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. Author manuscript; available in PMC: 2018 Dec 15.
Published in final edited form as: Oncogene. 2018 Jun 15;37(42):5618–5632. doi: 10.1038/s41388-018-0358-1

Fig. 3.

Fig. 3

CHD7 mediates ras-induced p16INK4A expression as a transcriptional coactivator.

(A–B) p16INK4A protein (A) and mRNA (B) levels were measured in BJ cells transduced with miR-30 or vector (Ctrl) or CHD7 shRNA or a scrambled shRNA (shSC) and HaRasV12 (Ras) or vector (WH). * p < 0.05 vs Ctrl or shSC by Student t test.

(C) Luciferase reporters for the 3kb (−3570~+306) and 1.2kb (−891~+306) p16INK4A promoters were transfected into 293T cells with pcDNA3-CHD7 or vector.

(D) BJ cells containing stable luciferase reporters for the 3kb- and 1.2kb-p16INK4A promoters were transduced with HaRasV12 (Ras) or vector (WH).

(C, D) ns, no statistical significance by Student t test.

(E) ChIP analysis of the p16INK4A promoter. Abundance of DNA between −3523~+2917 of p16INK4A promoter immnuoprecipitated from BJ cells transduced with HaRasV12 (Ras) or vector (WH) by CHD7, H3K4me3, H3K4me1 antibodies or IgG was determined by qRT-PCR, and normalized to input. Arrows, positions of the DNA binding peaks. *, regions lacking efficient primers.

(F) Diagrams of the p16INK4A promoter luciferase reporters used in (G) and (H), showing position of the deleted CHD7-binding site and transcriptional start site (TSS).

(G) The p16INK4A promoter luciferase reporter with (WT) or without the CHD7-binding site were transfected into 293T cells with pcDNA3-CHD7 or vector.

(H) BJ cells containing a stable p16INK4A promoter luciferase reporter with (WT) or without the CHD7-binding site (Δ−119~−12) were transduced with HaRasV12 or vector.

(G, H) * p < 0.05 vs WT by Student t test.

(I–J) BJ cells containing a stable luciferase reporter for the 3kb (−3570~+306) p16INK4A promoters were transduced with CRISPR/Cas9 for CHD7 or a scrambled sequence (SC), and then with HaRasV12 (Ras) or vector (WH). CHD7 protein (I) and luciferase activity (J) were determined. * p < 0.05 vs SC by Student t test.

(C–D, G–H, J) Luciferase activity was determined 48 hrs (C, G) or 7 days (D, H, J) later and normalized to protein concentrations.

(B, C–E, G–H, J) Values are mean±SD for triplicates.