HOXD Regulation of Neuroblastoma Differentiation Figure 6. HOXC9 is a direct target gene of HOXD8. (A) qRT-PCR analysis of HOXC9 mRNA levels in BE(2)-C/Tet-Off/Luc-sh and BE(2)-C/Tet-Off/ HOXD8sh-1 cells that were cultured in the absence of Doxy for 6 days and then either untreated or treated with 10 mM RA for 3 or 6 days. The HOXC9 mRNA levels in BE(2)-C/Tet-Off/Luc-sh cells cultured in the absence of Doxy without RA treatment were designated as 1.0 (dashed line). (B) qRT-PCR analysis of HOXC9 mRNA levels in BE(2)-C/Tet-Off/myc-HOXD8 cells cultured in the presence or absence of Doxy for 3 or 6 days. The HOXC9 mRNA levels in BE(2)-C/Tet-Off/myc-HOXD8 cells in the presence of Doxy were designated as 1.0. (C) Schematic representation of the HOXC9 promoter region with a potential HOX-binding site 1,361 bp upstream of the transcription start site (TSS,+1). (D) ChIP-qPCR analysis of HOXD8 interaction with the HOXC9 promoter region (-1,502 to -1,236, HOXC9_5Phox) in BE(2)-C/Tet-Off/myc-HOXD8 cells cultured in the presence or absence of Doxy for 6 days, presented as fold enrichment over IgG control (1.0, dashed line). HOXD8 did not bind to the region 6-kb upstream of the HOXC9 promoter (HOXC9_5P6K) or the TSS of the FBXW7 gene (FBXW7V1_0K), which is shown as negative control. (E) ChIP-qPCR analysis of H3K4me3 levels at the HOXC9 promoter in BE(2)-C/Tet-Off/myc-HOXD8 cells cultured in the presence or absence of Doxy for 6 days, presented as fold enrichment over the IgG control (1.0, dashed line). The primer pairs target the HOXC9 promoter region around 22,000, 21,500, and 21 bp. The data (A, B, D, and E) were from two independent samples with each being assayed in triplicate and analyzed using two-tailed Student’s t-test with the p values indicated. Error bars, SD. doi:10.1371/journal.pone.0040728.g006 during development, a phenomenon called homeotic transforma- tion [43]. However, in mammals, there is evidence that some Hox genes are interchangeable and redundant. For example, Hoxa32/2 mice die at or immediately after birth [44], which can be fully rescued by replacing the protein-coding sequence of Hoxa3 with that of Hoxd3 [45]. Also, replacing the homeodomain of Hoxa11 with that of Hoxa10 in mice can provide wild-type function for the missing Hoxa11 gene in the development of the axial skeleton and male reproduction track [46]. Our study has uncovered the same principle in a cell-based system. HOXD8, HOXD9, HOXD10 and HOXD12 proteins are functionally equivalent in inducing cell cycle arrest and neuronal differentiation of BE(2)-C cells. Also interestingly, other HOXD proteins are functionally distinct, even within the same cellular context: HOXD1 has no phenotypic effect; HOXD3 is able to induce cell cycle arrest and morphologic differentiation but fails to upregulate the neuronal marker NEFM; HOXD4, HOXD11 and HOXD13 are able to upregulate NEFM but cannot induce cell cycle arrest and morphologic differentia- tion. It is important to point out that in addition to the temporal regulation, the expression of HOX genes is spatially regulated, showing a direct correlation between their linear arrangement along the chromosomes and the anteroposterior boundaries of their expression [47]. This spatial colinearity is critical for the developmental function of HOX genes in specifying positional identities of tissues along the anteroposterior axis. Notably, the four human HOXD proteins (HOXD8, HOXD9, HOXD10, HOXD12) with both anti-growth and differentiation-inducing activities in neuroblastoma cells correspond to the Drosophila Hox proteins abd-A (HOXD8) and Abd-B (HOXD9, HOXD10, HOX12) that are responsible for specifying the abdomen [43]. Most of neuroblastomas occur in the adrenal gland and abdomen PLoS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e40728