HOXD Regulation of Neuroblastoma Differentiation Figure 3. Distinct functions of HOXD proteins in regulation of cyclin and CDK expression. Immunoblot analysis of cyclin A2, cyclin B1 and CDK1 levels in BE(2)-C/Tet-Off/myc-HOXD cells cultured in the presence or absence of Doxy for 3 or 6 days. Alpha-tubulin levels are shown as loading control. Shown are representative of two independent experiments with similar results. doi:10.1371/journal.pone.0040728.g003 the indirect evidence for a specific role of HOXD8 in medicating RA action in BE(2)-C cells. HOXC9 is a downstream target of HOXD8 in the RA signaling pathway The above finding that HOXD8 knockdown significantly inhibited the ability of RA to induce differentiation of BE(2)-C cells prompted us to investigate the connection between HOXD8 and HOXC9, which is also critical for RA action in neuroblas- toma cells [25]. HOXD8 knockdown significantly inhibited RA- induced HOXC9 expression (Figure 6 A). In addition, induction of HOXD8 alone was sufficient to upregulate HOXC9 expression (Figure 6 B). We further investigated the possibility of HOXC9 as a direct target gene of HOXD8. HOX proteins bind DNA sequences with a conserved TAATT/AA-motif [3,37]. Sequence examination revealed multiple potential HOX-binding sites within the 2-kb 59 flanking region of the human HOXC9 gene, with one matching closely to the consensus sequence located 21,361 bp upstream of the transcription start site (TSS,+1; Figure 6 C). ChIP- qPCR analysis revealed significant interaction of HOXD8 with the HOXC9 promoter region spanning from21,502 to21,236 bp (5.4-fold enrichment relative to IgG control, HOXC9_5Phox, Figure 6 D). By contrast, we observed no detectable association of HOXD8 with the region 6-kb upstream of the HOXC9 TSS (HOXC9_5P6K) or with the TSS of the ubiquitin ligase gene FBXW7 (FBXW7V1_0K, Figure 6 D). Together, these findings demonstrate that HOXD8 directly and specifically binds to the HOXC9 promoter-proximal region spanning from21,502 to21,236 bp in the context of chromatin. We next examined the chromatin state of the HOXC9 promoter by ChIP-qPCR using an antibody against trimethylated histone H3 at lysine 4 (H3K4me3), which marks a transcriptionally active state [38]. Consistent with the observed transcriptional activation of HOXC9, ChIP-qPCR assays revealed a significant increase in the levels of H3K4me3 (2.4–3.7 fold over the basal levels) at the HOXC9 promoter following HOXD8 induction (Figure 6 E). Together, these findings indicate that HOXD8 directly targets HOXC9 for transcriptional activation. Discussion In this report, we present three major findings from our comprehensive study of all HOXD genes for their functions in the control of neuroblastoma cell proliferation and differentiation in response to RA. First, all HOXD genes are induced by RA, which displays an expression pattern similar to what was originally observed in human embryonal carcinoma cells: HOX genes located at the 39 end are activated generally earlier by RA than those positioned more 59 within the cluster [11,12]. Second, several human HOXD proteins corresponding to the Drosophila Hox proteins abd-A (HOXD8) and Abd-B (HOXD9, HOXD10, HOX12) are functionally equivalent in promoting cell cycle arrest and neuronal differentiation of BE(2)-C cells, with the ability to recapitulate the phenotype of RA action. Finally, HOXD8 is a mediator of RA action in neuroblastoma cells by transcriptional activation of the HOXC9 gene, suggesting an RA-HOXD8- HOXC9 pathway in driving neuroblastoma cell differentiation. These findings have implications both in developing differentia- tion-based therapies for neuroblastoma and in employing neuro- blastoma cells as a model system for investigating the molecular basis of HOX genes in regulation of neuronal differentiation. RA has been used in clinic as a differentiation agent for treatment of high-risk neuroblastomas [23,24]. In combination with bone-marrow transplant, RA treatment can significantly improve the event-free survival of high-risk neuroblastoma patients [39,40]. However, resistance to RA presents a major barrier to successful RA-based therapy [23,24]. Identification of downstream genes that are mediators of RA-induced differentia- tion may offer opportunities to bypass resistance to RA. In this study, we show that individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 in neuroblastoma cells is able to recapitulate RA-induced differentiation phenotype. Moreover, we identify HOXD8 as a key component of the RA signaling pathway. We show that RA induction of HOXD8 is critical for the differentiation process at both the cellular and molecular levels: Downregulation of HOXD8 expression interferes with RA-induced morphologic differentiation and G1 arrest, as well as upregulation of neuronal genes and downregulation of cell cycle genes. We further present evidence that HOXC9, also a key mediator of RA action in neuroblastoma cells [25], is a direct target gene of HOXD8. Importantly, HOXD8 knockdown significantly inhibits the ability of RA to upregulate HOXC9 expression, providing both the physiological relevance and a molecular mechanism to the function of HOXD8 in the RA signaling pathway. Interestingly, HOXD9, despite its ability to induce neuronal differentiation and G1 arrest, is not essential for RA action, at least in BE(2)-C cells. The roles of HOXD10 and HOXD12 in the RA signaling pathway remain to be investigated. Nonetheless, our finding that induction of many HOXD genes can fully recapitulate RA action in neuroblastoma cells suggests that pharmacological activation of HOXD cluster genes may represent a therapeutic strategy for RA- resistant neuroblastomas. Early studies in the human embryonal carcinoma cell line NT2/D1 have shown that RA differentially activates HOX genes PLoS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e40728