Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma

Abstract

Amplification of the locus encoding the oncogenic transcription factor MYCN is a defining feature of high-risk neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of MYCN perturbation in neuroblastoma. At oncogenic levels, MYCN associates with E-box binding motifs in an affinity-dependent manner, binding to strong canonical E-boxes at promoters and invading abundant weaker non-canonical E-boxes clustered at enhancers. Loss of MYCN leads to a global reduction in transcription, which is most pronounced at MYCN target genes with the greatest enhancer occupancy. These highly occupied MYCN target genes show tissue-specific expression and are linked to poor patient survival. The activity of genes with MYCN-occupied enhancers is dependent on the tissue-specific transcription factor TWIST1, which co-occupies enhancers with MYCN and is required for MYCN-dependent proliferation. These data implicate tissue-specific enhancers in defining often highly tumor-specific 'MYC target gene signatures' and identify disruption of the MYCN enhancer regulatory axis as a promising therapeutic strategy in neuroblastoma.

Figure 1:. Deregulated MYCN binds active chromatin and amplifies transcription in neuroblastoma

Cropped western blot of MYCN protein levels in human neuroblastoma cell lines., a. Cropped western blot of MYCN protein levels upon MYCN shutdown in the SHEP21-N cell line. The percent of MYCN remaining versus 0hr is indicated and calculated based on pixel intensity quantification., b. Cropped western blot of MYCN protein levels upon MYCN expression in the tet-on engineered SHEP cell line. c. ChIP-seq signal (rpm/bp) of the indicated marks at the NPM1 locus across a panel of neuroblastoma cell line models (MYCN amplified cell lines and inducible MYCN systems). Meta track representation across four neuroblastoma cell lines shown for MYCN and H3K27ac (top). , d. (Left) Pie chart showing the genomic distribution of conserved MYCN binding regions across four neuroblastoma cell lines. (Middle) De novo motif analysis of conserved MYCN binding regions across four neuroblastoma lines. (Right) Heat map of conserved MYCN binding occupancy at E-box sequences. , e. Box plots of MYCN ChIP-seq signal at promoters (left) and enhancers (right) upon MYCN shutdown. Significant differences at 0, 2, and 24hrs denoted; Welch’s two tailed t-test: *** p-value < 1e-9. **p-value <1e-6., f. RNA Pol II meta gene across all active genes upon MYCN shutdown. The TSS and gene body is magnified and significance denoted. (Bottom right) Distribution plots of RNA Pol II TR for all active genes. Differences in the TR distribution at 0hr and 2hr are significant; Welch’s two tailed t-test: *** p-value < 1e-9. **p-value <1e-6., g. mRNA levels of the NPM1 transcript during MYCN shutdown. Units are cell count normalized fpkm for triplicate biological replicates. Error bars represent standard deviation (s.d.)., h. Box plots of log₂ fold changes in active gene expression at the indicated time points versus 0hr post MYCN shutdown. Differences between 2 vs. 8hrs and 4 vs. 24hrs are significant; Welch’s two tailed t-test: *** p-value < 1e-9. **p-value <1e-6.

Figure 2:. Enhancer invasion shapes MYCN transcriptional response in neuroblastoma

a. Schematic illustrating a model in which clusters of low affinity E-boxes at enhancers shape deregulated MYCN transcriptional amplification b. ChIP-seq tracks of MYCN and H3K27ac at 0, 2 and 24hrs post MYCN shutdown at RPL22, HAND2 and the upstream ID2 enhancer. c. Meta track representation of MYCN and H3K27ac ChIP-seq signal (rpm/bp) across (top) four neuroblastoma cell lines and (bottom) and three TH-MYCN tumors at the GATA2 locus. d. ChIP-seq tracks of MYCN and H3K27ac at 0, 2 and 6hrs post MYCN induction at RPL22, HAND2 and the upstream ID2 enhancer. e. (Top) Plot showing the top 5,000 genes (x-axis) in SHEP21 ranked by total proximal MYCN signal (y-axis). (Bottom) Dot plot of % enhancer contribution (enhancer/total MYCN signal) sampled across bins (100 genes/bin) with a best fit line superimposed (loess correlation).f. Box plots of the log₂ fold change of MYCN load at the TSS and distal enhancers of the top 5,000 genes ranked by proximal MYCN signal. (Top) log₂ fold change at 2hr and 24hr (versus 0hr) post MYCN shutdown. (Bottom) log₂ fold change at 2 and 6hr (versus 0hr) post MYCN induction. g. Cell count normalized log₂ fold change (versus 0hr) of gene expression changes during MYCN shutdown in the SHEP-21N system. Genes are grouped according to rank ordered MYCN proximal load (promoters and enhancers). Error bars represent 95% confidence interval (CI) of the mean.h. Box plots of the log₂ fold change (versus 0hr) of the amount of RNA Pol II (ChIP-Rx) at the TSS (left) and gene body (right) of genes grouped according to rank ordered MYCN proximal load. Significance is denoted by Welch’s two tailed t-test: *** p-value < 1e-9. **p-value <1e-6. *p-value <1e-3. i. Overall survival of patients ranked by expression (high or low) of the top 25 genes defined by total MYCN load for all neuroblastoma patients (top) and patients without MYCN amplification (bottom). Significance is denoted by a chi-squared test and p-values are shown.

Figure 3:. Enhancer invasion accounts for tumor specific MYC/MYCN signatures

a. (Top) Meta track representation of MYCN and H3K27ac ChIP-seg signal (rpm/bp) across four neuroblastoma cell lines at the indicated loci. (Bottom) ChIP-seq tracks of MYC and H3K27ac signal in the MM.1S cell line. mRNA expression levels are shown as a bar plot in each with expression in arbitrary units (A.U.). b. Differential MYCN signal contribution across NB lines for promoters (red) and enhancers (blue) of associated genes (y-axis) of the top 5,000 proximal MYCN bound regions are shown ranked by difference in MYCN enhancer to promoter contribution (x-axis). c. GSEA plots of MYCN bound promoter (red) versus enhancer (blue) dominant gene sets defined by leading edge analysis. d. Normalized enrichment score (NES) of target gene signatures (Molecular Signature Database) are plotted on the x-axis versus the FDR (false discovery rate) on the y-axis. e. Highly significant gene signatures from promoter (red) and enhancer (blue) bias gene sets are highlighted and tabulated.f. Normalized enrichment score (NES) of target gene signatures (Molecular Signature Database) are plotted on the x-axis versus the FDR (false discovery rate) on the y-axis at 0, 2 and 24hrs post MYCN shutdown. g. Clustering of MYC/MYCN ChIP-seq signal at promoters across MYC/MYCN deregulated cell lines.h. Clustering of MYC/MYCN ChIP-seq signal at enhancers across MYC/MYCN deregulated cell lines.i. Heat-map of gene signatures enriched across MYC/MYCN deregulated cell lines. Selected signatures are annotated at a FDR <0.01 & NES > 2 cutoff.

Figure 4:. TWIST1 co-occupies enhancers with MYCN and is required for expression of the MYCN enhancer axis

a. (Left) Schematic of the computational approach to identifying a core regulatory circuit based on TF enhancer binding. (Right) Histogram plot ranking core regulatory circuit TFs based on motif co-occupancy with MYCN. b. Pie chart showing the genomic distribution of overlapping TWIST1 and MYCN bound sites. c. De novo motif analysis of enhancer regions for MYCN and TWIST1 binding sites. d. Meta track representation of MYCN ChIP-seq signal, H3K27ac ChIP-seq signal, and ATAC-seq signal (rpm/bp) at the HAND2 locus. Corresponding ChIP-seq signal of TWIST1 in the BE(2)-C and SHEP-21N cell lines during MYCN shutdown is shown. e. Boxplots of TWIST1 signal at 0, 2 and 24hrs post MYCN shutdown. Significance is denoted, Welch’s two tailed t-test: *** p-value < 1e-9f. Cropped western blot of Vinculin, MYCN, and TWIST1 levels upon MYCN shut down. g. Cell count normalized log₂ fold change (versus 0hr) of gene expression changes during MYCN shutdown in the SHEP-21N system. Genes are grouped by MYCN and/or TWIST1 binding: MYCN and TWIST1 co-bound sites (yellow), MYCN highly bound sites alone (black) and MYCN and TWIST1 lowly co-bound sites (gray). Error bars represent 95% CI.h. Overall survival of patients ranked by expression (high or low) of genes highly loaded with MYCN and TWIST1 in all neuroblastoma patients (left) and patients without MYCN amplification (right). Significance is denoted by a chi-squared test and p-values are shown.i. CRISPR scan of the TWIST1 locus and its downstream enhancers. (Top) Illumina sequencing readout of log₂ fold enrichment/depletion (early versus late time point) of 3,351 sgRNAs. (Bottom) Simple moving average of log₂ fold enrichment/depletion is shown. j. ChIP-seq signal of H3K27ac (blue), ATAC-seq (green), MYCN (red), and TWIST1 (yellow) at the TWIST1 locus and enhancers with respect to CRISPR sgRNAs. Red shaded boxes highlight regions of marked log₂ fold depletion. k. (Top) Cropped western blots of Vinculin, MYCN, and TWIST1 levels upon TWIST1 knockdown. (Bottom) Viable cell counts at 3 and 7 days post inducible TWIST1 knockdown. l. Log₂ fold change (versus 0hr) of gene expression changes upon TWIST1 knockdown of genes ranked by MYCN promoter load (top) or MYCN distal enhancer load (bottom). Error bars represent 95% CI.