p53 Activation by Knockdown Technologies

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies

p53 Activation by Knockdown Technologies

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies

Rereplication in <em>emi1</em>-Deficient Zebrafish Embryos Occurs through a Cdh1-Mediated Pathway

<div><p>Disruption of early mitotic inhibitor 1 (Emi1) interferes with normal cell cycle progression and results in early embryonic lethality in vertebrates. During S and G2 phases the ubiquitin ligase complex APC/C is inhibited by Emi1 protein, thereby enabling the accumulation of Cyclins A and B so they can regulate replication and promote the transition from G2 phase to mitosis, respectively. Depletion of Emi1 prevents mitotic entry and causes rereplication and an increase in cell size. In this study, we show that the developmental and cell cycle defects caused by inactivation of zebrafish <em>emi1</em> are due to inappropriate activation of APC/C through its cofactor Cdh1. Inhibiting/slowing progression into S-phase by depleting Cdt1, an essential replication licensing factor, partially rescued <em>emi1</em> deficiency-induced rereplication and the increased cell size. The cell size effect was enhanced by co-depletion of cell survival regulator <em>p53</em>. These data suggest that the increased size of <em>emi1</em>-deficient cells is either directly or indirectly caused by the rereplication defects. Moreover, enforced expression of Cyclin A partially ablated the rereplicating population in <em>emi1</em>-deficient zebrafish embryos, consistent with the role of Cyclin A in origin licensing. Forced expression of Cyclin B partially restored the G1 population, in agreement with the established role of Cyclin B in mitotic progression and exit. However, expression of Cyclin B also partially inhibited rereplication in <em>emi1-</em>deficient embryos, suggesting a role for Cyclin B in regulating replication in this cellular context. As Cyclin A and B are substrates for APC/C-Cdh1 - mediated degradation, and Cdt1 is under control of Cyclin A, these data indicate that <em>emi1</em> deficiency-induced defects <em>in vivo</em> are due to the dysregulation of an APC/C-Cdh1 molecular axis.</p> </div

Knockdown of <i>cdt1</i> partially ablates rereplication and increased cell size in <i>emi1</i> morphants.

<p>(A) Cell death assay in 4–5 somite embryos injected with the indicated morpholinos. C- indicates the mismatch control morpholino for the indicated gene. We injected 2 ng <i>emi1</i> MO or C-<i>emi1</i> MO, 2.7 ng <i>cdt1</i> MO or C-<i>cdt1</i> MO, 7 ng <i>p53</i> MO or C-<i>p53</i> MO per embryo from a cocktail mix. Cell death was detected by immunoflurescence staining of activated Caspase 3. Note the high levels of activated Caspase 3 in <i>cdt1</i> morphants and <i>cdt1 emi1</i> double morphants. Co-knockdown of <i>p53</i> significantly alleviated the cell death in all cases. (B) Representative PI-based cell cycle analysis of total cells from pools of 4–5 somite embryos injected with the indicated morpholinos (same doses as in A). Note that <i>cdt1</i> morpholino partially, but significantly rescued the cell cycle defects in <i>emi1</i> morphants. The 4 panels on the right show no significant effect of <i>p53</i> knockdown on cell cycle distribution in embryos injected with <i>cdt1</i> or <i>emi1</i> morpholinos or both. (C) Normalized average cell size based on FACS analysis of total cells from the indicated morphants from 10 independent experiments. We removed the highest and lowest value for each sample and averaged data from 8 experiments. (D) Summary of the cell cycle and cell size distribution at different phases of the cell cycle. Top panels were obtained from <i>p53</i> wild-type embryos, bottom panels show data from <i>p53</i> morphant embryos. The legend indicates the morphant populations.</p

<i>CYCLIN A2</i> and <i>CYCLIN B1</i> expression partially inhibits rereplication in <i>emi1</i>-deficient cells.

<p>(A) Amino acid alignment of the dead box domain of wild-type human CYCLIN A2 and CYCLIN B1 and dead box (DB) mutant proteins. (B) Expression of <i>CYCLIN A</i>-DB and <i>CYCLIN B</i>-DB GFP fusion constructs at 5 somites, as visualized by fluorescence microscopy. Embryos injected with either DNA construct showed significant and pervasive mosaic expression at this stage and the level of expression was not altered by co-injection of <i>emi1</i> MO. (C) Representative example of FACS scanning of propidium iodide (PI)-stained total cells from embryos injected with the indicated morpholinos and DNA constructs. Note the partial decrease in >G2 population in embryos injected with <i>emi1</i> MO and <i>CYCLIN A</i>-DB as compared to <i>emi1</i> MO only (quantitation in panel D). Also, embryos injected with <i>emi1</i> MO and <i>CYCLIN B</i>-DB showed a partially decreased >G2 population and a partially increased G1 population as compared to <i>emi1</i> MO only (quantitation in panel D). (D) Quantitation of the cell cycle distribution in embryos injected with the indicated morpholinos and DNA constructs. There was a significant difference between the percentage of cells with >G2 DNA content in embryos injected with <i>emi1</i> MO and <i>CYCLIN A</i>-DB as compared to <i>emi1</i> MO only. Also, there was a significant difference between the percentage of cells with >G2 DNA content and in G1 phase in embryos injected with <i>emi1</i> MO and <i>CYCLIN B</i>-DB as compared to <i>emi1</i> MO only. The results shown are average of 3 independent experiments and error bars indicate standard deviation. (E) Normalized average cell size in embryos injected with the indicated morpholinos and DNA constructs, as indicated by FSC of total cells in the FACS analysis. There was no significant decrease in the size of cells in embryos injected with <i>emi1</i> MO and <i>CYCLIN A</i>-DB or <i>CYCLIN B</i>-DB as compared to <i>emi1</i> MO only. If anything, there was a slight but significant increase in cell size of embryos injected with <i>emi1</i> MO and <i>CYCLIN A</i>-DB as compared to <i>emi1</i> MO only.</p

Developmental time course of <i>emi1</i> morphant cell cycle defects.

<p>FACS scanning of propidium iodide (PI)-stained total cells from embryos injected with control (C, in black) or <i>emi1</i>-specific morpholinos (in red) (2 ng per embryo). Each panel shows an overlay of the distribution of control and <i>emi1</i>-morphant cells. Age and developmental stage of embryos is indicated. The insert shows the percent of cells with 2 n DNA content (G0/G1), cells replicating their DNA (S), cells with 4 n DNA content (G2/M) and cells with greater than 4 n DNA content. The percent of cells in each stage has been estimated with the Watson mathematical model in Flowjo software, except for the 4 hpf and 7 hpf time points for each we have assigned the gates for each cell cycle stage.</p

Cellular and developmental defects caused by <i>emi1</i> knockdown are due to <i>cdh1</i> activity.

<p>(A and B) Phase-contrast analysis of embryos at 24 hpf. (A) Morpholino injected embryos, where C- indicates the mismatch control morpholino for the indicated gene. We injected 1 ng of control and <i>cdh1</i> morpholinos per embryo. The <i>emi1</i> morpholino was injected at 1 ng (low) or 2.7 ng (high) per embryo. (B) Genotyped wild-type siblings (+/+) and homozygous <i>emi1</i> mutant embryos (m/m) injected with 2 ng control or <i>cdh1</i> morpholinos as indicated. Note the wild-type appearance of homozygous mutants and <i>emi1</i> morphants that were injected with <i>cdh1</i> morpholino. (C) Cell cycle analysis by FACS scanning of propidium iodide (PI)-stained cells. Total cells were analyzed from single cell suspensions of pools of the indicated morpholino-injected embryos at 4–5 somites. A representative experiment is shown. (D) The average cell size from FACS analysis of cells from morpholino-injected disaggregated embryos at 4–5 somites, normalized to the average control cell size (C, arbitrarily set at 1.00). Error bars indicate standard deviation (SD). Cell size data and SD were obtained from 3 independent experiments.</p

Genome-wide reverse genetics framework to identify novel functions of the vertebrate secretome

BACKGROUND: Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown. RESULTS: We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies. CONCLUSION#ENTITYSTARTX00028;S#ENTITYSTARTX00029;: This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.status: publishe