FIGURE 8.

O-GlcNAcylation and HAS2-AS1 induce chromatin remodeling. A, AoSMCs were left untreated or treated with 2 mm GlcN, 100 μm PUGNAC, 40 μm DON, 5 mm alloxan, or combinations of alloxan and GlcN. After 48 h, the nuclease accessibility index was calculated for the proximal promoter of HAS2 (red bars), HAS2-AS1 (blue bars), and HAS3 (gray bars). The nuclease accessibility index was set at 1 in control samples. Results are expressed as the mean ± S.E. in three different determinations. *, p < 0.01 untreated versus treatment, respectively. B, HAS2-AS1 abrogation prevented the aperture of chromatin in proximal promoter (Prom.) of HAS2. AoSMCs were nucleofected with siHAS2-AS1 (white bars) or a scrambled control (red bars). After 24 h of incubation, the cells were left untreated (Control) or treated with 2 mm GlcN or 5 mm alloxan or a combination of alloxan and GlcN. After 24 h nuclease accessibility assays were performed as described above. Results are expressed as the mean ± S.E. in three different determinations. *, p < 0.01 untreated versus treatment, respectively. C, overexpression of HAS2-AS1 S and L induced aperture of chromatin around HAS2 proximal promoter. AoSMCs were nucleofected with no DNA (Control) or with 3 μg of pcDNA3 vector or with 3 μg of pcDNA3 expressing either splice variant of HAS2-AS1 short (S) or long (L). After 48 h the accessibility of nuclease to HAS2 (red bars) or HAS2-AS1 (blue bars) proximal promoters was determined. Results are expressed as the mean ± S.E. in three different determinations. *, p < 0.01 control versus treatment, respectively.