly mediated by E2F through a novel E2F-responsive element that varies from the typical E2F site. The element responds to E2F activity resulting from ectopic E2F1 expression, inactivation of pRb by adenovirus E1a or shRNA, but not to phosphorylation of pRb by serum stimulation or ectopic cyclin D1/cyclindependent kinase-4 expression in normal human fibroblasts. The element has activity in various tumor cells with defective pRb, but not in normally growing cells. These results indicate that the distinct regulation constitutes the basis of p14 ARF function as a tumor suppressor, discriminating abnormal growth signals caused by defects in pRb function from normal growth signals.

SUMMARYAsymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. We used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. We identify the transcription factor Klumpfuss (Klu) as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led us to propose that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division.

We also identified that the BAP chromatin-remodeling complex probably functions cooperatively with Erm to restrict the developmental potential of immature INPs. Together, these data led us to conclude that the Erm-BAP-dependent mechanism stably restricts the developmental potential of immature INPs by attenuating their genomic responses to stem cell self-renewal factors. We propose that restriction of developmental potential by the Erm-BAP-dependent mechanism functionally distinguishes intermediate progenitor cells from stem cells, ensuring the generation of differentiated cells and preventing the formation of progenitor cell-derived tumor-initiating stem cells.

The transcription factor E2F mediates cell cycle-dependent expression of genes important for cell proliferation in response to growth stimulation. To further understand the role of E2F, we utilized a sensitive subtraction method to explore new E2F1 targets, which are expressed at low levels and might have been unrecognized in previous studies. We identified 33 new E2F1-inducible genes, including checkpoint genes Claspin and Rad51ap1, and four genes with unknown function required for cell cycle progression. Moreover, we found three groups of E2F1-inducible genes that were not induced by growth stimulation. At least, two groups of genes were directly induced by E2F1, indicating that E2F1 can regulate expression of genes not induced during the cell cycle. One included Neogenin, WASF1 and SGEF genes, which may have a role in differentiation or development. The other was the cyclin-dependent kinase inhibitor p27 Kip1 , which was involved in suppression of inappropriate cell cycle progression induced by deregulated E2F. E2F1-responsive regions of these genes were located more upstream than those of typical E2F targets and did not have typical E2F sites. These results indicate that there are groups of E2F1 targets, which are regulated in a distinct manner from that of typical E2F targets.

During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the selfrenewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification.

The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. In this study, we report that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.DOI: http://dx.doi.org/10.7554/eLife.03502.001

Self-renewal genes maintain stem cells in an undifferentiated state by preventing the commitment to differentiate. Robust inactivation of self-renewal gene activity following asymmetric stem cell division allows uncommitted stem cell progeny to exit from an undifferentiated state and initiate the commitment to differentiate. Nonetheless, how self-renewal gene activity at mRNA and protein levels becomes synchronously terminated in uncommitted stem cell progeny is unclear. We demonstrate that a multilayered gene regulation system terminates self-renewal gene activity at all levels in uncommitted stem cell progeny in the fly neural stem cell lineage. We found that the RNA-binding protein Brain tumor (Brat) targets the transcripts of a self-renewal gene, deadpan (dpn), for decay by recruiting the deadenylation machinery to the 3 ′ untranslated region (UTR). Furthermore, we identified a nuclear protein, Insensible, that complements Cullin-mediated proteolysis to robustly inactivate Dpn activity by limiting the level of active Dpn through protein sequestration. The synergy between post-transcriptional and transcriptional control of self-renewal genes drives timely exit from the stem cell state in uncommitted progenitors. Our proposed multilayered gene regulation system could be broadly applicable to the control of exit from stemness in all stem cell lineages.

During DNA replication, DNA becomes more vulnerable to certain DNA damages. DNA repair genes involved in repair of the damages may be induced by growth stimulation. However, regulation of DNA repair genes by growth stimulation has not been analysed in detail. In this report, we analysed the regulation of expression of mammalian MSH2, MSH3 and MLH1 genes involved in mismatch repair, and Rad51 and Rad50 genes involved in homologous recombination repair, in relation to cell growth. Unexpectedly, we found a clear difference in regulation of these repair gene expression by growth stimulation even in the same repair system. The expression of MSH2, MLH1 and Rad51 genes was clearly growth regulated, whereas MSH3 and Rad50 genes were constitutively expressed, suggesting differential requirement of the repair gene products for cell proliferation. MSH3 gene is located in a bidirectionally divergent manner with DHFR gene that is regulated by growth stimulation, indicating that bidirectionally divergent promoters are not necessarily coordinately regulated. Promoter analysis showed that the growth-regulated expression of MLH1 and Rad51 genes was mainly mediated by E2F that plays crucial roles in regulation of DNA replication, suggesting close relation between some of the repair genes and DNA replication.