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Deletion of etoposide-induced 2.4 kb transcript (ei24) reduced cell proliferation and aggregate-size in Dictyostelium
1School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Abstract:
The etoposide-induced 2.4 kb transcript (ei24) gene is induced both by p53 and etoposide, an anti-cancer tumour drug. There is no p53 gene present in Dictyostelium discoideum. Thus, the functions of ei24 in the absence of p53 were analysed. Both overexpressor (ei24OE) and knockout (ei24-) mutants were made to study its role during growth, development and differentiation. Additionally, cell cycle and its response to DNA-damage were also analysed. We identified, characterized and elucidated the functions of the ei24 gene in Dictyostelium. In silico analyses demonstrated the conservation across eukaryotes and in situ hybridization showed it to be prestalk-specific. ei24- cells showed reduced cell proliferation and cell-cohesive properties, ultimately forming small-sized aggregates that developed into miniature and stalky fruiting bodies. The ei24OE cells formed fruiting bodies with engorged or double-decker type sori with short stalks. The ei24- cells showed reduced cAMP signalling with lower intracellular cAMP levels resulting in diminished migration of cells along cAMP gradients. Deletion of ei24 resulted in mis-expression of prestalk-specific markers. Cell cycle analysis revealed an increased bias towards the stalk-pathway by ei24- cells and vice-versa for ei24OE cells. EI24 in Dictyostelium functions even in the absence of p53 and is induced in response to both UV-radiation and etoposide treatments. ei24OE cells showed enhanced DNA-damage repair mechanisms. Also, etoposide treatment and overexpression of ei24 caused G2/M arrest in the cell cycle. Our results indicate that EI24 is important for the growth, development and differentiation of Dictyostelium apart from being a DNA-damage response gene.
Insights
The ei24 gene in Dictyostelium, crucial for development and DNA repair, functions independently of the p53 gene. Its absence impairs cell proliferation and differentiation, while overexpression enhances DNA damage response.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- The etoposide-induced 2.4 kb transcript (ei24) gene is known to be induced by p53 and etoposide, an anti-cancer drug.
- The model organism Dictyostelium discoideum lacks the p53 gene, presenting a unique system to study ei24 function without p53 influence.
Purpose of the Study:
- To identify, characterize, and elucidate the functions of the ei24 gene in Dictyostelium discoideum.
- To investigate the role of ei24 in cell growth, development, differentiation, cell cycle, and response to DNA damage in the absence of p53.
Main Methods:
- In silico analysis for gene conservation.
- In situ hybridization to determine gene expression patterns.
- Creation and analysis of ei24 overexpressor (ei24^OE) and knockout (ei24^-) mutants.
- Cell cycle analysis and DNA-damage response assays.
Main Results:
- In silico analysis revealed ei24 conservation across eukaryotes; in situ hybridization showed it to be prestalk-specific.
- ei24^- mutants exhibited reduced proliferation, cohesion, and cAMP signaling, leading to smaller fruiting bodies with altered morphology.
- ei24^OE mutants formed fruiting bodies with enlarged sori and short stalks.
- ei24^- cells showed mis-expression of prestalk markers and a bias towards stalk-pathway differentiation.
- ei24^OE cells displayed enhanced DNA-damage repair and G2/M cell cycle arrest upon etoposide treatment or overexpression.
- ei24 functions in response to UV radiation and etoposide, independent of p53.
Conclusions:
- EI24 is essential for Dictyostelium growth, development, and differentiation.
- EI24 plays a significant role in the DNA-damage response pathway in Dictyostelium.
- The study elucidates novel functions of EI24 in a p53-independent manner, highlighting its conserved eukaryotic roles.
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