Naomi C. Mitchell

The Ecdysone-inducible zinc-finger transcription factor Crol regulates Wg transcription and cell cycle progression in Drosophila

The steroid hormone Ecdysone is crucial for developmental cell death, proliferation and morphogenesis in Drosophila. Herein, we delineate a molecular pathway linking Ecdysone signalling to cell cycle regulation in the Drosophila developing wing. We present evidence that the Ecdysone-inducible zinc-finger transcription factor Crol provides a crucial link between the Ecdysone steroid hormone pathway and the Wingless (Wg) signalling pathway in Drosophila. We identified Crol as a strong enhancer of a wing phenotype generated by overexpression of the Wg-inducible cell cycle inhibitor Hfp. We demonstrate that Crol is required for cell cycle progression: crol mutant clones have reduced cell cycles and are removed by apoptosis, while upregulation of Crol overrides the Wg-mediated developmental cell cycle arrest in the zone of non-proliferating cells in the wing disc. Furthermore, we show that Crol acts to repress wg transcription. We also show that overexpression of crol results in downregulation of Hfp, consistent with the identification of the crol mutant as a dominant enhancer of the Hfp overexpression phenotype. Taken together, our studies have revealed a novel mechanism for cell cycle regulation, whereby Crol links steroid hormone signals to Wg signalling and the regulation of crucial cell cycle targets.

Drosophila ribosomal protein mutants control tissue growth non-autonomously via effects on the prothoracic gland and ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.

Hfp inhibits Drosophila myc transcription and cell growth in a TFIIH/Hay-dependent manner

An unresolved question regarding the RNA-recognition motif (RRM) protein Half pint (Hfp) has been whether its tumour suppressor behaviour occurs by a transcriptional mechanism or via effects on splicing. The data presented here demonstrate that Hfp achieves cell cycle inhibition via an essential role in the repression of Drosophila myc (dmyc) transcription. We demonstrate that regulation of dmyc requires interaction between the transcriptional repressor Hfp and the DNA helicase subunit of TFIIH, Haywire (Hay). In vivo studies show that Hfp binds to the dmyc promoter and that repression of dmyc transcription requires Hfp. In addition, loss of Hfp results in enhanced cell growth, which depends on the presence of dMyc. This is consistent with Hfp being essential for inhibition of dmyc transcription and cell growth. Further support for Hfp controlling dmyc transcriptionally comes from the demonstration that Hfp physically and genetically interacts with the XPB helicase component of the TFIIH transcription factor complex, Hay, which is required for normal levels of dmyc expression, cell growth and cell cycle progression. Together, these data demonstrate that Hfp is crucial for repression of dmyc, suggesting that a transcriptional, rather than splicing, mechanism underlies the regulation of dMyc and the tumour suppressor behaviour of Hfp.

The Novel Zinc Finger Protein dASCIZ Regulates Mitosis in Drosophila via an Essential Role in Dynein Light-Chain Expression

The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. Here we report the identification of the Drosophila ortholog of ASCIZ (dASCIZ) and show that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, we show that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp–LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. We speculate that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, our data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light-chain levels and a novel regulator of mitosis in flies.

S6 Kinase is essential for MYC-dependent rDNA transcription in Drosophila

Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, we performed a transient expression screen for known oncogenic signalling pathways in Drosophila. Strikingly, from the broad range of pathways tested, we found that ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, we demonstrated that one of the mechanism(s) by which MYC and S6K cooperate is through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

The Ecdysone receptor constrains wingless expression to pattern cell cycle across the Drosophila wing margin in a cyclin B-dependent manner

BackgroundEcdysone triggers transcriptional changes via the ecdysone receptor (EcR) to coordinate developmental programs of apoptosis, cell cycle and differentiation. Data suggests EcR affects cell cycle gene expression indirectly and here we identify Wingless as an intermediary factor linking EcR to cell cycle.ResultsWe demonstrate EcR patterns cell cycle across the presumptive Drosophila wing margin by constraining wg transcription to modulate CycB expression, but not the previously identified Wg-targets dMyc or Stg. Furthermore co-knockdown of Wg restores CycB patterning in EcR knockdown clones. Wg is not a direct target of EcR, rather we demonstrate that repression of Wg by EcR is likely mediated by direct interaction between the EcR-responsive zinc finger transcription factor Crol and the wg promoter.ConclusionsThus we elucidate a critical mechanism potentially connecting ecdysone with patterning signals to ensure correct timing of cell cycle exit and differentiation during margin wing development.

Steroid Hormones in Drosophila: How Ecdysone Coordinates Developmental Signalling with Cell Growth and Division

1.1 The ecdysone pathway directs Drosophila development Ecdysone is the major steroid hormone in all holometabolous insects responsible for driving the metamorphosis of larval tissues into adult structures. During metamorphosis, ecdysone is essential for upregulating the genes required to control apoptosis and differentiation, essential processes for removal of larval structures which have become obsolete and for tissue remodelling. In addition, ecdysone directs cell growth and division in many tissues throughout the larval to pupal transition. This chapter will discuss the many diverse mechanisms reported for connecting the ecdysone pulse to the developmentally regulated cell growth and cycle progression required for tissue growth and for insects to reach their target body size. Like all other holometabolous insects, the size of Drosophila adult flies is set by the size of the larvae prior to metamorphosis, at the time of pupariation when feeding has ceased and growth can no longer occur. The major developmental hormone in Drosophila, the steroid hormone 20-hydroxyecdysone (20E), commonly known as ecdysone, is required for all the developmental transitions needed for metamorphosis (Figure 1-3; (Thummel 1995, 1996, 2001)). Ecdysone is produced in and released by the prothoracic gland (PG), a component of the ring gland, which also contains the corpora allata (CA) and corpora cardiaca (CC) (Figure 1; (Zitnan et al. 2007; McBrayer et al. 2007)). Ecdysone release is controlled by a complex combination of upstream factors, including peptide hormones and neuropeptide signals (see section 2.2). For example, Prothoracicotropic hormone (PTTH) from the central nervous system (CNS) is required to regulate the synthesis and release of ecdysone from the PG (McBrayer et al. 2007). Ecdysone pulses from the PG are required for all aspects of morphogenesis, starting with the formation of the body plan during late embryogenesis, hatching and development of the first larval instar, and for cuticle moulting at the end of the first and second instars. A large

Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth

Summary The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. Here, we use Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. We further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly.

Defining the essential function of FBP/KSRP proteins: Drosophila Psi interacts with the mediator complex to modulate MYC transcription and tissue growth

Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, we demonstrate the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and we demonstrate Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. Our data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, we predict interaction between MED and FBP1 might also have implications for cancer initiation and progression.

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

Nucleotide excision DNA repair (NER) pathway mutations cause neurodegenerative and progeroid disorders (xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD)), which are inexplicably associated with (XP) or without (CS/TTD) cancer. Moreover, cancer progression occurs in certain patients, but not others, with similar C-terminal mutations in the XPB helicase subunit of transcription and NER factor TFIIH. Mechanisms driving overproliferation and, therefore, cancer associated with XPB mutations are currently unknown. Here using Drosophila models, we provide evidence that C-terminally truncated Hay/XPB alleles enhance overgrowth dependent on reduced abundance of RNA recognition motif protein Hfp/FIR, which transcriptionally represses the MYC oncogene homologue, dMYC. The data demonstrate that dMYC repression and dMYC-dependent overgrowth in the Hfp hypomorph is further impaired in the C-terminal Hay/XPB mutant background. Thus, we predict defective transcriptional repression of MYC by the Hfp orthologue, FIR, might provide one mechanism for cancer progression in XP/CS.