Gene expression patterns in heterozygous Plk4 murine embryonic fibroblasts

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Cell cycle profile of Plk4 heterozygous and wild-type MEFs. Presented are typical cell cyle profiles of asynchronous populations of Plk4 heterozygous and wild-type MEFs based on flow cytometry of propidium iodide stained cells. The data is representative of three independent experiments.

Figure 2

Comparison of transcript levels between Plk4 heterozygous and wild-type MEFs. A) Confirmation of microarray results. Semi-quantitative RT-PCR was carried out to measure the relative difference in the expression of Plk4 and the candidate genes prohibitin, wisp1 and sap30bp in Plk4 wild-type and heterozygous MEFs. Values in each case were normalized to the levels of GAPDH transcript. Presented data are a result of three independent experiments. B) Relative levels of Plk4 transcript post ionizing radiation (IR). MEFs were exposed to 25 Gy IR and RNA was isolated at the indicated time points post exposure followed by RT-PCR to measure the relative abundance of Plk4 transcript (as above). C) Relative levels of Plk4 transcript post ultraviolet radiation (UV). MEFs were exposed to 40 J/Cm² UV and RNA was isolated at the indicated time points post exposure followed by RT-PCR to measure the relative abundance of Plk4 transcript (as above).

Figure 3

Comparison of protein levels upon DNA damage between heterozygous and wild-type MEFs. Heterozygous and wild-type Plk4 MEFs were exposed to 25 Gy IR or 40 mJ/cm² UV. Six hours post exposure cell extracts were subjected to Western Blot analysis. Shown are representative data from 3 repeats. GAPDH was used as a loading control to ensure equal protein loading.

Figure 4

Assessment of levels of apoptosis and cell cycle arrest in heterozygous and wild-type Plk4 MEFs. A.) Levels of apoptosis after UV induced DNA damage in Plk4 heterozygous and wild-type MEFs. Apoptosis was analyzed prior to and after UV induced DNA damage (40 mJ/cm²) with a TUNEL assay. The results are presented as a percentage of apoptosis positive cells and are representative of three independent experiments. B.) Effect of UV induced DNA damage in Plk4 heterozygous and wild-type MEFs on progression through the cell cycle. Cell cycle profiles were analyzed prior to and after UV induced DNA damage (40 mJ/cm²) by flow cytometry after staining of the DNA with propidium iodide. The population of cells in G₀/G₁, S, G₂/M and Sub G₀ are displayed as percentage and are representative of three independent experiments.

Figure 5

p53 activity and model of Plk4 and p53 interactions in Plk4 heterozygous MEFs. A.) Effect of Plk4 haploinsufficiency on p53 activity. p53 transcriptional activity was analyzed in Plk4 heterozygous and wild-type MEFs from nuclear extracts using a capture ELISA assay. The p53 specificity was confirmed upon incubation with competing labeled and unlabeled oligonucleotides. The results were normalized against wild-type control and are representative of three independent experiments. B.) Model of p53 and Plk4 regulation in Plk4 heterozygous and wild-type MEFs. Shown are proposed Plk4/p53 direct/indirect interactions in Plk4 wild-type and heterozygous MEFs with the resultant corresponding changes in protein levels, activity and gene expression. Solid lines indicate pathways that are functionally active, whereas dotted lines indicate pathways with reduced activity.