Fig. 6

HOXA-AS2 is required to achieve high expression levels of the proximal HOXA genes during RA induction in NT2-D1 cells. a Diagram of the HOXA gene cluster as described in Fig. 2b. The HOXA-AS2 gene is shown below the cluster and enlarged to better visualize the position of siAS2 used to deplete the lncRNA during RA induction (red line). b, c Analysis of proximal HOXA gene expression during RNAi knockdown of HOXA-AS2 with siAS2. Steady state levels of HOXA and lncRNA genes are measured by RT-qPCR in control (siGFP) and HOXA-AS2 (siAS2) knockdown cells induced 5 days with 10 μM RA. Fold change (c) is relative to the siGFP control set at 1. All measurements are from at least 3 PCRs, with error bars representing standard deviations.d RNAi depletion of HOXA-AS2 during RA induction leads to lower long-range contacts at the proximal HOXA region, and higher contact frequencies within the subdomain where it resides. Data is shown in heatmap form according to the color scale on the left and as described in Fig. 5. 2C-ChIP analysis of H3K4me3 (Additional file 6: BED file 37, 38), and H3K27me3 (Additional file 6: BED file 39, 40) in control and HOXA-AS2-knockdown cells is shown below the heatmaps. e Changes in the frequency of chromatin contacts occurring upon RNAi depletion of HOXA-AS2 during a 5-day RA induction. Heatmap values are IF differences between the HOXA-AS2 and control knockdown samples that are color-coded according to the scale on the left, with blue indicating a loss of contact and red showing a gain. Tracks under the differential heatmap are changes in chromatin mark levels or bound proteins detected with 2C-ChIP. Regions highlighted in heatmaps are as described in Fig. 5. The dashed black box identifies a HOXA cluster region slow to lose H3K27me3 signal