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Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models
Published on: January 12, 2015
Gene expression patterns in heterozygous Plk4 murine embryonic fibroblasts
Alan Morettin1, Alejandra Ward, Jordan Nantais
1Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada. morettia@uwindsor.ca
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.
Insights
Reduced polo-like kinase 4 (Plk4) levels in mouse cells disrupt cell division and activate DNA damage responses. This haploinsufficiency increases p53 protein levels and alters gene expression, impacting cellular proliferation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Polo-like kinases (Plks) regulate mitosis and stress responses.
- Plk4, a divergent Plk, is crucial for cell proliferation.
- Altered Plk4 levels cause mitotic defects, including abnormal centrosome duplication and spindle formation.
Purpose of the Study:
- To investigate the effects of reduced Plk4 levels on cellular function.
- To analyze transcript profiles of Plk4 wild-type and heterozygous mouse embryonic fibroblasts (MEFs).
- To examine the protein levels of key DNA damage response regulators.
Main Methods:
- Comparative transcript profiling of Plk4 wild-type and heterozygous MEFs.
- Analysis of gene promoter regions for p53 responsive elements.
- Quantification of key cell cycle and apoptosis regulator protein levels (p53, Chk1, Chk2, Cdc25C, p21).
Main Results:
- 143 genes upregulated, 9 downregulated in heterozygous MEFs.
- Upregulated genes are enriched in p53-dependent pathways.
- Increased protein levels and activity of p53, Chk2, and p21 in heterozygous MEFs.
Conclusions:
- Plk4 haploinsufficiency alters RNA accumulation and protein levels of cell cycle/DNA damage proteins.
- Reduced Plk4 induces elevated p53 levels.
- A model is proposed where reduced Plk4 increases p53, leading to global transcription changes.
