Elizabeth A. Monckton

A Role for DEAD Box 1 at DNA Double-Strand Breaks

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

The early isoform of disabled-1 functions independently of Reelin-mediated tyrosine phosphorylation in chick retina.

ABSTRACT The Reelin-Disabled-1 (Dab1) signaling pathway plays a key role in the positioning of neurons during brain development. Two alternatively spliced Dab1 isoforms have been identified in chick retina and brain: Dab1-E, expressed at early stages of development, and Dab1-L (commonly referred to as Dab1), expressed at later developmental stages. The well-studied Dab1-L serves as an adaptor protein linking Reelin signal to its downstream effectors; however, nothing is known regarding the role of Dab1-E. Here we show that Dab1-E is primarily expressed in proliferating retinal progenitor cells whereas Dab1-L is found exclusively in differentiated neuronal cells. In contrast to Dab1-L, which is tyrosine phosphorylated upon Reelin stimulation, Dab1-E is not tyrosine phosphorylated and may function independently of Reelin. Knockdown of Dab1-E in chick retina results in a significant reduction in the number of proliferating cells and promotes ganglion cell differentiation. Our results demonstrate a role for Dab1-E in the maintenance of the retinal progenitor pool and determination of cell fate.

Interferon-gamma gene expression during acute graft-versus-host disease: relationship to MHC induction and tissue injury

The pathogenetic role of interferon‐γ (IFN‐γ) in acute graft‐versus‐host disease (GVHD) was examined in a murine model. IFN‐γ gene expression was evaluated by northern blotting and mRNA in situ hybridization. The temporal and tissue specific patterns of IFN‐γ gene expression were related to the patterns of major histocompatibility complex (MHC) antigen induction and of tissue injury. Markedly increased levels of IFN‐γ transcripts were seen in the spleen during the early lymphoproliferative phase and coincided with widespread MHC induction in non‐lymphoid tissues. Increased IFN‐γ transcripts were also found in the non‐lymphoid target tissues during the phase of subsequent tissue injury. These findings support a role for IFN‐γ in leading to widespread MHC induction during acute GVHD and suggest that IFN‐γ may also contribute to target tissue injury during acute GVHD. Copyright

Ddx1 knockout results in transgenerational wild-type lethality in mice

DEAD box 1 (DDX1) is a member of the DEAD box family of RNA helicases which are involved in all aspects of RNA metabolism. DDX1 has been implicated in a variety of biological processes, including 3’-end processing of mRNA, DNA repair, microRNA processing, tRNA maturation and mRNA transport. To study the role of DDX1 during development, we have generated mice carrying a constitutive Ddx1 knock-out allele. Ddx1+/− mice have no obvious phenotype and express similar levels of DDX1 as wild-type mice indicating compensation from the intact Ddx1 allele. Heterozygote matings produce no viable Ddx1−/− progeny, with Ddx1−/− embryos dying prior to embryonic day (E) 3.5. Intriguingly, the number of wild-type progeny is significantly decreased in heterozygote crosses, with two different heterozygote populations identified based on parental genotype: (i) normal Ddx1+/− mice which generate the expected number of wild-type progeny and (ii) Ddx1*/− mice (with * signifying a non-genetically altered allele) which generate a significantly reduced number of wild-type mice. The transgenerational inheritance of wild-type lethality observed upon crossing Ddx1*/− mice is independent of parental sex and occurs in cis through a mechanism that is different from other types of previously reported transgenerational epigenetic inheritance.

Effects of gold thioglucose on food intake, growth and forebrain histology in goldfish, Carassius auratus.

Abstract The effects of gold thioglucose (GTG) injected intraperitoneally or into the ventricles of the brain on food intake, body weight gain, and forebrain histology in the goldfish were investigated. GTG caused a dose-dependent decrease in food intake, and usually initially caused loss of body weight, or at least slowed the rate of weight gain for up to 4 weeks. Over a long term the tendency was for recovery of normophagia and recovery of the normal rate of body weight gain. No significant effects on plasma glucose concentrations or growth hormone concentrations were detected following GTG treatment. No lesions were observed in the ventromedial hypothalamus of goldfish following intraperitoneal or brain injection of GTG. A dose dependent hypertrophy of the ependyma of the forebrain was found following brain injection of GTG. However, ependymal hypertrophy was found in only about half of the fish injected intraperitoneally with GTG at a dosage causing hypophagia. There was disruption of the ependyma lining the lateral-dorsal aspect of the lateral recess of the third ventricle, and some hypertrophy of the adjacent nucleus recessus lateralis, following brain injection, but not intraperitoneal injection, of a dose of GTG effective in causing decreased weight gain and hypophagia. Since no consistent histological effects of GTG were observed, its site of action in causing decreased weight gain and hypophagia in the goldfish is unknown. Fish injected with gold chloride all died soon after injection. Gold thiomalate injection caused weight loss and slowed the rate of weight gain, similar to GTG. The mechanism by which GTG causes hypophagia and decreased growth in goldfish is probably not related to the specific uptake of glucose by some brain center.

Loss of AP-2delta reduces retinal ganglion cell numbers and axonal projections to the superior colliculus.

BackgroundAP-2δ is the most divergent member of the Activating Protein-2 (TFAP2) family of transcription factors. AP-2δ is restricted to specific regions of the CNS, including a subset of ganglion cells in the retina. Retinal ganglion cells (RGCs), the only output neurons of the retina, are responsible for transmitting the visual signal to the brain.ResultsAP-2δ knockout results in loss of Brn3c (Pou4f3) expression in AP-2δ -positive RGCs. While AP-2δ-/- mice have morphologically normal retinas at birth, there is a significant reduction in retinal ganglion cell numbers by P21, after eye opening. Chromatin immunoprecipitation indicates that Brn3c is a target of AP-2δ in the retina. Using fluorochrome-conjugated cholera toxin subunit B to trace ganglion cell axons from the eye to the major visual pathways in the brain, we found 87 % and 32 % decreases in ipsilateral and contralateral projections, respectively, to the superior colliculus in AP-2δ-/- mice. In agreement with anatomical data, visually evoked responses recorded from the brain confirmed that retinal outputs to the brain are compromised.ConclusionsAP-2δ is important for the maintenance of ganglion cell numbers in the retina. Loss of AP-2δ alters retinal axonal projections to visual centers of the brain, with ipsilaterial projections to the superior colliculus being the most dramatically affected. Our results have important implications for integration of the visual signal at the superior colliculus.

NFIB promotes cell survival by directly suppressing p21 transcription in p53-mutated triple-negative breast cancer: Role of NFIB in breast cancer cell survival

Triple‐negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options and poor prognosis. There is an urgent need to identify and understand the key factors and signalling pathways driving TNBC tumour progression, relapse, and treatment resistance. In this study, we report that gene copy numbers and expression levels of nuclear factor IB (NFIB), a recently identified oncogene in small cell lung cancer, are preferentially increased in TNBC compared to other breast cancer subtypes. Furthermore, increased levels of NFIB are significantly associated with high tumour grade, poor prognosis, and reduced chemotherapy response. Concurrent TP53 mutations and NFIB overexpression (z‐scores > 0) were observed in 77.9% of TNBCs, in contrast to 28.5% in non‐TNBCs. Depletion of NFIB in TP53‐mutated TNBC cell lines promotes cell death, cell cycle arrest, and enhances sensitivity to docetaxel, a first‐line chemotherapeutic drug in breast cancer treatment. Importantly, these alterations in growth properties were accompanied by induction of CDKN1A, the gene encoding p21, a downstream effector of p53. We show that NFIB directly interacts with the CDKN1A promoter in TNBC cells. Furthermore, knockdown of combined p21 and NFIB reverses the docetaxel‐induced cell growth inhibition observed upon NFIB knockdown, indicating that NFIBs effect on chemotherapeutic drug response is mediated through p21. Our results indicate that NFIB is an important TNBC factor that drives tumour cell growth and drug resistance, leading to poor clinical outcomes. Thus, targeting NFIB in TP53‐mutated TNBC may reverse oncogenic properties associated with mutant p53 by restoring p21 activity. Copyright

Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma

Glioblastomas (GBMs) are highly aggressive brain tumors with a dismal prognosis. Nuclear factor I (NFI) is a family of transcription factors that controls glial cell differentiation in the developing central nervous system. NFIs have previously been shown to regulate the expression of astrocyte markers such as glial fibrillary acidic protein (GFAP) in both normal brain and GBM cells. We used chromatin immunoprecipitation (ChIP)–on-chip to identify additional NFI targets in GBM cells. Analysis of our ChIP data revealed ~400 putative NFI target genes including an effector of the Notch signaling pathway, HEY1, implicated in the maintenance of neural stem cells. All four NFIs (NFIA, NFIB, NFIC, and NFIX) bind to NFI recognition sites located within 1 kb upstream of the HEY1 transcription site. We further showed that NFI negatively regulates HEY1 expression, with knockdown of all four NFIs in GBM cells resulting in increased HEY1 RNA levels. HEY1 knockdown in GBM cells decreased cell proliferation, increased cell migration, and decreased neurosphere formation. Finally, we found a general correlation between elevated levels of HEY1 and expression of the brain neural stem/progenitor cell marker B-FABP in GBM cell lines. Knockdown of HEY1 resulted in an increase in the RNA levels of the GFAP astrocyte differentiation marker. Overall, our data indicate that HEY1 is negatively regulated by NFI family members and is associated with increased proliferation, decreased migration, and increased stem cell properties in GBM cells.