Abstract
The epigenetic factor Methyl-CpG-Binding Protein 2 (MeCP2) is a nuclear protein that binds methylated DNA molecules (both 5-methylcytosine and 5-hydroxymethylcytosine) and controls gene transcription. MeCP2 is an important transcription factor that acts in a dose-dependent manner in the brain; thus, its optimal expression level in brain cells is important. As such, its deregulated expression, as well as gain- or loss-of-function mutation, lead to impaired neurodevelopment, and compromised structure and function of brain cells, particularly in neurons. Studies from others and us have characterized two well-recognized MeCP2 isoforms: MeCP2E1 and MeCP2E2. We have reported that in Daoy medulloblastoma brain cells, MeCP2E2 overexpression leads to MeCP2E1 protein degradation. Whether MeCP2 isoforms regulate the Mecp2 promoter regulatory elements remains unexplored. We previously showed that in Daoy cells, metformin (an anti-diabetic drug) induces MECP2E1 transcripts. However, possible impact of metformin on the Mecp2 promoter activity was not studied. Here, we generated stably transduced Daoy cell reporters to express EGFP driven by the Mecp2 promoter. Transduced cells were sorted into four EGFP-expressing groups (R4-to-R7) with different intensities of EGFP expression. Our results confirm that the Mecp2 promoter is active in Daoy cells, and that overexpression of either isoform inhibits the Mecp2 promoter activity, as detected by flow cytometry and luciferase reporter assays. Interestingly, metformin partially relieved the inhibitory effect of MeCP2E1 on the Mecp2 promoter, detected by flow cytometry. Taken together, our data provide important insight towards the regulation of MeCP2 isoforms at the promoter level, which might have biological relevance to the neurobiology of the brain.
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Acknowledgements
The authors would like to thank Dr. Frederic Jones for the Mecp2 promoter (MeP-Luc) plasmid Adachi et al. (2005), also used in our former studies Rastegar et al. (2009); Dr. Carolyn Schanen and team for the Mecp2E1-EGFP and Mecp2E2-EGFP plasmids Kumar et al. (2008); and Dr. James Ellis for the lentiviral vector Lenti-MeP-EGFP that was reported in previous studies Rastegar et al. (2009).
Funding
This research study is supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2016–06035 to M.R., University of Manitoba NSERC Bridge Funding, and Ontario Rett Syndrome Association (ORSA) to M.R.
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S.L., M.G., and A.A.S. performed promoter assay studies. M.B. and K.S. maintained, cultured, and prepared transduced cells for flow cytometry sorting. S.L., A.A.S., K.S., and D.K. contributed to the primary optimization of metformin concentration. C.O.O. completed lentiviral production and transduction in Daoy cells. B.T. helped analyzing and organizing the raw data. S.L., B.T., and M.R. prepared the Figures. M.R. contributed to the conception and design of experiments, provided reagents, materials, analysis tools, and research facilities. M.R., S.L., M.G., and B.T. wrote the manuscript. All authors read and agreed to the final version of the published manuscript.
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Lockman, S., Genung, M., Sheikholeslami, K. et al. Transcriptional Inhibition of the Mecp2 Promoter by MeCP2E1 and MeCP2E2 Isoforms Suggests Negative Auto-Regulatory Feedback that can be Moderated by Metformin. J Mol Neurosci 74, 14 (2024). https://doi.org/10.1007/s12031-023-02177-0
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DOI: https://doi.org/10.1007/s12031-023-02177-0