Figure 8. c-MYC is an upstream inducer of RBPJ expression in BTICs.

(A) Cross-linked chromatin was prepared from 2 BTIC models (3691 and 4121) and then immunoprecipitated with an anti-MYC antibody or IgG control, followed by qPCR using primers specific for the RBPJ promoter. CCND2 was used as a positive control (t test, *P < 0.05, **P < 0.01, n = 3). (B) Lysates of 3691 and 4121 BTICs expressing shCONT, shMYC-1, or shMYC-2 were immunoblotted with the indicated antibodies. shRNA-mediated knockdown of MYC decreased RBPJ levels. (C) 3691 and 4121 non-BTICs were transduced with either MYC or vector control, and then lysates were prepared and immunoblotted with the indicated antibodies. MYC expression in non-BTICs induced increased RBPJ levels. (D) 3691 and 4121 BTICs were treated with the BET domain inhibitor, JQ1 (1 μM), or vehicle control (DMSO). Lysates were immunoblotted with the indicated antibodies. JQ1 treatment decreased MYC and RBPJ levels. (E) 3691 BTICs were treated with JQ1 (1 μM) or DMSO and cellular proliferation was measured sequentially with CellTiter-Glo. (F) Global analysis of the RBPJ landscape reveals that RBPJ binds nearly exclusively to active promoters and enhancers. Transcription factor motif enrichment analysis found that RBPJ binding sites were the most enriched transcription factor–binding motif found under RBPJ ChIP-seq peaks, followed by MYC binding motifs, suggesting an interaction between these two transcription factors. (G) An example of RBPJ binding at enhancers and promoters of OLIG2 and OLIG1. The bottom two rows demonstrate called promoters and enhancers.