Figure 6. Histone deacetylation contributes to stable gene silencing.

(A) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. (B) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. (C) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. (D) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit).

DOI: http://dx.doi.org/10.7554/eLife.22767.021