MYCN and HDAC2 are recruited to the miR-183 promoter region in the same complexes. (A) qRT-PCR analysis of endogenous miR-183 expression levels in neuroblastoma cell lines. Western blots for HDAC2 and MYCN expression with the GAPDH loading control are shown below each cell line. Statistical analysis compared cell lines with (green-tone bars) and without (blue-tone bars) MYCN amplifications. (B) Schematic representation of genomic localization and organization of the miR-183 cluster (based on the UCSC genome browser, GRCh37/hg19 assembly, February 2009) with distances to neighboring protein-coding genes. TSS, region of predicted putative transcription start site; arrows, direction of transcription. (C) ChIP-Seq analysis from Kelly cell lysates using an antibody detecting MYCN showing MYCN association in the promoter region of miR-183 cluster (black bars) superimposed on the miR-183 cluster genomic organization. (D) Enrichment of H3K27 tri-methylation at the miR-183 promoter region in MYCN-amplified cell lines. Heat map representing ChIP-on-chip data for the H3K4me3, H3K27me3 and H3K36me3 epigenetic marks at the miR-183 cluster in neuroblastoma cell lines superimposed on the genomic probe localization. Enrichment was calculated as the log₂ ratio of antibody (ab) to control, and is shown as graded color bars ranging from no binding (blue) to strong binding (red). (E, F) ChIP analysis in BE(2)-C cell lysates showing MYCN (E) and HDAC2 (F) recruitment to the miR-183 promoter region and no recruitment of HDAC1 (F). Bars represent mean relative enrichment above the IgG control (±SD), detected by qRT-PCR of the miR-183 promoter region. (G) ChIP showing increased histone H4 acetylation at the miR-183 promoter region after HDAC2 knockdown. BE(2)-C cells were transiently transfected with siRNA against HDAC2 (siRNA#2) or negative control (siNC#1), and ChIP analysis with a pan-acetyl-histone H4 antibody was performed 96 h after transfection. Bars show mean fold enrichment above siNC#1 control (±SD) measured by miR-183 promoter region qRT-PCR. (H) Re-ChIP showing recruitment of MYCN and HDAC2 in the same complexes. First ChIP conducted on BE(2)-C cell lysates with an anti-HDAC2 antibody. Chromatin precipitates used in the second ChIP with the indicated antibodies (x-axis). Bars represent mean enrichment above the Re-ChIP IgG control (±SD) detected by qRT-PCR of the miR-183 promoter region. *P ≤ 0.05; **P ≤ 0.001; n.s., not significant.