Identification with SILAC Proteomics of Novel Short Linear Motifs in Demethylase Enzymes Regulated During Myoblast Differentiation

Abstract

Methylation and demethylation of Lysine and Arginine on proteins is quantitatively an extensive and functionally a significant post-translational modification yet there is a dearth of information at a functional systems level. Using SILAC proteomics and high resolution mass spectrometry we have identified eight demethylase and two methylase proteins whose levels are regulated during myogenic differentiation in a mouse myoblast-to-myocyte model. Using the general methylation inhibitor adenosine dialdehyde (AdOX) we established that methylation is required for differentiation. Whole proteome analysis revealed that 1134 out of 4600 proteins identified were differentially expressed, of which 488 were up-regulated and 646 down-regulated. Of these, two were methylases and eight were demethylases. Notably, five of the eight enzymes demethylate Lysine 9 on histone 3 (H3K9) whereas two also demethylate H3K4. Lastly, we have identified short linear motifs (SliMs) in the demethylase enzymes that are enriched in differentiation. We briefly discuss the significance of our findings within a developmental/epigenomics framework.

The Dynamic and Crucial Role of the Arginine Methylproteome in Myoblast Cell Differentiation

“…Significant differences were observed in the overall protein arginine methylation status for several To assess the role of protein methylation in myoblast differentiation, we treated the C2C12 cells grown in culture to confluency, which had begun the process of differentiation, with AdOX. No visible effects were observed on the cells at the low end of the concentration range [60]; however, the cells treated with 50 µM AdOX visibly exhibited reduced numbers of differentiated myoblasts compared to the controls (Figure 2A, day 3 and day 5, bottom panels). Notably, the differentiated cells in the 50 µM group were markedly smaller than those in the control group, indicating that the process was interrupted at a specific stage of the cell cycle.…”

“…We and others have previously established [59,60] that adenosine-2 ,3 -dialdexyde (AdOX), an adenosine analog and a global S-adenosylmethionine-dependent methyltransferase inhibitor with an IC50 of 40 nM, inhibits C2C12 cell differentiation in the range of 5 to 50 µM. AdOX inhibits transmethylation through the accumulation of Sadenosylhomocysteine (SAH), a negative feedback inhibitor of methylation, through the suppression of SAH hydrolase (SAHH).…”

Characterization of histone modification patterns and prediction of novel promoters using functional principal component analysis

“…Similarly, there is also evidence showing that active genes are characterized by high level of acetylation of the H3K9, H3K9ac [15][16][17][18][19]. On the other hand, the monomethylated H4K20me1 has been found to be associated with gene silencing, although there are recent studies that indicate that H4K20me1 is enriched in active gene promoters or gene body [10,[20][21][22][23], and increased level of H4K20me1 on the X chromosome has also been observed [24].…”