Fig. 1: Interactions of RBPJ with cofactors and DNA determine transactivation activity and binding specificity.
From: Effective in vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding
a Surface representation of a RBPJ-SHARP complex structure (light gray/blue) bound to DNA (dark gray) (PDB entry: 6DKS). Amino acid mutations in interfaces of cofactor binding (green) and DNA-binding (orange) are highlighted. In vivo luciferase activity assays. Relative luciferase activity of RBPJ-specific reporter constructs in RBPJ knockout cell line transfected with b RBPJ-VP16 fusion variants or c RBPJ variants and co-transfected with NICD (mean values ± s.d.; n = 4 independent experiments). P-values were determined using two-tailed, unpaired Student’s t-test. Insets: Scheme of the reporter constructs and activating moieties. For details of variants see explanation in the text. -RBPJ: control with untransfected RBPJ knockout cell line, −/+NICD: control with untransfected/NICD-transfected RBPJ knockout cell line. d Heatmap of ChIP-seq reads from RBPJ knockout cell line transduced with HT-RBPJ variants with reads centered around RBPJ binding sites called in an untransduced HeLa cell line control. ChIP was performed with an antibody against RBPJ. Read number is color-coded. For details of variants see the explanation in the text. S-KO: ChIP-seq reads from SHARP knockout cell line transfected with HT-RBPJ. e Number of binding sites called for HT-RBPJ variants in RBPJ knockout cell line or SHARP knockout cell line. f, g Venn diagrams depicting common binding sites of indicated HT-RBPJ variants. Color code for RBPJ variants in b, c, e–g: RBPJ WT: light blue, R/H: yellow, K/E: orange, KRS/EHD: red, FL/AA: dark green, RFL/HAA: light green, S-KO RBPJ-WT: dark blue. Source Data are provided as a Source Data file for Fig. 1b, c, e.
