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. Author manuscript; available in PMC: 2015 Feb 20.
Published in final edited form as: Mol Cell. 2014 Jan 30;53(4):672–681. doi: 10.1016/j.molcel.2013.12.029

Figure 2. ChIP-indirect peaks reflect long-range interaction sites.

Figure 2

A. Genomic view from our Gbrowser showing the tsp39D and mio/crc loci from chromosome 2L that harbor direct and indirect peaks, respectively. Also indicated are ChIP data for Beaf32 (red), CP190 (green) and GAF (blue; from (Negre et al., 2010); see also Figure S2, S3). The arrow represents the interactions as detected by 3C (see panel B). All genomic features are shown in the flipped orientation.

B. 3C analysis of long-range interactions between direct Beaf32 peak at tsp39D and indirect peaks at mio/crc. The graph represents the relative frequency chimera products as measured by qPCR from Beaf32- (red), CP190-(orange) or control mock- (blue) depleted cells (see Experimental procedures). Chimera (ligation product) were measured using HindIII restriction (2L21342022) site prior to the direct Beaf32 peak as anchor site (reverse primer and Taqman-MGB probe) and primers spanning the whole locus. Variations were tested by Student test (see Extended Experimental procedures). Error bars are standard errors to the mean from three independent preparation of 3C DNA (see Supplementary Figure S2F for 3C using ChIP-indirect peaks as anchor sites).

C. Normalized Hi-C profiles obtained from Hi-C data (Sexton et al., 2012) probing the long-range interactions of distant/direct Beaf32 peaks (> 15 kbp away; orange triangle/green oval) over ChIP indirect peaks (position 0; x-axis) as compared to control windows. Long-range interaction profiles were aggregated over ChIP indirect peaks or control neighbor windows (see Extended Experimental procedures; as adapted from (Jee et al., 2011)) resulting in the amplification of the signal allowing us to accurately probe interactions over 500 bp windows. The relative enrichment in interactions (y-axis) was obtained by normalizing interaction levels between direct and indirect peaks over background interaction levels between the same direct peaks and the 26 neighboring bins (+/− 3; 3.5; up to 9 kbp). The error bar represents the variation of the measures (see Extended Experimental procedures for details).

D. Normalized Hi-C profiles through aggregation plots as in panel (C) except that enrichment in long-range interactions were measured over CP190 binding sites (position 0) as compared to neighbor control sites.