Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo
© Dickey-Sims et al; licensee BioMed Central Ltd. 2005
Received: 25 May 2005
Accepted: 02 August 2005
Published: 02 August 2005
Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1.
We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription.
Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.
The Runt domain (Runx) is a highly conserved 128 amino acid sequence that defines a metazoan family of sequence-specific DNA binding proteins important for the developmental control of cell fate . Vertebrates have three Runx genes that are essential for the development of specific tissues (blood, bone, and proprioceptive neurons of the dorsal root ganglia, respectively, for Runx1, Runx2, and Runx3), each of which is associated with leukemogenic or carcinogenic mutations [1–4]. Some of these mutations cause loss of Runx function while others are gain-of-function; hence, mammalian Runx genes display attributes of both tumor suppressors and proto-oncogenes . Runx proteins have been shown to regulate cell cycle transit via their function as context-dependent transcriptional regulators. For example, Runx1 stimulates the G1 to S phase transition in cultured mammalian cells, which may in part reflect its transcriptional activation of cyclin D3 [5–7]. Runx1 is also an transactivator of the p14ARF tumor suppressor , while both Runx1 and Runx2 are transcriptional repressors of the p21Cip1/Waf1 cyclin-dependent kinase inhibitor [9, 10]. These findings indicate that Runx is a critical node in the gene regulatory network that controls cell proliferation and/or survival during animal development. Since many of the core linkages in this network are likely to be conserved throughout animal evolution, identification and cis-regulatory analysis of relevant Runx target genes in experimentally tractable animal models should help illuminate how Runx genes contribute to human development and health.
The sea urchin embryo is optically accessible and highly amenable to experimental manipulations such as gene transfer and morpholino antisense mediated knockdown, making it a useful system for discovering fundamental roles played by Runx proteins in the cell biology of animal development. Whole genome sequence data  as well as genomic Southern blot analysis  suggest that, contrary to previous reports [13–15], the sea urchin Strongylocentrotus purpuratus has two Runx genes (A. Poustka, personal communication; AJR and JAC, unpublished results). Only one of these (SpRunt-1, previously referred to as SpRunt) is expressed in the embryo [12, 14]. SpRunt-1 is required for embryogenesis beyond blastula stage  and contributes to transcriptional activation of the CyIIIa actin gene, a marker of aboral ectoderm differentiation . SpRunt-1 mRNA is expressed globally in the early embryo, whereas in the larva it accumulates predominantly in oral ectoderm and endomesoderm, tissues associated with continued growth and cell proliferation . Embryos depleted of SpRunt-1 by morpholino antisense oligonucleotides (MASOs) that sequence-specifically block either translation or pre-mRNA splicing fail to gastrulate normally, display global defects in cell differentiation and proliferation and underexpress a number of genes including that encoding the conventional protein kinase C SpPKC1 .
Here we provide evidence that the gastrulation defect and abnormal proliferation associated with SpRunt-1 deficiency are secondary effects of extensive apoptosis that occurs in the post-blastula stage embryo. We further demonstrate that apoptosis is caused by a deficit in SpPKC1 activity, and that cell survival in SpRunt-1 morphant embryos can be rescued by exogenous SpPKC1 mRNA. Finally, we show that the SpPKC1 promoter region contains SpRunt-1 target sequences that are required for its transcriptional activation.
Results and Discussion
SpRunt-1 deficiency leads to apoptosis and secondary proliferation
Rescue of gastrulation in SpRunt-1 morphants by Ac-DEVD-CHO. Embryos injected with SpRunt-1 MASO were divided into two groups, one of which was treated with Ac-DEVD-CHO as described in the Methods. At 48 h the embryos were observed under a stereomicroscope and scored as gastrulation defective (with a typical morphant phenotype similar to that depicted in Figure 2A), rescued gastrulation (with guts but no skeletons, a phenotype similar to that depicted in Figure 2D), or normal (indistinguishable from uninjected control embryos with skeletons). The latter group probably represents embryos that were under injected; note that this particular experiment had an unusually high proportion of such embryos (which more typically are in the range of 10–15%).
SpRunt-1 MASO alone (n = 259)
SpRunt-1 MASO + Ac-DEVD-CHO (n = 158)
Apoptosis associated with SpRunt-1 deficiency is caused by loss of PKC expression
Rescue of gastrulation in SpRunt-1 knockdown embryos by SpPKC1 mRNA. Zygotes were coinjected with SpRunt-1 MASO and varying amounts of SpPKC1 mRNA in two different experiments. At 48 h embryos were observed under a stereomicroscope and scored for successful gastrulation (fully invaginated guts, with a phenotype similar to that depicted in Figure 2F). The SpPKC1 [mRNA] refers to the concentration of the injection solution, approximately 2–5 pl of which was injected into each zygote.
SpPKC1 [mRNA] (ng/μl)
SpRunt-1 target sequences in the SpPKC1promoter region are required for transcriptional activation
The relatively weak activation of the reporter conferred by the 1.9 kb SpPKC1 promoter fragment, together with our previous results showing that endogenous SpPKC1 expression is reduced approximately seven fold in SpRunt-1 morphant embryos , suggests that there may be additional enhancers outside of the 1.9 kb fragment that collaborate with SpRunt-1 in activating transcription of the endogenous gene. Testing this possibility would require cis-regulatory analysis using a larger genomic fragment that contains the SpPKC1 locus. Nevertheless, the data presented here support the proposition that the direct interaction of SpRunt-1 with its target sequences in the SpPKC1 cis-regulatory domain is required for normal levels of SpPKC1 transcription in the post-blastula stage embryo, which in turn is required to maintain PKC activity at levels sufficient to antagonize caspase activity and suppress apoptosis (Figure 4E).
Our results support the idea that, in animal development, cells do not survive by default but rather by actively suppressing apoptosis in response to permissive cell signalling [25, 26]. Furthermore, we find that deregulated apoptosis can lead to secondary cell proliferation, similar to the compensatory cell proliferation described by others [27, 28]. In contrast to the latter phenomenon however, we do not observe overproliferation caused by apoptotic cell signalling in the caspase-3 inhibited embryos. While the specific role of SpPKC1 in suppressing apoptosis is not known, there are several plausible pathways based on well-known functions of mammalian PKCα, most notably its role in phosphorylating and thereby activating the anti-apoptotic protein Bcl-2. [29–31]. Identification of the relevant substrate(s) and pathway(s) in sea urchins awaits further analysis, as does the basis for the secondary proliferation observed in SpRunt-1 morphants.
In addition to the conventional PKCs (cPKCs), the PKC family also includes non-conventional and atypical PKCs (nPKCs and aPKCs, respectively) . We do not yet know how many PKC family members are in S. purpuratus, although available expressed sequence tag (EST) and genomic sequence data suggest that SpPKC1 is the only cPKC gene expressed in the embryo, and possibly the only cPKC gene encoded in the genome (JAC, unpublished results). We note in closing that mammals have three cPKC genes, PKCα, PKCβ, and PKCγ. PKCβ is actually pro-apoptotic in myeloid cells, and its gene was recently shown to be directly activated by a human Runx homolog, RUNX1 . This suggests that Runx-mediated control of cPKC gene expression may be a primitive conserved character (i.e., plesiotypic) in animals, whereas the functional output of this interaction (cell death or survival) varies with context, something that would bear on whether a given Runx gene is an oncogene or tumor suppressor. It will therefore be interesting to determine whether the other mammalian cPKC genes are also direct Runx regulatory targets, and whether the Runx-dependency of cell survival in Drosophila  is a function of PKC.
Animals and gametes
Adult Strongylocentrotus purpuratus were purchased from either Charles Hollahan (Santa Barbara, California, USA) or Pat Leahy (Corona del Mar, California, USA). Gametes were obtained by vigorous shaking of animals or by intracoelomic injection of 0.5 M KCl.
Morpholino antisense oligonucleotides
Morpholino antisense oligonucleotides (MASOs) were purchased from GeneTools, LLC (Philomath, Oregon, USA). Nonspecific control and splice-blocking SpRunt-1 (m5) MASOs were as described previously . The sequence of the SpPKC1 MASO, which targets the AUG start codon of SpPKC1 mRNA, is: CATCTCAAACGACGAATCCGACATT.
Microinjection and embryo culture
Microinjection of zygotes was carried out by the method of McMahon et al. , essentially as described in Kirchhamer et al. . MASO injection solutions contained 500 μM MASO and 120 mM KCl. Embryos were cultured in artificial seawater at 15°C.
BrdU labeling and antibody staining
Late gastrula stage embryos (44 h) were incubated in 300 μg/ml BrdU (Sigma-Aldrich; St. Louis, Missouri, USA) for four hours in artificial seawater (ASW) at 15°C then fixed in 4% formaldehyde in ASW for 90 min on ice. After the embryos were washed three times in ASW, they were treated with 2 M HCl on ice for 30 min, neutralized in 100 mM Tris, pH 9.5, 100 mM NaCl for 2 min on ice, and washed three times in phosphate buffered saline containing 0.2% Tween-20 (PBST). The embryos were post-fixed in 4% formaldehyde in PBST for 20 min on ice, and washed three times in ice-cold PBST. BrdU was detected by staining with an AlexaFluor 647-conjugated anti-BrdU antibody (Molecular Probes, Eugene, Oregon, USA) diluted 1:20 in PBST and incubated overnight at 4°C. Digital images were collected using a Leica TCS SP2 confocal microscope.
Antibodies directed against activated caspases 3 and 6 were obtained from Calbiochem (San Diego, California, USA). Embryos were stained with 5 μg/ml antibody in PBST with 5% bovine serum albumin (BSA) overnight at 4°C and then washed three times in PBST. Antibody labeling was detected by staining with AlexaFluor 488 goat anti-mouse IgG from Molecular Probes in PBST with 5% BSA for 1 h at room temperature. Following three washes in PBST and nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI), the digital images were obtained with the Leica TCS SP2 confocal microscope.
Terminal transferase-mediated dUTP nick end labeling (TUNEL) assay
Late gastrula stage embryos (48 h) were fixed for 1 h in 4% paraformaldehyde at 4°C and washed for 1 h in PBST. Staining was performed using the In Situ Fluorescein Detection of Cell Death Kit from Roche (Indianapolis, Indiana, USA). Stained embryos were washed three times in PBST and labeled once in DAPI. Digital images were collected using a Leica TCS SP2 confocal microscope.
DNA quantitation by fluorometry
The DNA content of isolated nuclei was quantitated by fluorometry as described previously .
The caspase-3 selective inhibitor Ac-DEVD-CHO (BD Biosciences; San Jose, California, USA) was added to SpRunt-1 morphant cultures at a final concentration of 50 μM at the 1–2 cell stage.
The DNA polymerase inhibitor aphidicolin (Sigma-Aldrich) was added to SpRunt-1 morphant cultures at a final concentration of 0.2–0.5 μg/ml at mesenchyme blastula stage (24 h post-fertilization).
The PKC inhibitor chelerythrine (Sigma-Aldrich) was added to embryo cultures at concentrations ranging from 2–4 μM from the mesenchyme blastula stage on. Higher concentrations tended to be embryonic lethal whereas lower ones gave no phenotype at all. Since the efficacy of the drug in producing the gastrulation-defective phenotype varied from culture to culture, the optimal concentration for producing the gastrulation-defective phenotype at a frequency close to 100% was determined empirically for each experiment. For example, in one experiment, 85% of embryos treated with 3.5 μM chelerythrine were viable but gastrulation defective (the remaining 15% appearing normal) whereas 100% of embryos treated with 4 μM chelerythrine were viable but gastrulation defective. For other cultures the effective dose for a similar effect was somewhat lower.
Full-length SpRunt-1 mRNA for rescue experiments was synthesized as previously described . SpPKC1 mRNA for rescue experiments was obtained as follows: an EST (National Center of Biotechnology Information (NCBI) accession number CD290393) containing the SpPKC1 start codon was obtained by BLAST using the Genbank entry of Lytechinus pictus PKC1 (Genbank:U02967; ) as a query sequence. The SpPKC1 coding sequence was then assembled from the S. purpuratus genome traces using the LpPKC1 sequence as a scaffold. The assembled SpPKC1 sequence was used to design the following primers: GTGGATAATTTTTGCTGGATTTGCGCCTTGA, (forward; ends 13 bp upstream of start codon) and GTTCTACAGACAGACTGGCACTAGCT (reverse; reverse complement of stop codon underlined). These primers were used in a reverse transcription (RT)- PCR reaction using the Platinum Quantitative RT-PCR ThermoScript One-Step System from Invitrogen (Carlsbad, California, USA) and 200 ng total RNA (purified using the RNeasy Mini Kit from Qiagen; Valencia, California, USA) from 60 h embryos. An amplicon of the expected size was gel purified and inserted to pGEM-T Easy (Promega; Madison, Wisconsin, USA). Sequencing was performed to determine the insert orientation and also to confirm the identity of the PKC1 sequence. An Apa1-Spe1 restriction fragment containing the full length SpPKC1 sequence with some flanking sequence was then transferred into a pBluescript (Stratagene; LaJolla, California, USA) expression construct containing the Xenopus BetaGlobin 3' UTR and polyA sequence (pGloBlu) . Pst1 was used to linearize the resulting construct for in vitro transcription of SpPKC1 using the T7 mMessage Machine from Ambion (Austin, Texas, USA).
Assembly of the SpPKC15' flanking sequence
A contiguous sequence of over 2,500 bases extending in the upstream direction relative to the start codon of SpPKC1 was assembled from trace sequences obtained from the Sea Urchin Genome Project  using the ContigExpress program in the InforMax Vector NTI suite (Invitrogen). Genotrace , which supports the reassembly of short spans of genomic DNA sequence from trace files, was used to generate independently a contig for comparison with the manually assembled one in ContigExpress. The sequence was verified by PCR amplification from genomic DNA (see below).
Electrophoretic mobility shift assay
Cy5 5'-modified oligonucleotides containing the Runx target sequences from the SpPKC1 gene were used as probes (see Additional file 2). The binding reaction contained 25 mM HEPES, pH 7.5, 75 mM NaCl, 10 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 0.3 mg/ml BSA, 10 μg/ml poly dIdC/dIdC and 8% glycerol. Approximately 10 nmol of purified recombinant protein was incubated for 15 min at room temperature with or without competitor and then incubated an additional 15 min at room temperature upon addition of the probe. Specificity of binding was determined by the addition of 100-fold molar excess of unlabeled competitor DNA corresponding to Runx target sequence from CyIIIa . DNA-protein complexes were resolved on a 5% (w/v) nondenaturing polyacrylamide gel using 0.5× TBE running buffer at 150 V for 3 h at room temperature. Gels were imaged on a Typhoon 8700 (APBiotech; Piscataway, New Jersey, USA).
Chromatin immunoprecipitation (ChIP)
Late gastrula stage embryos were dissociated into single cells by a washing once in dissociation buffer (1 M glycine pH 8, 2 mM EDTA) and then three times in calcium-free ASW. The dissociated cells were resuspended in a hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl and protease inhibitors) and incubated for 15 min on ice. Igepal (10% solution) was then added to a final concentration of 0.6%, and the suspension was vortexed to release nuclei. Nuclei were recovered by centrifugation and resuspended in 20 mM HEPES, pH 7.9, 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, 20% glycerol and protease inhibitor cocktail (Roche). Formaldehyde was then added to a concentration of 1%, and the nuclear suspension was incubated for 10 min at room temperature to crosslink the chromatin. After stopping the crosslinking reaction with the addition of glycine to 125 mM, the nuclei were recovered by centrifugation, resuspended in a nuclear lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS and protease inhibitors) and sonicated with a force empirically determined to produce chromatin with an average fragment size of ~500 base pairs. The sonicated chromatin solution was then precleared with Staphylococcus aureaus protein A positive cells (Staph A) and aliquoted.
Prepared aliquots of chromatin were diluted with immunoprecipitation (IP) buffer (16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS and protease inhibitors) and incubated with equivalent amounts of either nonimmune or anti-SpRunt-1 IgG overnight at 4°C. The antibody complexes were recovered by precipitation with Staph A, and, following several washes of the precipitated Staph A with IP wash buffer (100 mM Tris-HCl, pH 9, 500 mM LiCl, 1% NP-40, 1% deoxycholic acid, and protease inhibitors), eluted with an SDS elution buffer (50 mM NaHCO3, 1% SDS). Following RNAase A addition (to 33 μg/ml), crosslink reversal by heat treatment (67°C for 5 h), ethanol precipitation and proteinase K treatment (240 μg/ml for 2 h at 45°C), the DNA from the immunoprecipitates was purified using the QIAquick PCR purification Kit (Qiagen), and used as template in PCR reactions with the following primers: CyIIIa: GTAGCACACGGAGAGATTGTGGGACAT (forward) and GGATCGGGGTTAGAGTTACATTTGGCTT (reverse); SpPKC1: CGAAACACACCACCAGCAACTTGCA (forward) and CCAATGGTCTTCAACACACACTTCGCTA (reverse); and H4: GTCGAGGAAAAGGAGGAAAGGGACTCGGAA (forward) and TGCTCGCAGTAGGTGACTGCATCACGGAT (reverse).
SpPKC1:luciferase reporter gene construction and analysis
A 2.1 kb fragment of the upstream region of SpPKC1 was PCR amplified from genomic DNA, subcloned into pGEM-TEasy (Promega), and sequenced (see Additional file 2). The fragment was subsequently inserted into the pGLII Luciferase reporter plasmid (Promega) to generate the SpPKC1-Luc reporter. The primers used for PCR, GGGGTACC TGCACCCAATCCACACCCACTAAGA (forward) and CGGG ATCCACTGTCAAGGCGCAAATCCAGCAA (reverse), contain Kpn1 and BamH1 sites, respectively, which are compatible with the multiple cloning sequence in pGLII. The Quikchange Site Directed Mutagenesis Kit (Stratagene) was used to mutate each of the SpRunt-1 target sites within the SpPKC1-Luc reporter, using primers with the following sequences: ATCTCACCATAACgAtATAATTAGGGTTAGGAA (distal site forward), TTCCTAACCCTAATTATaTcGTTATGGTGAGAT (distal site reverse), TCATTCCTTTACATTTACgttAGTAGAAGTCAA (proximal site forward) and TTGACTTCTACTaacGTAAATGTAAAGGAATGA (proximal site reverse). The base substitutions introduced by these primers (shown in lower case) are known to abolish Runx binding  and were verified by sequencing of the resulting clones. Expression of SpPKC1-Luc and each Runx-site mutant was measured by quantitative real time RT-PCR essentially as described previously , and normalized to luciferase DNA content using the method of Revilla-i-Domingo et al . The primers used to detect luciferase mRNA were GGGATTTCAGTCGATGTACACGTTCGTCACATCT (forward) and GGCATGCGAGAATCTGACGCAGGCAGTTCTAT (reverse).
We thank Drs. Robb Krumlauf and Chunying Du for helpful critiques of the manuscript prior to its submission, the two anonymous reviewers for comments that improved the quality of the manuscript, and the Molecular Biology Core of the Stowers Institute for DNA sequencing. This work was supported by the Stowers Institute for Medical Research and by a grant from the National Institutes of Health (GM070840).
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