Figure 2.

Alteration of histone acetylations at Region P of GM2-synthase gene directly regulate its transcription. A and C, butyrate increases GM2-synthase mRNA expression. Total RNA was isolated from NKE and SK-RC-45 cell lines upon treatment with indicated concentration of NaBu for different time points and reverse-transcribed. cDNAs were subjected to semi-quantitative PCR using GM2-synthase primer (786 bp). β-actin (687 bp) was used as an internal control for the above PCR experiments (bp). B and D, butyrate increases ganglioside GM2 levels. Immunostaining was performed with hamster anti-human GM2 antibody or hamster IgG antibody (isotype control) as indicated upon time-dependent NaBu treatment on NKE, SK-RC-45. E, ChIP assay performed with different cell lines using antibodies specific for H3, acetyl-H3K9, and acetyl-H3K14. Precipitated chromatin DNA was estimated by qPCR. Error bars represent mean ± S.E. of two independent determinations. F, butyrate decreases chromatin compaction at Region P. Following time-dependent treatment of 1 mm NaBu in NKE and SK-RC-45 cells, loss of chromatin compaction was measured by MNase protection assay. Data from three independent determinations represented as relative -folds with respect to Region P of GM2-synthase gene of untreated control cells (average ± S.E.; Student's t test; *, p < 0.05; ***, p < 0.001). ns, not significant.