Differential methylation of the gene encoding myo-inositol 3-phosphate synthase (Isyna1) in rat tissues

Abstract

Aims:Myo-inositol levels are frequently altered in several brain disorders. Myo-inositol 3-phosphate synthase, encoded by the Isyna1 gene, catalyzes the synthesis of myo-inositol in cells. Very little is known about the mechanisms regulating Isyna1 expression in brain and other tissues. In this study, we have examined the role of DNA methylation in regulating Isyna1 expression in rat tissues. Materials & methods: Transfection analysis using in vitro methylated promoter constructs, Southern blot analysis of genomic DNA from various tissues digested with a methylation-sensitive enzyme and CpG methylation profiling of genomic DNA from different tissues were used to determine differential methylation of Isyna1 in tissues. Transfection analysis using plasmids harboring mutated CpG residues in the 5‘-upstream region of Isyna1 was used to identify critical residues mediating promoter activity. Results: The -700 bp to -500 bp region (region 1) of Isyna1 exhibited increased methylation in brain cortex compared with other tissues; it also exhibited sex-specific methylation differences between matched male and female brain cortices. Mutation analysis identified one CpG residue in region 1 necessary for promoter activity in neuronal cells. A tissue-specific differentially methylated region (T-DMR) was found to be localized between +450 bp and +650 bp (region 3). This DMR was comparatively highly methylated in spleen, moderately methylated in brain cortex and poorly methylated in testis, consistent with mRNA levels observed in these tissues. Conclusion: Rat Isyna1 exhibits tissue-specific DNA methylation. Brain DNA was uniquely methylated in the 5‘-upstream region and displayed gender specificity. A T-DMR was identified within the gene body of Isyna1. These findings suggest that Isyna1 is regulated, in part, by DNA methylation and that significant alterations in methylation patterns during development could have a major impact on inositol phosphate synthase expression in later life.

KEYWORDS::

• CpG island
• CpG methylation
• epigenetics
• Isyna1
• mean methylation index
• myo-inositol
• myo-inositol 3-phosphate synthase
• T-DMR
• tissue-specific differentially methylated region

Author disclosure

The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of National Center for Research Resources (NCRR), NIH or Office of Research and Development (ORD)/ Medical Research Service (MRS) Department of Veterans Affairs (DVA).

Financial & competing interests disclosure

This work was made possible by grant #5P20RR017702 from the COBRE program of the NCRR, a component of the NIH, to Robert M Greene, grant #MH69991 from the NIH to Manuel F Casanova and by the ORD, MRS, DVA, Washington DC, USA, to Ranga N Parthasarathy. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

This work was made possible by grant #5P20RR017702 from the COBRE program of the NCRR, a component of the NIH, to Robert M Greene, grant #MH69991 from the NIH to Manuel F Casanova and by the ORD, MRS, DVA, Washington DC, USA, to Ranga N Parthasarathy. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.