Fig 1: Fos eRNA is transcribed independently of but interacts with the histone acetyltransferase CBP. (A) shRNA-mediated knockdown of Crebbp mRNA resulted in deceased Fos mRNA but not eRNA expression (n = 18 per group, Mann–Whitney for Crebbp mRNA U = 0, P < 0.0001, Fos mRNA U = 55, P = 0.0004, eRNA1 U = 140, P = 0.5010, eRNA3 U = 132, P = 0.3550). (B) CREB inhibition (666–15; 1 µM) blunted the KCl response of Fos mRNA but not eRNA (n = 6 per group, two-way ANOVA for mRNA F(1,20) = 37.79, P < 0.0001, eRNA1 F(1,20) = 0.2461, P = 0.8769, eRNA3 F(1,20) = 0.1592, P = 0.6941, with Tukey's post hoc test for multiple comparisons). (C) CRISPR dCas-HAT targeting in C6 cells, in which dCas9 carrying a histone acetyltransferase domain is expressed with sgRNAs to target selected enhancers (left) induced Fos mRNA transcription (right, n = 8–9 per group, one-way ANOVA F(2,23) = 6.151, P = 0.0072). (D) Illustration of CREB-binding protein (CBP) domains (top), and recombinant glutathione-S-transferase (GST) tag-containing CBP-histone acetyltransferase (HAT) domain and CBP-bromodomain (Bromo) used in mobility shift assays. (E–G) RNA electrophoretic mobility shift assay (REMSA) with escalating concentrations of recombinant protein (0 = free probe (FP), 0.05–0.6 µM) reveal complete binding of synthetic Fos eRNA1 (151 bp, 20 nM), eRNA3 (153 bp, 20 nM), and control RNA (150 bp, 20 nM) to CBP-HAT. (H) Unlabeled eRNA1 competes for CBP-HAT binding with labeled eRNA1 (LP) in competition assay. (I) No binding of eRNA1 to CBP-Bromo domain was observed. For all REMSA experiments, n = 6 per group. (J) Model of enhancer function at promoters with associated eRNAs interacting with CBP. Data expressed as mean ± s.e.m. Multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplier Page from Abcam for Recombinant Human CREBBP protein