Figure 1.
The open chromatin at the Cga gene is enriched with phosphorylated H3S10 compared with the more closed chromatin at the β-subunit genes. A, basal mRNA levels of Cga, Lhb, and Fshb in αT3-1 partially differentiated gonadotrope cells were analyzed by qPCR and normalized to those of Rplp0. The mRNA levels were measured against standard curves, comprising purified and quantitated cDNA of each gene, which was serially diluted. The corresponding Ct value was plotted against the log of the amount of DNA (repeated three times and averaged) and these values (normalized to Rplp0) are presented relative to the mean level of the Cga, with the gray bar indicating the mean. Analysis of variance followed by Bonferroni t test were used to determine significantly different means: those marked with the same letter are not significantly different (p > 0.05). B–D, ChIP analysis was carried out to determine the levels of (B) H3S10p and (C) total H3 at the promoter/5′ end of each of the three gonadotropin subunit genes. In D, levels of H3S10p are shown normalized to those of total H3 in the same sample. For all, an aliquot from the same cells before precipitation was designated as input, and the levels in input and precipitated DNA samples were quantitated by qPCR. The precipitated DNA was normalized against the respective input samples and these values were compared with the mean level at the Cga gene; presented and statistical analysis as above.