Abstract
Metal-binding inducible proteins called metallothioneins (MTs) protect cells from heavy-metal toxicity. Their transcription is regulated by metal response element (MRE)-binding transcription factor-1 (MTF1), which is strongly recruited to MREs in the MT promoters, in response to Zn and Cd. Mouse Mt1 gene promoter contains 5 MREs (a–e), and MTF1 has the highest affinity to MREd. Epigenetic changes like DNA methylation might affect transcription and, therefore, the cytoprotective function of MT genes. To reveal the CpG site(s) critical for Mt1 transcription, we analyzed the methylation status of CpG dinucleotides in the Mt1 gene promoter through bisulfite sequencing in P1798 mouse lymphosarcoma cells, with high or low MT expression. We found demethylated CpG sites near MREd and MREe, in cells with high expression. Next, we compared Mt1 gene-promoter-driven Lucia luciferase gene expression in unmethylated and methylated reporter vectors. To clarify the effect of complete and partial CpG methylation, we used M.SssI (CG→5mCG) and HhaI (GCGC→G5mCGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of a CpG site near MREd and MREe strongly inhibited Mt1 gene expression. Our results suggest that the methylation status of this site is important for the regulation of Mt1 gene expression.
Highlights
Metallothioneins (MTs) are low-molecular-weight, cysteine-rich, metal-binding proteins [1]
We found that methylation status of a CpG site near MREd and MREe is important for the regulation of Mt1 gene expression
Bisulfite sequencing revealed that cytosines at CpG sites in some regions of the MT1 promoter tended to be methylated in cells with low levels of MT1 expression but unmethylated in cells with high levels
Summary
Metallothioneins (MTs) are low-molecular-weight, cysteine-rich, metal-binding proteins [1]. The expression of ubiquitous MT1 and MT2 isoforms is induced by heavy metals. These isoforms protect against toxic heavy metals by sequestering them and lowering their concentrations at critical intracellular sites [2,3,4]; they provide protection against reactive oxygen species (ROS) [5,6,7]. Epigenetic modifications regulate gene expression through various epigenomic marks; for example, methylation of cytosine residues at CpG sites with histone modifications, to regulate gene expression [10,11]. CpG methylation or demethylation in the MT promoters might cause individual differences in MT expression levels, and differences in susceptibility to heavy metal toxicity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
