To understand how Brm and CBP, in conjunction with EcR-B1, activate the expression of their common target gene, sox14, we examined whether the levels of the transcriptionally active chromatin mark H3K27Ac are elevated at the sox14 region in an ecdysone-dependent manner. The expression of EcR-B1, Sox14, and Mical proteins is significantly upregulated in S2 cells upon treatment with ecdysone, similar to that seen in ddaC neurons during the larval-to-pupal transition ( Kirilly et al., 2009). We used nontreated and ecdysone-treated S2 cell extracts to perform chromatin check details immunoprecipitation (ChIP)
assays with an anti-H3K27Ac antibody, examined the H3K27Ac levels at the sox14 locus using quantitative real-time polymerase chain reaction (qRT-PCR) assays
with ten sox14 genomic primer sets ( Figure 7A), and subsequently normalized them against the H3K27Ac level at the internal control actin5C. Upon ecdysone treatment, the level of H3K27Ac increased more than 3-fold at the first intron of the sox14 gene (I1-3 and I1-4; Figure 7B), as compared to those in nontreated PR-171 cells. To confirm whether ecdysone signaling facilitates the enrichment of the H3K27Ac levels at the sox14 locus, we knocked down the EcR-B1 receptor in ecdysone-treated cells using EcR-B1 dsRNA fragments ( Figure 7H) and performed ChIP assays. Although total H3K27Ac levels in EcR-B1 RNAi S2 cells remained the same ( Figure 7H), the enrichment of H3K27Ac at the sox14 locus decreased significantly, as compared to the GFP RNAi ecdysone-treated S2 cells ( Figure 7C). Hence, local enrichment of H3K27Ac at the sox14 region is drastically elevated in response to ecdysone signaling. We then investigated whether the Metalloexopeptidase enrichment of H3K27Ac at the sox14 locus is mediated by CBP, the major HAT for H3K27 acetylation in ddaC neurons. Indeed, upon CBP knockdown in ecdysone-treated S2 cells ( Figure 7G), H3K27Ac enrichment at
the sox14 locus was drastically reduced ( Figure 7D). Thus, CBP facilitates H3K27 acetylation at the sox14 locus in response to ecdysone, thereby activating Sox14 expression. Given that Brm, like CBP, is specifically required for activation of Sox14 expression during ddaC pruning, we next examined whether Brm-mediated chromatin remodeling promotes local enrichment of H3K27Ac at the sox14 gene region. Strikingly, the knockdown of Brm also resulted in strong reduction of H3K27Ac enrichment at the sox14 locus ( Figure 7E) without affecting overall H3K27Ac levels ( Figure 7H). The relative levels of H3K27Ac were reduced to a lesser extent in CBP RNAi S2 cells than in brm RNAi S2 cells because CBP RNAi, rather than brm RNAi, also led to reduction of the H3K27Ac levels in the locus of the internal control actin5C (data not shown).