Ccdc94 Protects Cells from Ionizing Radiation by Inhibiting the Expression of p53

DNA double-strand breaks (DSBs) represent one of the most deleterious forms of DNA damage to a cell. In cancer therapy, induction of cell death by DNA DSBs by ionizing radiation (IR) and certain chemotherapies is thought to mediate the successful elimination of cancer cells. However, cancer cells often evolve to evade the cytotoxicity induced by DNA DSBs, thereby forming the basis for treatment resistance. As such, a better understanding of the DSB DNA damage response (DSB–DDR) pathway will facilitate the design of more effective strategies to overcome chemo- and radioresistance. To identify novel mechanisms that protect cells from the cytotoxic effects of DNA DSBs, we performed a forward genetic screen in zebrafish for recessive mutations that enhance the IR–induced apoptotic response. Here, we describe radiosensitizing mutation 7 (rs7), which causes a severe sensitivity of zebrafish embryonic neurons to IR–induced apoptosis and is required for the proper development of the central nervous system. The rs7 mutation disrupts the coding sequence of ccdc94, a highly conserved gene that has no previous links to the DSB–DDR pathway. We demonstrate that Ccdc94 is a functional member of the Prp19 complex and that genetic knockdown of core members of this complex causes increased sensitivity to IR–induced apoptosis. We further show that Ccdc94 and the Prp19 complex protect cells from IR–induced apoptosis by repressing the expression of p53 mRNA. In summary, we have identified a new gene regulating a dosage-sensitive response to DNA DSBs during embryonic development. Future studies in human cancer cells will determine whether pharmacological inactivation of CCDC94 reduces the threshold of the cancer cell apoptotic response

Doctor of Philosophy

dissertationMany cancers are curative with surgery, chemotherapy, radiotherapy, or a combination thereof. However, a subset of patients' cancers return and form a refractory cancer, which is often resistant to therapies, difficult to treat, and create a poor prognosis for the patient. Therefore, it is necessary to develop therapies that target refractory cancers. To this end, we utilize the forward and reverse genetics of the zebrafish to identify and validate mutations in genes that sensitize cells to ionizing radiation (IR)- induced apoptosis. We determine that loss-of-function mutations in ribonucleic acid (RNA) splicing factors cause an increase in IR-induced apoptosis, specifically in the neural tissue. Curiously, despite the ubiquitous requirement of RNA splicing in all cells, the neural tissue is the most sensitive to disruption of RNA splicing. Previous studies in yeast and cell culture have shown that the disruption of RNA processing, including RNA splicing, can cause R-loops, which are RNA:DNA hybrids that form when the nascent RNA base pairs to the complementary DNA strand. Aberrant R-loop accumulation causes an increase in DNA double-strand breaks (DSBs), and can be resolved by RNaseH1, an enzyme that specifically cleaves the RNA in an RNA:DNA hybrid. Here, we show that disruption of RNA splicing in the zebrafish causes R-loops and DNA DSBs. Expression of a conditional RNaseH1 mitigates the increase in DNA DSBs and sensitivity to IRinduced apoptosis. While we hypothesized that radiosensitivity was through a mechanism iv of increased p53 expression, we show that the increase in p53, seen in most RNA splicing mutants, is neither necessary nor sufficient for radiosensitivity. Our study further emphasizes the link between RNA splicing and the DNA damage response pathway. Furthermore, whereas previous single-cell models of R-loop formation have been unable to recognize tissue-specificity, the zebrafish provides a robust model to study why the loss of a cell-essential gene causes tissue-specific IRsensitivity. Future studies will be necessary to determine mechanisms of tissue-specific radiosensitivity, as well as determining whether targeting RNA splicing factors can be of clinical therapeutic value

Spliceosomal components protect embryonic neurons from R-loop-mediated DNA damage and apoptosis

RNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels

Loss of <i>prp19</i> or <i>plrg1</i> phenocopies, loss of <i>ccdc94</i>.

<p>(A) Wild-type embryos were injected with the indicated morpholinos. RNA was harvested at 30 hpf, reverse transcribed using oligo-dT primers and analyzed for the expression of <i>p53</i> mRNA by qPCR. Expression of the <i>gapdh</i> gene was also analyzed to normalize <i>p53</i> mRNA levels. All values were then compared to the average value from uninjected embryos, which was adjusted to one. (B) RNA from (A) was reverse transcribed with random hexamers, and intron 9 of the <i>p53</i> gene was analyzed by qPCR to determine levels of <i>p53</i> pre-mRNA. Expression of <i>28S</i> RNA was also analyzed to normalize <i>p53</i> pre-mRNA levels. All values were then compared to the average value from uninjected embryos, which was adjusted to one. (C, D) Siblings and mutants from the <i>plrg1(hi3174aTg)</i> line were distinguished by morphology at 30 hpf and collected for analysis. RNA was harvested and analyzed by qPCR as in (A, B). For panels (A–D), error bars represent the standard error of the mean from at least three independent experiments. (E) Wild-type embryos were injected with the indicated morpholinos and analyzed at 30 hpf for p53 protein similar to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g003" target="_blank">Figure 3E</a>. (F) Wild-type embryos were injected with the indicated morpholinos, irradiated (or not) at 24 hpf with 8 Gy and analyzed three hours later by whole-mount immunofluorescence to detect activated Caspase-3. Three independent experiments showed that knockdown of <i>prp19</i> or <i>plrg1</i> radiosensitized the embryos. (G) Activated-Caspase-3-specific immunofluorescence from (F) was quantified as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g001" target="_blank">Figure 1D</a>. (H) Genetic diagram showing that the Ccdc94 and core members of the Prp19 complex inhibit the transcription of <i>p53</i>, and therefore p53-mediated induction of <i>puma</i> expression, and normally restrict IR-induced mitochondrial apoptosis, upstream of Bcl-2. For all relevant panels in this figure, morpholinos were injected at the same concentrations as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g004" target="_blank">Figure 4B-4D</a>. RT; reverse transcriptase, mm; mismatch, <i>ccdc; ccdc94, prp; prp19</i>. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s008" target="_blank">Figure S8</a>.</p

The <i>rs7</i>-mediated radiosensitizing phenotype is caused by an increase in <i>p53</i> mRNA expression.

<p>(A) Thirty hpf <i>rs7</i> siblings and mutants were identified based on morphology (similar to 1C). RNA was isolated, reverse transcribed using oligo-dT primers and analyzed for the expression of <i>p53</i> mRNA by qPCR. Expression of the <i>gapdh</i> gene was also analyzed to normalize <i>p53</i> mRNA levels. All data was then compared to sibling data, which was adjusted to a value of one. (B) <i>rs7</i> siblings and mutants were grown to 24 hpf and analyzed by whole-mount <i>in situ</i> hybridization with a probe complementary to <i>p53</i> mRNA. High levels of <i>p53</i> mRNA expression are evident in neural tissue (arrowheads) and the ICM (arrows). Pictures were taken first, and the embryos were subsequently genotyped to identify mutants and heterozygous or wild-type siblings. (C) RNA from (A) was reverse transcribed with random hexamers, and intron 9 of the <i>p53</i> gene was analyzed by qPCR to determine levels of <i>p53</i> pre-mRNA. Expression of <i>28S</i> RNA was also analyzed to normalize <i>p53</i> pre-mRNA levels. Similar results were obtained from an analysis of intron 4 (data not shown). All data was then compared to sibling data, which was adjusted to a value of one. (D) <i>rs7^+/−;p53^e7/e7</i> fish were incrossed to analyze <i>rs7</i> siblings and mutants in a <i>p53</i> homozygous mutant background. <i>Rs7</i> mutants and siblings were distinguished by morphology since loss of p53 does not prevent the <i>rs7</i>-mediated “curly-up” tail phenotype. RNA was harvested at 30 hpf and analyzed as in (C). (E) Protein was harvested from <i>rs7</i> siblings and mutants, and p53 and control morphants (injected at 400 µM) at 30 hpf and analyzed for p53 and Tubulin (as a loading control). ImageJ software was used to quantify band intensity from film. Shown below the blot are values for p53 divided by values for Tubulin (corresponding to above lanes) with <i>rs7</i> siblings normalized to one. (F) <i>rs7</i> sibling or mutant embryos were irradiated at 24 hpf with 8 Gy IR and analyzed 2 h later by whole-mount <i>in situ</i> hybridization with a probe complementary to <i>puma</i> mRNA. Arrowheads point to <i>puma</i> expression in neural tissue. (G) <i>rs7</i> sibling or mutant embryos in the <i>p53</i> wild-type or homozygous mutant background were irradiated at 24 hpf with 8 Gy IR and analyzed three hours later by immunofluorescence to detect activated Caspase-3. For panels (A), (C), and (D), error bars represent the standard error of the mean from at least three independent experiments. Panels (B) and (F–G) show representative data from at least three independent experiments. RT; reverse transcriptase. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s004" target="_blank">Figures S4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s005" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s006" target="_blank">S6</a>.</p

Identification of <i>radiosensitizing mutation #7</i>.

<p>(A) Wild-type AB strain embryos were exposed to the indicated amount of IR at 24 hpf and visualized by brightfield microscopy 6 hours later. A threshold of IR exists between 8 and 15 Gy that gives rise to obvious cell death (seen as opaque tissue, marked by arrowheads) in the brain. (B) Wild-type embryos (derived from crossing wild-type parents) or <i>rs7</i> mutant embryos (derived from crossing <i>rs7</i> heterozygous parents) were exposed to 8 Gy IR at 24 hpf and visualized by brightfield microscopy 6 hours later. Arrowheads in the <i>rs7</i> mutant mark cell death reminiscent of exposure of wild-type embryos to 15 Gy (as shown in A) (C) Embryos in (B) were sorted by phenotype, fixed at 6 hpIR and analyzed by immunofluorescence to detect activated Caspase-3. The “curly-up” tail phenotype and opaque tissue in the head were used to identify <i>rs7</i> mutants. These phenotypes are present (to different degrees of severity) in both unirradiated and irradiated mutants such that they can be readily distinguished from siblings and wild-type at 30 hpf. After fixation of mutants, tails were clipped to distinguish them from wild-type embryos, which were analyzed in the same tube for Caspase-3 activity. Embryos were grouped for analysis according to whether embryos were irradiated or not, and <i>rs7</i> mutants were identified based on the presence of tail-clips. Arrowheads point to the enhanced apoptosis in the spinal cord of an <i>rs7</i> mutant, and arrows point to increased apoptosis in the ICM region of an <i>rs7</i> mutant. (D) Immunofluorescence in the spinal cords from at least 10 embryos per group in (C) was quantified. wt; wild-type. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s001" target="_blank">Figure S1</a>.</p

The <i>rs7</i> phenotype is caused by a mutation in the <i>ccdc94</i> gene.

<p>(A) The <i>rs7</i> mutation was localized to a 2.1 cM region (compared to the Massachusetts General Hospital genetic map for linkage group 2) as defined by flanking markers z58296 and z7358. (B) Sequencing of candidate genes (based on gene expression profiles) revealed a premature stop codon in the <i>ccdc94</i> gene. Sequence from the <i>ccdc94</i> gene exon 4 is shown for an <i>rs7</i> mutant, an unrelated wild-type AB embryo, and a heterozygous parent of the <i>rs7</i> mutant. Asterisk indicates the <i>rs7</i> point mutation, and box illustrates mutation to a TGA nonsense codon. (C) Wild-type AB strain or <i>rs7</i> heterozygous fish were incrossed, and the progeny were injected with mRNA encoding the indicated genes at the one-cell stage of development. At 24 hpf, embryos were exposed to 8 Gy IR and visualized by brightfield microscopy 6 hours later. (D) Embryos were injected similar to (C) but were not irradiated. Instead, they were left to develop until 48 hpf. (C and D) Because <i>rs7</i> mutant and sibling embryos injected with wild-type <i>ccdc94</i> mRNAs were morphologically indistinguishable, pictures were taken first, and the embryos were subsequently genotyped to identify mutants. (E) Wild-type embryos were injected with 400 µM mismatch morpholino (mm) as a negative control, or a translation-blocking <i>ccdc94</i> morpholino (<i>ccdc94 atg</i>). Embryos were then irradiated at 24 hpf with 8 Gy IR and analyzed three hours later by immunofluorescence to detect activated Caspase-3. (F) Embryos in (E) were quantified similar to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen-1002922-g001" target="_blank">Figure 1D</a>. Comparisons between mismatch morpholino-injected embryos plus and minus IR generated a <i>p</i> value of 0.0114. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s002" target="_blank">Figures S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s003" target="_blank">S3</a>.</p