Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

Abstract

Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic–exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the transcription start site (−40 to +42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (−40 to +42).

Developmental exposure to 50 parts-per-billion arsenic influences histone modifications and associated epigenetic machinery in a region- and sex-specific manner in the adult mouse brain

“…We chose to assess H3K4me3 and H3K9me3 levels, as both of these modifications are influenced by arsenic (Zhou et al, 2008) and each is present on either actively transcribed or repressed genes, respectively (Kouzarides, 2007). Surprisingly, we found no differences in H3K9me3 levels, as anticipated based on cell culture studies; potential reasons for this include the dose and timing of arsenic exposure or the cell type, as in vitro studies use direct application of high concentrations of arsenite on cancer cells (Chervona et al, 2012a, Hong and Bain, 2012). We did observe an increase in H3K4me3 levels in the dentate gyrus of adult males, and a decrease in levels in adult females in the same region; this is directly opposite of the expression patterns for this HPTM measured in PBMCs isolated from humans chronically exposed arsenic-contaminated drinking water (Chervona et al, 2012b).…”

Arsenic induces sustained impairment of skeletal muscle and muscle progenitor cell ultrastructure and bioenergetics

“…However, these and other recent studies indicate direct actions of As(III) on muscle progenitor cell functioning and skeletal muscle homeostasis (9, 10, 38). The in vivo effects of As(III) are evident with low to moderate exposure in drinking water, and result in an in vivo imprinting of a maladaptive and dysfunctional muscle progenitor cell phenotype.…”

“…Since there is no As(III) added to the cultures as the cells go through multiple population doublings, the sustained altered bioenergetics may be caused by As(III)-induced epigenetic repression that does not revert once As(III) has been removed. This epigenetic repression has been suggested by studies demonstrating increased repressive DNA and histone hypermethylation in and near the myogenin and Igf-1 promoters in C2C12 cells following acute in vitro As(III) exposures (10, 38). These increased methylations and decreased histone acetylation were associated with increased expression and recruitment of the histone methyltransferase EzH2, as well as recruitment of the DNA methyltransferase, DNMT3a, to the myogenin promoter (38).…”

“…This epigenetic repression has been suggested by studies demonstrating increased repressive DNA and histone hypermethylation in and near the myogenin and Igf-1 promoters in C2C12 cells following acute in vitro As(III) exposures (10, 38). These increased methylations and decreased histone acetylation were associated with increased expression and recruitment of the histone methyltransferase EzH2, as well as recruitment of the DNA methyltransferase, DNMT3a, to the myogenin promoter (38). More extensive studies are needed to determine whether these arsenic-epigenetic marks are responsible for the sustained impairment of progenitors isolated from As(III)-exposed mice and to determine the breadth of epigenetic change that produces the muscle progenitor cell phenotype with impaired regenerative capacity and dysfunctional bioenergetics.…”

Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression

“…Arsenic has been demonstrated to silence genes by increasing DNA methylation and altering histone modifications at the promoter of genes (21,22). The mechanisms underlying arsenic-induced epigenetic alterations are vague, but various investigations have reported on the effect of arsenic on a number of epigenetic enzymes, such as EZH2 (23,24) and G9a (25). In this study, we identified a new response to arsenic exposure causing the depletion of SLBP via epigenetic regulation at its promoter and enhanced proteasomal degradation.…”