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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 4. Synthetic Beaf32 mutants impair its interaction to ChIP-indirect sites.

Figure 4

A. Scheme representing WT- and mutant- Beaf proteins (green and orange ovals, respectively) with respect to their predicted binding to direct or indirect peaks (orange and blue triangles, respectively) as tested by 3C and ChIP (see panels B–C).

B. Relative frequency chimera products (obtained from 3C data) measured by qPCR analyses in multiple independent 3C assays in Beaf32 depleted cells expressing mutant-Beaf as compared to WT control (orange and green curves, respectively). The x-axis represents the distance from Anchor site (direct Beaf32 peak at tsp39D). Chimera (ligation product) were measured with a set of primers spanning the whole locus relative to 3 control sites using Taqman-MGB probe (see Extended Experimental procedures). Error bars are standard errors from 2 independent 3C DNA preparation.

C. Box plots showing the binding levels of WT/mutant -Beaf proteins (see panel A) as measured by qPCR of ChIP samples precipitated with anti-Beaf32 or IgG control antibodies, from Beaf32 depleted cells stably transfected with WT/mutant -Beaf. The binding levels were measured corresponding to 10 direct Beaf32 sites and 10 nearby ChIP-indirect peaks (see Extended Experimental procedures; Figure S3D).

D. Scheme representing the constructs used to measure the interactions of WT- or mutant- Beaf proteins to the CGATA-less promoter region (blue triangle) of hsp27 in the presence or absence of an upstream Beaf32 site (orange triangle), as tested by ChIP (panels E–F).

E. Histogram showing the binding of Beaf proteins to ChIP-indirect peaks (blue) in the presence or absence of an upstream direct Beaf32 peak (orange triangle +/−) as measured by qPCR of ChIP with anti-Beaf32 or IgG control antibodies.

F. Histogram showing the binding of WT/mutant -Beaf proteins to ChIP direct (orange) or indirect (blue) peaks as measured by ChIP with anti-Beaf32 or IgG control antibodies, from Beaf32 depleted cells stably transfected with WT/mutant -Beaf. See also Supplementary Figure S4.