Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation

Abstract

Background: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors.

Methodology: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions.

Conclusion: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the activity of individual genes, and that of a mediator of global chromatin structure.

Figure 1. MYCN ChIP-chip.

(A) Identification of MYCN binding upstream of NME2. The scale across the top of the panel indicates the base pair position on chromosome 17. Fluorescent intensity of probes from experiments using 2 independent MYCN antibodies around the NME2 promoter are expressed as log₂ ratios (green bars) and high confidence MYCN peaks (red bars) as identified by the NimbleScan peak finding algorithm. Position of the NME2 transcript and the region tiled on the array are indicated by the bottom two panels. (B) Total number of MYCN peaks identified across neuroblastoma cell lines. Venn diagrams (i–v) display the number of MYCN peaks which is shared and unique to each cell line. Statistical significance of overlap for all comparisons was determined to be statistically significant (P<0.001) by Fisher's Exact Test.

Figure 2. Distribution of MYCN transcription factor binding and E-box preference.

(A) Percentage of MYCN binding sites around transcription start sites. X-axis displays the distance in base pairs from UCSC annotated transcriptional start sites. Y-axis represents the percentage frequency of positive MYCN binding sites identified in each neuroblastoma cell line. (B) Prevalence of E-box motifs within MYCN binding sites detected by ChIP-chip across all cell lines and both antibodies. Statistically significant bars are indicated with their respective p-values (C) Prevalence of E-box motifs within the MYCN binding sites detected by ChIP-chip in the MYCN-amplified state only. Statistically significant bars are indicated with their respective p-values. (D) Association of E-box frequency with raw fluorescent ratios in MYCN binding sites on the promoter array which were detected in the Kelly NB cell line. The number of E-boxes per kilobase (y-axis) is plotted against the raw array fluorescent ratio intensities.

Figure 3. Association of MYCN binding and DNA hypermethylation around the let 7g and miR-135a-1 locus in SK-N-AS NB cell line.

(A) Array-CGH profile of chromosome 3p displaying a large 60.5 Mb terminal deletion in SK-N-AS. (B) Detailed view of the hemizygously deleted let-7 g/miR135a-1 locus. The upper two panels display the MYCN ChIP-chip raw log₂ ratios and identified peaks for both MYCN antibodies (NCMII-100 and B84b). Red and orange peaks represent regions of enrichment with an FDR of <0.05 and 0.05–0.1, respectively. MeDIP results for SK-N-AS are displayed in the lower panels in blue, which include log₂ ratios of MeDIP/Input, Kolmogorov-Smirnov test p-values (−log₁₀) and the merged statistically significant peaks of hypermethylation across the region. The position of CpG islands and orientation of miRNAs are displayed in the two bottom panels.