Figure 6. Heterodimer formation with C/EBPβ is necessary for ATF4 binding and is sufficient to alter C/EBPβ-sequence specificity.
(A) Browser shot comparing C/EBPβ peaks from the in vitro cistromics assay (IVC) and hMSC ChIP-seq. 0.1 and 1 μl C/EBPβ molarity equivalents in the initial binding reaction are ∼60 and 600 nM, respectively. Tracks are RPM normalized, y-axes scaled from 0–3 (in vitro) and 0–5 (hMSC). (B) In vitro ChIP-seq peaks total vs a 1 RPM threshold, plotted as a function of C/EBPβ titration (0.1, 0.25, 1 and 10 μl). Total C/EBPβ peaks found in hMSCs (red line) is included for point of comparison. (C) Box plot of peak strength in the 10 μl C/EBPβ cistrome comparing gained vs co-bound sites. The co-bound fraction met a 0.5 RPM threshold in all C/EBPβ homodimer cistromes. (D) Motif enrichment for the strongest- and weakest-1000 sites of the C/EBPβ cistromes. (E) Browser shots of C/EBPβ peaks upon titration of ATF (0.1, 1 and 10 μM) with C/EBPβ (60 nM). Tracks are RPM normalized, 0–3 RPM scale on the left panel, 0–20 RPM on the right. (F) K-means clustered density heat maps of C/EBPβ occupancy as a function of ATF4 titration. Titration of ATF4 (0.1, 1 and 10 μM, C/EBPβ IP; 1 and 10 μM, ATF4 IP) with C/EBPβ (60 nM) is indicated. ATF4 homodimer binding was examined with 250 μM protein. Peaks thresholded to meet 1 RPM in at least two C/EBPβ cistromes. Three discrete clusters were identified and the de novo motif enrichment was based on all sites in each cluster.
Figure 6—figure supplement 1. Characterization of binding associated with either in vitro-selective C/EBPβ sites or ATF4 homodimers.
