Gene expression patterns in heterozygous Plk4 murine embryonic fibroblasts
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The polo-like kinases (Plks) are a group of serine/threonine kinases which have roles in many aspects of cellular function including the regulation of mitotic activity and cellular stress responses. This study focuses on Plk4, the most divergent member of the Plk family, which is necessary for proper cellular proliferation. More specifically, alterations in Plk4 levels cause significantly adverse mitotic defects including abnormal centrosome duplication and aberrant mitotic spindle formation. We sought to clarify the effect of reduced Plk4 levels on the cell by examining transcript profiles of Plk4 wild-type and heterozygous mouse embryonic fibroblasts (MEFs). Subsequently, the levels of several key proteins involved in the DNA damage response were examined.
143 genes were found to be significantly up-regulated in the heterozygous MEFs compared to their wild-type counterparts, while conversely, 9 genes were down-regulated. Numerous genes with increased transcript levels in heterozygous MEFs were identified to be involved in p53-dependent pathways. Furthermore, examination of the promoter regions of all up- and down-regulated genes revealed that the majority contained putative p53 responsive elements.
An analysis of transcript levels in MEFs after exposure to either ionizing or ultraviolet radiation revealed a significant change between wild type and heterozygous MEFS for Plk4 transcript levels upon only UV exposure. Furthermore, changes in protein levels of several important cell check-point and apoptosis regulators were examined, including p53, Chk1, Chk2, Cdc25C and p21. In heterozygous MEFs, p53, p21 and Chk2 protein levels were at significantly higher levels. Furthermore, p53 activity was increased 5 fold in the Plk4 heterozygous MEFs.
Global transcript profiles and levels of key proteins involved in cellular proliferation and DNA damage pathways were examined in wild-type and Plk4 heterozygous MEFs. It was determined that Plk4 haploinsufficiency leads to changes in the levels of RNA accumulation for a number of key cellular genes as well as changes in protein levels for several important cell cycle/DNA damage proteins. We propose a model in which reduced Plk4 levels invoke an increase in p53 levels that leads to the aforementioned changes in global transcription profiles.
KeywordsIonize Radiation Cell Cycle Profile Supernumerary Centrosome Mouse Embryonic Fibroblast Cell Line Plk4 Activity
Cell division cycle 25 homolog C
Cyclin dependent kinase 8
Casein Kinase II
glyceraldehyde 3-phosphate dehydrogenase
Loss of heterozygosity
Mouse embryonic fibroblasts
Microarray Analysis System
Online Mendelian Inheritance in Man
Polo-like Kinase 4
reverse trancription polymerase chain reaction; Sak-Snk/Plk-akin kinase
Sap 30 binding protein
Squamous cell carcinoma antigen recognized by T-cells
The Institute for Genomic Research
TdT mediated dUTP Nick-End Labeling
University Health Network
Wnt Inducible Signaling Pathway protein.
Plk4 (Sak), is a member of the polo-like kinase (Plk) family of serine/threonine kinases which are involved in the regulation of the cell cycle, cellular response to stress such as DNA damage, and the duplication and maturation of centrosomes [1, 2, 3, 4]. Deregulation of the Plks by overexpression, depletion via epigenetic silencing or loss of heterozygosity (LOH) has implicated them in the development of centrosome abnormalities and has been associated with a CIN (chromosomal instability) phenotype and malignancy. Plk4 is a major regulator of centriole duplication as indicated first by an increase in the number of supernumerary centrosomes correlated with Plk4 overexpression, and second, by a reduction in centriole duplication with the eventual development of mono-polar spindles upon repeated cell divisions observed after RNA interference for Plk4 [5, 6, 7, 8]. Homozygous null Plk4 mice are embryonic lethal at ~E7.5 of development, with an increase in the proportion of mitotic cells, whereas Plk4 heterozygous mice are phenotypically normal . Interestingly aged Plk4 heterozygous mice display haploinsufficiency with tumours developing at a high frequency in major sites such as the liver and lung . Haploinsufficiency for Plk4 affects normal progression through the cell cycle and maintenance of the genome. For example, in a two thirds liver hepatectomy model, Plk4 heterozygous hepatocytes had an increased rate of tri- and tetra-polar spindle complexes with frequent mitotic errors as compared to those form wild-type regenerating livers . At 9–12 months post-hepatectomy all the Plk4 heterozygous mice had abnormal liver morphology and there was an increased rate of tumourigenesis . These results suggest that Plk4 haploinsufficiency potentially leads to increased aneuploidy a likely tumour promoting event. Plk4 loss also has implications in human malignancy, where LOH for Plk4 was found in the majority of a small sample of hepatocellular carcinomas .
Plks 1–3 in general all play important roles in the regulation of the cell cycle and the DNA damage response. Furthermore, several of their respective substrates are in common, with the individual Plks likely placing their substrate under tighter or opposing control. For example, both Plk3 and Plk1 phosphorylate Cdc25C and p53 by targeting different residues in each case. Plk3 phosphorylates Cdc25C on serine 216 , a site that is also targeted by Chk1 and Chk2 [4, 12]. Phosphorylation of serine 216 of Cdc25C is inhibitory, which is due to sequestration of the protein phosphatase in the cytoplasm by 14-3-3 protein, thus blocking mitotic entry . Human Cdc25C is phosphorylated on Ser-198 by Plk1, part of an activation amplification loop that increases the phosphatases activity to allow mitotic entry . Polo-like kinase 1 (Plk1) is known to inhibit p53 function by physical interaction , while phosphorylation of p53 at Ser 20 by Plk3 serves to functionally link DNA damage with increased p53 activity . Chk2 is another protein that is phosphorylated by the Plks. Plk1 interacts with, phosphorylates and colocalizes with Chk1 , Plk3 phosphorylates Chk2 at two residues, which results in subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage, thus upregulating Chk2 activity [18, 19].
Similar to the other Plk family members, which have established roles in DNA damage pathways, Plk4 likely functions within or is a target of DNA damage pathways. This is supported by the observation that Plk4 interacts with and phosphorylates p53 [10, 20]. Plk4 expression is repressed in a p53 dependent manner in response to DNA damaging agents, with the p53 repression of Plk4 activity occurring through the recruitment of a histone deacetylase (HDAC) transcription repressor . Additionally, Cdc25C, a key regulator of the entrance into mitosis and target of DNA damage proteins, is a substrate for Plk4 . Significant phenotypic differences are also observed between Plk4 wild-type and heterozygous mouse embryonic fibroblasts (MEFs) . Contrary to what would be expected, heterozygous Plk4 MEFs display a phenotype typified by multiple centrosomes which lead to multipolar spindles, mitotic failure and delayed proliferation .
All the evidence published to date is consistent with a model as suggested by Habendanck et al (2005) in which reduced Plk4 activity causes occasional cellular division failure as a result of aberrant centrosome duplication and subsequent mitotic spindle malformation, This cell division failure can lead to either aneuploidy or polyploidy, which could in turn contribute to the higher incidence of tumors in heterozygous mice. As an initial step in further characterizing the effect of lower Plk4 levels on the cell, we utilized microarrays to provide a general survey of differences in the transcript profiles of Plk4 wild-type and heterozygous MEFs. Here, we report on a spectrum of genes that are upregulated or downregulated in the Plk4 heterozygous MEFs, including the key cell cycle regulators p53, p21 and chk2 and the presence of increased p53 levels/activity as a result of Plk4 haploinsufficiency.
Establishment of primary mouse embryonic fibroblasts (MEFs)
Mouse embryonic fibroblast cell lines were established from 12.5 day old wild-type and heterozygous Plk4 embryos as previously described . All experiments utilizing mice as well as embryos and cell lines derived from them were performed in accordance to CCAC guidelines and approved by the University of Windsor Animal Care Committee. The MEFs were cultured in Dulbecco's Modified Eagles Medium (Sigma) containing 20% fetal bovine serum (Sigma), 1% penicillin-streptomycin (Gibco) and 250 ug/ml gentamicin (Gibco) and maintained at 37°C with 5% CO2. All experiments were performed with MEFs at passage 2–3.
Wild type and heterozygous mouse embryonic fibroblasts were grown to approximately 80% confluency. The MEFs were then harvested, fixed in 80% ice-cold ethanol, stained with PI and the cell cycle profiles were determined by flow cytometry on a Beckman Coulter Cytomics FC 500 flow cytometer. Flow cytometry results were analyzed using Cytomics RXP Analysis software (Beckman Coulter). Presented results are based on three independent experiments.
MEF cells were grown asynchronously to a confluency of 70–80% with total RNA isolation performed using the RNeasy Mini Kit (Qiagen). In order to confirm the integrity and quality of the RNA, the RNA was run on the 2100 Bioanalyzer (Agilent) using the RNA 6000 Nano Assay Kit. Total RNA extracted from MEFs was subjected to microarray analysis at the University Health Network (UHN) Microarray Centre in Toronto. The samples were labeled using the UHN's standard indirect labeling protocol and hybridized to a Mouse 22.4K chip. Results are based on three independent replicates with subsequent analysis performed using "The Institute for Genomic Research (TIGR) microarray software suite".
Oligonucleotide primer sequences for RT-PCR analysis.
Exposure of MEFs to DNA damaging agents
Wild-type and heterozygous MEFs were exposed to ultraviolet light (UV) at 40 mJ/cm2 using a GS Gene Linker UV Chamber (Biorad) or ionizing radiation (IR) of 25 Gy using a RX-650 Cabinet X-ray System (Faxitron) and RNA or protein was isolated from the MEFs at the specified time points.
Western blot analysis
Cells were lysed in lysis buffer (50 mM Tris-Cl, 100 mM Nacl, 500 mM EDTA, 1% Triton-X), the cell lysate was cleared by centrifugation and equal amount of total protein was loaded into 8% (or 12%) SDS-PAGE gels. Following separation, proteins were transferred onto a PVDF membrane, and Western blot analysis was performed using standard methods. The primary antibodies were as follows, anti-p53 (Sigma), anti-Chk2 (Sigma), anti-Chk1, (Sigma), anti-p21 (BD Pharmingen), anti-Cdc25C (Santa Cruz) and anti-GAPDH (Cell Signalling). The secondary antibodies were as follow: anti-mouse HRP (Amersham), anti-rabbit HRP (Amersham) and were used at dilutions recommended by manufacturers.
The level of apoptosis was determined in heterozygous and wild-type Plk4 MEFs using a TdT-mediated dUTP Nick-End Labeling (TUNEL) assay as per the manufacturer's provided protocol (Promega). Cells were exposed to 40 mJ/cm2 to induce UV mediated DNA damage and analyzed 1 hr, 2 hr, 4 hr, 6 hr, and 8 hr post radiation. Results are based on three independent experiments.
X-gal senescence staining
MEFs were grown to 70–80% confluency and then stained using a β-Galactosidase Staining Kit (Cell Signalling). Cells were washed in PBS, fixed in a 2% formaldehyde solution and incubated overnight in 20 mg/ml X-gal staining solution. Senescent cells were identified by the presence of a typical perinuclear blue stain.
p53 activity assay
The activity of p53 from Plk4 heterozygous and wild-type MEFs was analyzed with the Active p53 Activity Assay Kit (R*#38;D Systems). Cells were grown to 70–80% confluency and 5 ug of nuclear extracts (equal amounts of protein were determined by Bradford assay, Biorad) were subjected to the capture ELISA assay as per the manufacturer's protocol. Absorbance measurements were performed at 450 nm on a Victor 1420 Spectrophotometer. Results are based on three independent experiments and normalized to the wild-type controls.
Results and discussion
Comparison between transcript profiles in wild-type and heterozygous Plk4 MEFs using microarray
The main focus of the present study was to examine global changes in transcript profiles between Plk4 wild type and heterozygous MEFs. In order to accomplish this, we utilized independent cultures of asynchronously growing age matched Plk4 wild-type and heterozygous MEFs in three replicates. Quantification and normalization of the data was performed using "The Institute for Genomic Research" (TIGR) TM4 microarray data analysis suite. Normalization and filtering of the data was performed using the TIGR Microarray Data Analysis System (MIDAS) application. Analysis of all microarray data sets for the different microarray experiments (ex. Wild-type Plk4 MEFs vs Heterozygous Plk4 MEFs) were performed independently.
K-means clustering analysis was performed using TIGR Multiexperiment Viewer (MEV). Within each cluster, genes having a log ratio value greater than 1 or less than -1 on each microarray chip were identified. Genes having a log ratio greater than 1 represented genes in the heterozygous MEFs that have at least a two fold increase in gene expression. As the wild-type MEFs was used as the control, genes with a log ratio greater than 1 were classified as up-regulated in the heterozygous MEFs and genes with a log ratio less -1 represented genes in the wild-type MEFs that have at least a two fold increase in gene expression or are down-regulated in the heterozygous MEFs.
Transcripts up and down regulated in Plk4 heterozygous MEFs.
Putative p53 site
Putative p53 site
Casein kinase II (Csnk2a1)
N-acylsphingosine amidohydrolase (acid ceramidase) like (Asahl)
Protein phosphatase 1F (PP2C domain containing) (Ppm1f)
Leucyl/cystinyl aminopeptidase (Lnpep)
Squamous cell carcinoma antigen recognized by T-cells 1 (Sart1)
Origin recognition complex, subunit 4-like (Orc4l)
L-2-hydroxyglutarate dehydrogenase (L2hgdh)
Inhibitor of DNA binding 2 (Id2)
Fatty acid desaturase 3 (Fads3)
Protein phosphatase 5 (Ppp5c)
Carbohydrate sulfotransferase 2 (Chst2)
heme binding protein 2 (Hebp2)
Stearoyl-Coenzyme A desaturase 1 (Ankrd13c)
CCR4 carbon catabolite repression like 4 (Ccrn4l)
protein kinase, cAMP dependent regulatory, type I beta (Prkar1b)
Cyclin dependent kinase 8 (Cdk8)
Heterogeneous nuclear ribonucleoprotein C (Hnrpc)
Uracil-DNA glycosylase (Ung)
TVMSFG fibroblast growth factor receptor 1 precursor (Fgfr1)
MutS homolog 6 (Msh6)
Phosphatidylinositol 3-kinase (Pic3c2a)
Thymine DNA glycosylase (Tdg)
Pituitary tumor-transforming 1 (Pttg1)
Sal-like 3 (Sall3)
Cysteinyl-tRNA synthetase (Cars)
T-cell factor 4 (Rab27b)
Zinc Finger Protein 451 (Zfp451)
Nuclear receptor co-repressor 1 (Ncor1)
Tetratricopeptide repeat domain 1 (Ttc1)
Inositol 1,4,5-triphosphate receptor 5 (Itpr2)
Highly similar to CBP_MOUSE CREB-binding protein (Crebbp)
Procollagen, type VI, alpha 3 (Col6a3)
Zinc finger protein 689 (Zfp689)
Fetal Alzheimer antigen (Bptf)
Glutamyl-prolyl-tRNA synthetase (Eprs)
WNT1 inducible signaling pathway protein 1 (Wisp1)
GC-rich sequence DNA-binding factor homolog isoform 1 (C21orf66)
T-box transcription factor Tbx15 (Tbx15)
Phenylalanine-tRNA synthetase 2 (Fars2)
Nuclear factor I/X (Nfix)
GLIS family zinc finger 3 (Glis3)
Thrombospondin 2 (Thbs2)
Transmembrane and tetratricopeptide repeat containing 2 (Tmtc2)
Transcription factor A (Tfam)
Fukuyama type congenital muscular dystrophy homolog (Fktn)
SAP30 binding protein (Sap30bp)
SET domain ERG-associated histone methyltransferase (Olfml3)
Syntaxin 18 (Stx18)
Calcium binding and coiled coil domain 1 (Calcoco1)
Solute carrier family 6 (Slc6a6)
Miscellaneous Cellular Functions
Exocyst complex component 3 (Exoc3)
Coiled Coil domain containing 131 (Ccdc131)
Protein-coupled receptor 19 (Gpr19)
Thyroid hormone receptor interactor 11 (Trip11)
Solute carrier family 39 (Slc39a10)
Smg-6 homolog (Smg6)
Frequenin homolog (Freq)
Talin 2 (Tln2)
Serine Hydrolase like (Serhl)
Tomoregulin 1 (Tmeff1)
Solute carrier family 14 (Slc14a2)
Channel-interacting PDZ domain protein (Inadl)
Translocator of inner mitochondrial membrane (Timm17b)
WD repeat domain 50 (Utp18)
Similar to crooked neck protein (Ipo7)
Inositol hexaphosphate kinase 1 (Ip6k1)
Oxysterol binding protein like protein 9 (Osbpl9)
Spetex-2E protein (100040875)
ATPase, Ca++ transporting, plasma membrane 2 (Atp2b2)
Multiple PDZ domain protein (Mpdz)
Transient receptor potential cation channel, subfamily M, member 7 (Trpm7) 58800
Myosin heavy chain 10 (Myh10)
CDC42 effector protein (Rho GTPase binding) 2 (Cdc42ep2)
Zinc finger protein 507 (Zfp507)
aarF domain containing kinase 1 (Adck1)
Procollagen, type III, alpha 1 (Col3a1)
AHNAK nucleoprotein (Ahnak)
Procollagen, type V, alpha 2 (Col5a2)
Ring finger protein 11 (Arhgdia)
Oral-facial-digital syndrome 1 gene homolog (Ofd1)
Procollagen, type I, alpha 2 (Col1a2)
3-phosphoglycerate dehydrogenase (Phgdh)
Arginine/serine-rich coiled-coil 1 (Rsrc1)
Stearoyl-Coenzyme A desaturase 2 (Scd2)
Olfactory receptor 202 (Olfr202)
Mus musculus mVL30-1 retroelement mRNA sequence (mVL30-1)
Discs, large homolog 5 (Dlg5)
Transmembrane protein 34 (Tmem184c)
Mus musculus 0 day neonate cerebellum cDNA (E430024C06Rik)
HD domain containing 3 (Hdcc3)
Hypothetical protein LOC639390 (LOC639390)
Heat shock protein 1(Heatr1)
Myotubularin related protein 7 (Mtmr7)
Mitochondrial ribosomal protein L50 (Mrpl50)
Proteasome(macropain)26S subunit, non-ATPase (Psmd4)
NICE-5 protein (AA414768)
The presumed major cellular function for each down or up-regulated gene was identified using annotation data from the PubMed database, and/or the Online Mendelian Inheritance in Man (OMIM) database. Furthermore, we utilized GenMAPP 2  and Panther  to identify global biological trends in our gene expression data. The few genes that were downregulated in Plk4 heterozygous MEFs functionally included genes involved in development and metabolism. Far more genes were upregulated than were downregulated in the heterozygous MEFs. These included genes with a spectrum of known functions such as cell cycle control, the DNA damage response, DNA repair, epigenetic modification, development, and transcription/translation. In particular, several key genes involved in p53 dependent pathways, Rho signaling, Wnt signaling and the proteasome were upregulated in the heterozygous MEFs. Several of these genes have been implicated in malignancy and are of particular interest given the increased rate of malignancy previously identified in Plk4 heterozygous mice. This includes securin (Pttg1) which serves to prevent premature chromosome separation through inhibition of separase activity. Securin is involved in several key cellular events including mitosis, cell cycle progression, DNA repair and apoptosis. Furthermore, securin (Pttg1) is upregulated in several malignancies and in particularly, pituitary adenomas . Casein Kinase II (Csnk2a1), a serine/threonine kinase is a positive regulator of Wnt signalling pathway that is also upregulated in most cancers . Phosphatidylinositol 3-kinase (Pic3c2a) is an upstream regulator of Akt , both of which are aberrantly regulated in many cancer types and as such are prime targets for intervention , Wisp1 overexpression has been implicated in cellular morphological transformation  and hepatocellular carcinoma .
The observation that the expression levels of genes involved in p53 dependent pathways were altered, coupled with the known interaction of p53 with Plk4, and since changes in p53 levels, like Plk4, may also contribute to centrosome abnormalities [24, 31, 32], led us to further analyze this result. We therefore analyzed the promoter region of both the up and downregulated genes utilizing the MAPPER search engine [33, 34] and found that the majority of these genes contained numerous p53 responsive elements within the first 5 kilobases upstream of the transcriptional start site. Furthermore, several of these upregulated genes are known p53 targets (including msh2 ) or affect the p53 transcriptional machinery (like CDK8 ).
The effect of ionizing and ultraviolet radiation on the Plk4 transcript and protein profiles in MEFs
The effect of UV induced DNA damage on apoptosis and cell cycle profiles in Plk4 MEFs
Senescence and p53 activity in Plk4 MEFs
In most cell types p53 is a potential key regulator of senescence growth arrest, the maintenance of senescence growth arrest and the initiation of the senescence response following DNA damage . In order to address the possibility that elevated p53 levels may be correlated with a senescent phenotype in the Plk4 heterozygous MEFs, we stained the cells for β-galactosidase activity from passages 2–5 (see additional file 1). We found no evidence of increased β-galactosidase activity in the Plk4 heterzoygous MEFs relative to the wild type MEFs; thus suggesting that the elevated p53 protein levels in the Plk4 heterozygous MEFs were not correlated with an increase in cellular senescence.
We propose that the increased p53 protein levels and activity that occur as a result of Plk4 haploinsufficiency may in turn contribute to the over-expression of numerous genes containing p53 responsive elements within their promoters (Figure 5B). The function of these genes encompasses a spectrum of cellular activities including cell cycle control and the response to DNA damage. The results suggest that one function of Plk4 phosphorylation of p53 may be with respect to p53 protein stability and/or activity. In this scenario the possibility exists that this arises through a direct effect in which lower Plk4 levels result in reduced phosphorylation of p53 by Plk4 thus leading to an increase in protein p53 stability and activity. Alternatively, the presence of supernumerary centrosomes seen in Plk4 heterozygous MEFs may lead to an increase in genomic instability and the induction of checkpoints to deal with the ensuing DNA damage. Very few targets for Plk4 have been identified thus far. However, the possibility exists that the increased levels of p53 and phenotypic changes observed occur as a result of indirect consequences of Plk4 haploinsufficiency and targeting of other substrates. For example, one known plausible indirect effect of Plk4 haploinsufficiency could be through Chk2. Plk4 both interacts with and phosphorylates Chk2, a key regulator of the DNA damage response and p53 . Conceivably, reduced phosphorylation of Chk2 as a result of lower Plk4 levels may result in altered Chk2 levels and/or activity towards p53 thus resulting in p53's increased stability or activity. This is consistent with the observation that Chk2 levels are greatly increased in the heterozygous MEFs and the observation that the cell cycle profiles of heterozygous MEFs are altered.
In conclusion, our results demonstrate that Plk4 haploinsufficiency leads to changes in the levels of RNA accumulation for a number of key cellular genes as well as changes in protein levels for several important cell cycle/DNA damage proteins. The majority of the upregulated genes have numerous p53 responsive elements within their promoter regions, thus suggesting that Plk4 haploinsuficiency directly or indirectly leads to an increase in p53 activity in MEFs. Further studies should reveal the nature of the relationship between Plk4 levels, p53 and the down and upregulated genes found in Plk4 heterozygous MEFs.
This work is supported by operating grants to JWH from NSERC and NCIC (Terry Fox Foundation) as well as research equipment grants to JWH from CFI, OIT, NSERC, and NCIC. JN is a recipient of an NSERC undergraduate scholarship. The authors wish to thank A. Swan, B. Crosby and A. Kozarova for helpful suggestions.
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