Figure 7.

Pol II disruption in vivo reduces aNDRs only moderately and results in an increase of the +1 nucleosome. (A) To investigate the effect of Pol II presence on aNDRs, we stripped Pol II from the chromatin by treating the human Raji B-cell line with α-amanitin for the indicated time points. Western blot samples were analyzed at 12 h, 18 h, 24 h, 36 h, and 48 h and showed Pol II disappearance in the latter two points. Tubulin (lower panel) was used as an internal control to check equal protein loading. At t₀, t₂₄, and t₃₆, Pol II ChIP-seq and MNase-seq experiments were performed as indicated. (B) Genome-wide analysis of nucleosome occupancy and Pol II recruitment following treatment at 1504 Pol II–enriched promoters (top 20%). As expected, Pol II signals around the TSS decreased over time (left). Nucleosome density slightly increased downstream from the TSS, indicating narrowing of the effective aNDR area (right) and an increase of the +1 nucleosome. (C) Examples showing the difference of Pol II occupation and nucleosome density across the TRIM4 and PPIA genes. ChIP-seq and MNase-seq data were scaled such that the represented units correspond to an equivalent amount of sequenced tags. (Arrow) Position of the +1 nucleosome. (D) Effect of GC content on aNDRs after Pol II removal. Three groups of GC content were built for the group of Pol II–enriched promoters shown in B. CpG scores, nucleosome densities (bottom panels), and Pol II ChIP-seq signal (top panels), before and after treatment are displayed. A stronger aNDR reduction, but not disruption, is observed for the group with the highest GC (and CpG) content.