Dose- and time-dependent expression patterns of zebrafish orthologs of selected E2F target genes in response to serum
Ceren Sucularli1, Serif Senturk, Mehmet Ozturk
1Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, 06800, Ankara, Turkey.
Abstract:
Targets of E2F transcription factors effectively regulate the cell cycle from worms to humans. Furthermore, the dysregulation of E2F transcription modules plays a highly conserved role in cancers of human and zebrafish. Studying E2F target expression under a given cellular state, such as quiescence, might lead to a better understanding of the conserved patterns of expression in different taxa. In the present study, we used literature searches and phylogeny to identify several targets of E2F transcription factors that are known to be serum-responsive; namely, PCNA, MYBL2, MCM7, TYMS, and CTGF. The transcriptional serum response of zebrafish orthologs of these genes were quantified under different doses (i.e., 0, 0.1, 1, 3, and 10% FBS) and time points (i.e., 6, 24 and 48 hours, h) using quantitative RT-PCR (qRT-PCR) in the zebrafish fibroblast cells (ZF4). Our results indicated that mRNA expression of zebrafish pcna, mybl2, mcm7 and tyms drastically decreased while that of ctgf increased with decreasing serum levels as observed in mammals. These genes responded to serum starvation at 24 and 48 h and to the mitogenic stimuli as early as 6 h except for ctgf whose expression was significantly altered at 24 h. The zebrafish Mcm7 protein levels also were modulated by serum starvation/replenishment. The present study provides a foundation for the comparative analysis of quantitative expression patterns for genes involved in regulation of cell cycle using a zebrafish serum response model.
Insights
This study investigated serum-responsive E2F target genes in zebrafish fibroblasts, revealing conserved expression patterns with mammals. Zebrafish pcna, mybl2, mcm7, and tyms decreased with lower serum, while ctgf increased, offering insights into cell cycle regulation.
Area of Science:
- Comparative genomics
- Cell cycle regulation
- Cancer biology
Background:
- E2F transcription factors regulate the cell cycle across species.
- Dysregulation of E2F pathways is conserved in human and zebrafish cancers.
- Understanding E2F target gene expression in quiescence aids in comprehending conserved expression patterns.
Purpose of the Study:
- To identify serum-responsive E2F target genes in zebrafish.
- To quantify the transcriptional serum response of zebrafish orthologs of PCNA, MYBL2, MCM7, TYMS, and CTGF.
- To establish a zebrafish model for comparative analysis of cell cycle gene expression.
Main Methods:
- Literature searches and phylogenetic analysis to identify E2F targets.
- Quantitative RT-PCR (qRT-PCR) to measure mRNA expression in zebrafish fibroblast cells (ZF4).
- Exposure to varying fetal bovine serum (FBS) concentrations (0-10%) and time points (6, 24, 48 hours).
Main Results:
- Zebrafish pcna, mybl2, mcm7, and tyms mRNA levels decreased with reduced serum, similar to mammals.
- Zebrafish ctgf mRNA levels increased with reduced serum.
- Gene expression changes were observed at 24 and 48 hours for starvation and 6 hours for stimulation, with ctgf altered at 24 hours.
- Zebrafish Mcm7 protein levels were modulated by serum conditions.
Conclusions:
- Zebrafish exhibit conserved serum-responsive expression patterns for key cell cycle regulatory genes targeted by E2F.
- The study validates zebrafish as a model for comparative analysis of cell cycle gene regulation.
- Findings contribute to understanding conserved mechanisms in cell cycle control and cancer development.
