Mitochondrial dysfunction induces SESN2 gene expression through Activating Transcription Factor 4
Alisa A Garaeva1,2, Irina E Kovaleva3, Peter M Chumakov2
1a Department of Bioengineering and Bioinformatics , Lomonosov Moscow State University , Moscow , Russia.
Abstract:
We found that inhibitors of mitochondrial respiratory chain complexes III (myxothiazol) and I (piericidin A) in some epithelial carcinoma cell lines induce transcription of the p53-responsive SESN2 gene that plays an important role in stress response and homeostatic regulation. However, the effect did not depend on p53 because i) there was no induction of p53 after the treatment with piericidin A; ii) after the treatment with myxothiazol the peak of SESN2 gene upregulation occurred as early as 5h, before the onset of p53 activation (13h); iii) a supplementation with uridine that abolishes the p53 activation in response to myxothiazol did not abrogate the induction of SESN2 transcripts; iv) in the p53 negative HCT116 p53 -/- cells SESN2 transcription could be also induced by myxothiazol. In response to the respiratory chain inhibitors we observed an induction of ATF4, the key transcription factor of the integrated stress response (ISR). We found that the induction of SESN2 transcripts could be prevented by the ISR inhibitory small molecule ISRIB. Also, by inhibiting or overexpressing ATF4 with specific shRNA or ATF4-expressing constructs, respectively, we have confirmed the role of ATF4 in the SESN2 gene upregulation induced by mitochondrial dysfunction. At a distance of 228 bp upstream from the SESN2 transcription start site we found a candidate sequence for the ATF4 binding site and confirmed its requirement for the induction of SESN2 in luciferase reporter experiments. We suggest that the upregulation of SESN2 by mitochondrial dysfunction provides a homeostatic feedback that attenuates biosynthetic processes during temporal losses of energy supply from mitochondria thereby assisting better adaptation and viability of cells in hostile environments.
Insights
Mitochondrial dysfunction induces SESN2 gene transcription via ATF4, independent of p53. This stress response aids cell adaptation to low energy environments.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The SESN2 gene is crucial for cellular stress response and homeostasis.
- Mitochondrial dysfunction can impact cellular signaling pathways.
- The role of p53 in stress-induced gene expression is well-established.
Purpose of the Study:
- To investigate the mechanism by which mitochondrial respiratory chain inhibitors induce SESN2 gene transcription.
- To determine the involvement of p53 and the integrated stress response (ISR) in this process.
- To identify the transcription factors and regulatory elements controlling SESN2 expression under mitochondrial stress.
Main Methods:
- Treatment of epithelial carcinoma cell lines with mitochondrial respiratory chain inhibitors (myxothiazol, piericidin A).
- Analysis of p53 and ATF4 activation using Western blotting and qPCR.
- Assessment of SESN2 transcript levels via qPCR.
- Inhibition of ISR using ISRIB and manipulation of ATF4 expression via shRNA and overexpression constructs.
- Luciferase reporter assays to confirm ATF4 binding site functionality.
Main Results:
- Myxothiazol and piericidin A induced SESN2 transcription independently of p53 activation.
- SESN2 induction occurred upstream of p53 activation and in p53-deficient cells.
- Respiratory chain inhibitors triggered the integrated stress response (ISR) and induced ATF4.
- ATF4 was confirmed as the key transcription factor for SESN2 upregulation via ISR.
- A functional ATF4 binding site upstream of the SESN2 gene was identified.
Conclusions:
- Mitochondrial dysfunction upregulates SESN2 transcription through the ATF4-mediated integrated stress response, independent of p53.
- This pathway represents a homeostatic feedback mechanism to attenuate biosynthesis during energy deficits.
- SESN2 induction enhances cellular adaptation and viability in hostile environments characterized by mitochondrial dysfunction.
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