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. 2010 Jul 25;38(21):7472–7484. doi: 10.1093/nar/gkq644

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© The Author(s) 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — PcG is partially required for chromatin looping and transcription silencing of the human HoxA cluster. (A) RNAi knockdown specifically reduces EZH2 protein levels. Extracts from control (CTL) and EZH2 siRNA-treated cells treated with DMSO or RA were analyzed by western blotting with antibodies against EZH2 (top) or actin (bottom). EZH2 levels remaining after RNAi knockdown are indicated below each lane and are normalized to actin. (B) Loss of HoxA chromatin loops in EZH2 knockdown cells. Fixed 3C interaction profiles of HoxA2 (top) and HoxA5 (bottom) in control (CTL) and EZH2 knockdown cells. A schematic representation of the HoxA cluster is presented to scale above each graph. Vertical orange lines indicate position of fixed 3C primers and shaded yellow areas highlight looping contacts. Individual interaction frequencies were derived from at least three PCRs. Error bars show SEM. (C) 3′end HoxA genes are derepressed in EZH2 knockdown cells. Quantitative real-time PCR analysis of HoxA genes in control (CTL) and EZH2 siRNA-treated cells. Steady-state HoxA mRNA levels were normalized relative to actin (left). Fold induction after EZH2 knockdown is relative to levels in corresponding siRNA control, which was set at 1 (right). (D) 3′end genes are activated in partially RA-induced HoxA clusters. Quantitative real-time PCR analysis of HoxA genes in control siRNA-treated cells treated with DMSO (CTL silent) or RA (CTL induced). Fold induction after RA induction is relative to levels in corresponding siRNA control treated with DMSO, which was set at 1. (E) Loss of chromatin loops in partially RA-induced HoxA clusters. Fixed 3C interaction profiles of control siRNA-treated cells treated with DMSO (CTL silent) or RA (CTL induced) are as described in (B). (F) 3′end genes activation in partially RA-induced HoxA clusters depleted of EZH2. Quantitative real-time PCR analysis of HoxA genes in control siRNA-treated cells treated with DMSO (CTL silent) in EZH2 knockdown cells treated with RA (EZH2 induced). Fold induction after RA induction is relative to levels in corresponding siRNA control treated with DMSO, which was set at 1. (G) Loss of chromatin loops in partially RA-induced HoxA clusters depleted of EZH2. Fixed 3C interaction profiles of EZH2 siRNA-treated cells treated with DMSO (EZH2 silent) or RA (EZH2 induced) are as described in (B).