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. Author manuscript; available in PMC: 2014 Apr 10.
Published in final edited form as: Oncogene. 2011 Oct 17;31(24):3002–3008. doi: 10.1038/onc.2011.470

Figure 2.

Figure 2

Myc and HDAC3 are tethered to the miR-15a/16-1 promoter regions and Myc downregulates the transcription activity of miR-15a/16-1 promoter through HDAC3. (a) Knockdown of Myc or HDAC3 increased miR-15a/16-1 gene expression. Myc or HDAC3 was knocked down by siRNA (Dharmacon RNA Technologies, Lafayette, CO, USA) in Mino or Jeko-1 cells transfected by using Nucleofector (Amaxa, Cologne, Germany), and DLEU2 (primers sequence seen in Supplementary Table 2) and pri-miR-15a/16-1 expression levels (primers purchased from Applied Biosystems, Foster City, CA, USA) were measured by quantitative RT–PCR. Data were normalized to GAPDH. For relative mRNA expression, mRNA expression levels of cells treated with siCtrl were arbitrarily set as 1. (b) Schematic representation of miR-15a/16-1 host gene, DLEU2 promoter regions. (c) ChIP assay was performed using Myc or HDAC3 antibody to detect binding at the DLEU2 promoter, E1A and E1B regions. DLEU2 EX2, 6.3 kb distal from transcription start of promoter A, was used as a negative control. Pecentage input was calculated with 2(Ct [1% of input]–Ct [ChIP]). (d) ChIP assay using acetylated histone 4 (H4ac) antibody or RNA polymerase II antibody to detect binding of DLEU1A and 1B sites after HDAC3 was knocked down by shRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Jeko-1 cells. Jeko-1 cells treated with 2 μg/ml puromycin and selected for 4 days after 72 h of transduction were used to perform ChIP. (e) HDAC3 is involved in Myc-induced miR-15a/16-1 transcriptional regression. HEK 293 cells were transfected with either 200 ng of wild-type or mutant-type of DLEU1A or DLEU1B promoter luciferase reporter, 20 ng pcDNA3-cmyc or control pCDNA, 50 nm HDAC3 siRNA (Dharmacon RNA Technologies) or non-targeting siRNA, or 30 ng pCMV-β-galactosidase per well in 24-well plates. At 48 h posttransfection, cells were subjected to luciferase reporter assay using the luciferase assay system (Promega, Madison, WI, USA) and normalized to β-galactosidase activities. Each experiment was repeated three times in triplicate. ChIP, chromatin immunoprecipitation.