Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNAunwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR. DEAD box proteins are a family of putative RNA helicases that function by altering RNA secondary structure. This protein family has been implicated in all aspects of RNA metabolism. The DEAD box 1 gene (DDX1) is a widely expressed gene that is misexpressed in a number of cancers, including retinoblastoma, neuroblastoma, and breast cancer (1, 2). Knockout of DDX1 leads to early embryonic lethality in mice and severely reduced fertility in flies (3, 4). DDX1 is involved in the transport of RNAs from the nucleus to the cytoplasm and regulates cytoplasmic localization of the splicing-regulatory protein KSRP (5). In neurons, DDX1 resides in RNA-transporting granules, cytoplasmic organelles that regulate the localization and expression of target mRNAs (6, 7). DDX1 has also been identified as a core subunit of the human tRNA ligase complex which is essential for tRNA splicing (8).In addition to its roles in RNA metabolism, DDX1 has been implicated in the cellular response to DNA double-strand breaks (DSBs). Upon treatment of cells with ionizing radiation (IR), DDX1 rapidly accumulates at a subset of DNA DSBs (ϳ30%), where it forms IR-induced foci that colocalize with ␥-H2AX, a marker for DSBs (9). DDX1 coimmunoprecipitates with the MRN (MRE11-RAD50-NBS1) complex, the early sensor of DNA DSBs, and ATM (ataxia telangiectasia mutated) protein, the key transducer of the signaling cascade in response to DSBs (10, 11). DSBs induce DDX1 phosphorylation in an ATM-dependent manner. Notably, IR-induced DDX1 foci are lost when cells are treated with RNase H, an enzyme that specifically digests RNA from RNA-DNA hybrids (9). These results suggest that RNA-DNA double-stranded structures are required for the presence and/or retention of DDX1 at DSBs. In line with this observation, biochemical analysis has shown that DDX1 can unwind both...

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 ␥-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-DNAand 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.DEAD box proteins, classically defined as putative RNA helicases, have been implicated in all aspects of RNA metabolism involving the modulation of RNA secondary structure (38, 47). These proteins share nine conserved motifs (including the D-E-A-D motif) required for RNA binding, RNA-dependent ATP binding/hydrolysis, and ATP-dependent RNA unwinding. Although Ͼ35 DEAD box proteins in higher eukaryotes have been identified, we still have a poor understanding of their biological roles (1). The best-characterized mammalian DEAD box protein is the translation initiation factor eukaryotic initiation factor 4A (eIF4A), which unwinds RNA-RNA and RNA-DNA duplexes in vitro. eIF4A is believed to facilitate translation initiation by removing secondary structures from the 5Ј ends of transcripts (24).Analyses of DEAD box proteins in lower eukaryotes and prokaryotes suggest roles in RNA processing, RNA stability, RNA transport, and RNA remodeling. DEAD box proteins (and related DEAH box proteins) have recently been implicated in the DNA damage response, with Saccharomyces cerevisiae DHH1 playing a role in G 1 /S DNA damage checkpoint recovery (10) and yeast MPH1 proposed to function in a branch of homologous recombination (HR) involved in errorfree bypassing of DNA lesions (52). With an estimated Ͼ20,000 DNA lesions per cell each day, the effective repair of genomic DNA is critical to the survival of the cell. Of all DNA lesions, double-strand breaks (DSBs) are the most serious threat to the genome, as they can lead to the loss of genetic information, chromosome abnormalities, and cell death. DNA DSBs can be caused by exogenous agents, such as ionizing radiation (IR), or endogenous agents, such as reactive oxygen species (30). DNA DSBs trigger a sequence of events which include DNA damage sensing, the am...

No abstract

Brain fatty acid-binding protein (B-FABPMalignant gliomas are believed to be derived from the astrocytic cell lineage because they contain bundles of cytoplasmic glial fibrillary acidic protein (GFAP), 1 an intermediate filament protein specifically expressed in differentiated astrocytes.There is an inverse relationship between the number of GFAPpositive cells and aggressive behavior in glioma tumors. Glioblastoma multiforme, the most common and aggressive glioma, often have low GFAP expression, while low grade astrocytomas usually have high levels of GFAP (1-4). In vitro studies directly correlate GFAP expression with a less aggressive behavior (5). Transfection of a GFAP expression vector into GFAP(Ϫ) malignant glioma cells results in decreased cell proliferation and decreased growth in soft agar (6, 7). Conversely, transfection of a GFAP antisense vector into a GFAP(ϩ) line results in undetectable GFAP expression and increased proliferation rate, anchorage-independent growth, and invasiveness (8).We have previously shown that GFAP(ϩ) malignant glioma lines express a second glial cell marker, brain fatty acid-binding protein (B-FABP) (9). Of 15 malignant glioma lines tested, 5 co-expressed B-FABP and GFAP, 8 expressed neither gene, while 2 had low levels of B-FABP and undetectable levels of GFAP. B-FABP is a 15-kDa protein normally found in the radial glial cells of the developing central nervous system as well as in select glial cell populations of the adult brain including glia limitans cells and Bergmann glial cells (10, 11). B-FABP expression has been implicated in the establishment of the radial glial fiber system which serves to guide immature migrating neurons to their correct location in the central nervous system (10, 12). Addition of anti-B-FABP antibody to primary cultures of cerebellar cells prevents both the extension of radial glial processes and the migration of neuronal cells along these processes, suggesting a role for B-FABP in relaying inductive signals required for glial cell differentiation.It is generally believed that radial glial cells are converted into astrocytes once neuronal migration in the developing brain is complete (13). Co-expression of GFAP and B-FABP in the same malignant glioma cells (9) therefore suggests that these tumors are derived from cells that have the potential of expressing proteins that are normally produced at different stages in the glial differentiation pathway. We are studying the regulation of the B-FABP gene in order to identify transcription factors involved in the regulation of glial genes in malignant glioma and understand the basis for the variation in B-FABP expression in different malignant glioma lines. By sequencing and DNase I footprinting, we have identified two NFI-binding sites in the promoter region of the B-FABP gene. We present evidence that a phosphatase specifically expressed in B-FABP(ϩ) cells is responsible for differential expression of the B-FABP gene in malignant glioma lines.

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.

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 NFIB's 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 © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

No abstract

Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5 flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5 flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5 flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Sp1 binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.The fatty-acid-binding protein (FABP) family consists of a number of structurally related proteins with characteristic cellular, tissue, and developmental distribution patterns. Members of this family include heart FABP, intestinal FABP, adipocyte lipid-binding protein, myelin P 2 , cellular retinoicacid-binding proteins, and cellular retinol-binding proteins (1, 51). Some FABPs, such as adipocyte FABP and myelin P 2 , are restricted to one tissue or organ type while others are more broadly expressed. For example, intestinal FABP is found in both intestine and stomach while liver FABP is present in liver, intestine, kidney, and stomach (reviewed in reference 51). Roles proposed for these proteins include the uptake, intracellular solubilization, storage, and/or delivery of fatty acids and retinoids (1). A role in signal transduction has also been proposed for heart FABP and adipocyte lipid-binding protein via phosphorylation of a tyrosine residue at position 19 by the insulin receptor (8, 41). Furthermore, mammary-derived growth inhibitor (also called heart FABP [46]) and liver FABP appear to be involved in the differentiation of mammary epithelial cells and in the control of hepatocyte cell proliferation, respectively (35,36,60). Some FABPs are located in both the nucleus and the cytoplasm, suggesting that the...