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. 2015 Aug 28;43(20):9742–9756. doi: 10.1093/nar/gkv825

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — H2A.Z helps the recruitment of RNA polymerase II and cohesin complex as well as transcription at active enhancers. (A) Average profiles of strand-specific GRO-seq signal over intergenic ERα-active enhancers at 0 (grey), 10 (orange) and 40 (blue) min after E2 stimulation. (B) Distribution of the changes in the expression of E2-regulated genes, specifically associated with ERα w/ or w/o H2A.Z (considered to be their direct E2-regulated target genes, common targets were discarded from this analysis), following a 40 min of E2 stimulation. The transcription of the E2-regulated genes specifically associated with ERα w/ H2A.Z is more induced following E2 stimulation (P-value < 0.05, Mann-Whitney test). (C) ChIP-qPCR of RNAPII (8WG16 antibody) before and after E2 stimulation (30 min) and with or without H2A.Z depletion by shRNA. Five loci of both ERα w/ H2A.Z (loci (A) to (E)) and ERα w/o H2A.Z (loci (F) to (J)) were studied. The results are normalized to shCTL +E2 and represent the mean ± SEM of two independent biological replicates. The coordinates of each locus as well as the sequences of the primers are available in the Supplementary Table 3. (D) RT-qPCR analyses using the same conditions and primers than panel (C). The expression levels are relative to the expression of RPLP0 and are normalized to shCTL -E2. The results represent the mean ± SEM of four independent biological replicates. (E) Normalized RAD21 signal distribution at intergenic ERα-active enhancers in the presence of E2. The signal of RAD21 is significantly higher in ERα w/ H2A.Z (P-value < 0.0001, Mann-Whitney test). (F) Same analysis as (C), but using RAD21 antibody.