The regulation of the DNA methylation in the ovaries of mice under 23-days antiorthostatic suspension
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1
Institute of Biomedical Problems (RAS), Russia
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2
I.M. Sechenov First Moscow State Medical University, Russia
Introduction
Weightlessness has a negative impact on various systems of the human body, including on the cardiovascular, muscle and skeletal systems, which can impede the deep space exploration (Watenpaugh D.E., Hargens A.R., 1996; Riley D.A. et al., 2000; Vico L., Hargens A., 2018). Despite the long history of research, the mechanisms of perception and transduction of mechanical stresses at the cellular level have not been adequately studied. In particular, the effect of microgravity is changes in the expression of various genes (Gershovich P.M. et al., 2008; Pan Z. et al., 2008), however, the reasons for this are still unclear. In mammals, one way to regulate expression is to change the DNA methylation level. Therefore, the aim of this work was to estimate the total DNA methylation level, the content of the intermediate product 5-hydroxymethylcytosine (5hmC) and the enzymes controlling these processes (DNA methylases DNMT1, DNMT3a, TET demethylases and HDAC1 deacetylase) in the ovaries of mice after long-term antiorthostatic suspension.
Materials and methods
Microgravity effects were simulated using the Ilyin-Novikov standard model of antiorthostatic suspension modified by Morey-Holton (Morey-Holton et al., 2005). During the experiment, the animals were kept in standard vivarium conditions with food and water ad libitum. The animals were randomly divided into two groups: C (n=7), control group; HS (n=7), the 23-day suspension group. At the end of the suspension, after euthanizing the animals, the ovaries were isolated, weighed, and immediately frozen for subsequent isolation of nucleic acids and proteins. All the procedures conducted with animals were approved by the Commission on Biomedical Ethics of the Institute of Biomedical Problems.
Total DNA was isolated from the frozen tissues using a DNA isolation kit (Synthol, Russia) based on a phenol/chloroform method. The total methylation level was estimated through restriction analysis (MspI/HpaII) by EpiJET DNA Methylation Kit (Thermo Scientific, USA), the content of 5hmC – by dot-blotting with specific antibodies and proteins content – by Western blot with specific antibodies (all primary antibodies – Abcam, UK).
Results
The total methylation level in the ovaries was reduced after 23 days of hindlimb suspension by 20% (p <0.05) for the internal cytosine and by 10% (p <0.05) for the external cytosine in the 5’-CCGG-3’ loci (Figure 1, A). The content of 5hmC in the ovaries did not change in the HS group compared to the group C (Figure 1, B).
The content of S-phase DNA methylase DNMT1 and de novo methylase DNMT3a did not change after antiorthostatic suspension (Figure 2, A). But the relative content of the active demethylase TET2 increased after suspension by 16% (p <0.05), although there were no changes in TET3 content (Figure 2, B).
Content of histone acetylase HAT1 did not change, but the histone deacetylase HDAC1 content decreased after disuse by 17% (p <0.05) in comparison with control level (Figure 2, C).
Discussion
The obtained results indicate that in the ovarian cells of mice, after a 23-day antiorthostatic suspension, a hypomethylated state was established, in the absence of changes in the content of 5hmC. However, Western blot data indicate that the content of DNMT1 and DNMT3a methylases did not change, while the TET2 demethylase content increased. In this case, it can be assumed that the establishment of a hypomethylated state is due to active complete demethylation of the target sites, without accumulation of an intermediate 5hmC product. At the same time, the activity of TET2 demethylase may be related to the observed decrease of the deacetylase HDAC1 content, which, in turn, is capable of deacetylating TET proteins, inhibiting their activity in complex with DNMT1 (Zhang Y.W. et al., 2017).
Acknowledgements
The study was supported by program of the fundamental research SSC RF – IBMP RAS and program of RAS presidium “Molecular and cell biology”.
References
References
1. Watenpaugh DE, Hargens AR. The cardiovascular system in microgravity. In: Handbook of Physiology. Environmental Physiology. Bethesda, MD: Am. Physiol. Soc., 1996, sect. 4, vol. I, chapt. 29, p. 631–674.
2. Riley DA, Bain JLW, Thompson JL, Fitts RH, Widrick JJ, Trappe SW, Trappe TA. Decreased thin filament density and length in human atrophic soleus muscle fibers after spaceflight. J Appl Physiol. 2000; 88: 567–572.
3. Vico L., Hargens A. Skeletal changes during and after spaceflight. Nat Rev Rheumatol. 2018. 14(4): 229–245.
4. Gershovich PM, Gershovich JG, Buravkova LB. Simulated microgravity alters actin cytoskeleton and integrin-mediated focal adhesions of cultured human mesenchymal stromal cells. J Grav Physiol. 2008; 15(1): 203–204
5. Pan Z, Yang J, Guo C, Shi D, Shen D, Zheng Q, Chen R, Xu Y, Xi Y, Wang J. Effects of hindlimb unloading on ex vivo growth and osteogenic/adipogenic potentials of bone marrow derived mesenchymal stem cells in rats. Stem Cells and Development. 2008. 17(4): 795–804.
6. Zhang Y.W., Wang Zh., Xie W., Cai Y., Xia L., Easwaran H., Luo J., Chiu Yen R.-W., Li Y., Baylin S.B. Acetylation enhances TET2 function in protecting against abnormal DNA methylation during oxidative stress. Molecular Cell. 2017; 65: 323–335.
Keywords:
Epigenetic regulation,
DNA Methylation,
histone deacetylase (HDAC) 1,
Microgravity (μg),
Ovary
Conference:
39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.
Presentation Type:
Extended abstract
Topic:
Animal Models
Citation:
Usik
MA and
Ogneva
IV
(2019). The regulation of the DNA methylation in the ovaries of mice under 23-days antiorthostatic suspension.
Front. Physiol.
Conference Abstract:
39th ISGP Meeting & ESA Life Sciences Meeting.
doi: 10.3389/conf.fphys.2018.26.00004
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Received:
24 Nov 2018;
Published Online:
16 Jan 2019.
*
Correspondence:
Dr. Irina V Ogneva, Institute of Biomedical Problems (RAS), Moscow, Moscow Oblast, Russia, iogneva@yandex.ru