WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via β-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver.
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All processed Nodewalk data have been deposited at GEO (GSE76049). The ChIP and DamId-seq data were retrieved from GEO as follows: NUP98 (GSE48996), NUP133 (GSE87831), cLADs (GSE22428), BRD4 (GSM2058664), TCF4 and TCF7L2 (GSM782123), H3K9me2 (GSE58534), CTCF (GSM749690), H3K27ac (GSM946854) and H3K4me1 (GSM1240111). Source data are available online for Figs. 1–5 and Extended Data Figs. 1–3, 5, 8, 10.
The code used for the Nodewalk pipeline is available on request, and the code used to calculate the levels of cytoplasmic MYC mRNA in HCT-116 cells and HCECs over time is deposited at: https://github.com/Anita-Rolf-lab/Nature-Genetics-2019.
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This work was supported by the Swedish Research Council (VR 2017-04670/A.G and VR 2016-03108 to R.O.), the Swedish Childhood Cancer Fund (PR2017-0132 to R.G.), the Swedish Cancer Society (CAN2017/515 /A.G. and CAN 2016/708 to R.O.), the Lundberg Foundation (2018-0138 to A.G.), Karolinska Institutet (to A.G.), the Novo Nordisk Foundation (NNF16OC0021512 to A.G.), The Cancer Society in Stockholm (Cancerföreningen, 2018–2019 (to A.G.) and 2019–2020 (to R.O.)), China Scholarship Council (CsC), the MARIE Skłodowska-CURIE ACTIONS (Chromatin 3D, to A.G.) and the KA Wallenberg Foundation (KAW 2017.0077 to A.G. and R.O.). The authors acknowledge the ENCODE consortium and the ENCODE production laboratories for generating the extensive datasets.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a) Schematic representation of the Nodewalk procedure (see also31). Region of interest (Bait: blue) and an interacting locus (Interactor: green) are represented with lines depicting the restriction site (Hind III). Horizontal arrows indicate primers. b) Schematic representation of oligo DNAs and primers designed for the Nodewalk protocol. c) Principle to generate cDNAs from 3 C RNA. d) Assay to evaluate the fold enrichment of specifically primed 3 C cDNAs. The DNA band indicated by an arrow represents the enriched bait fragment. These experiments were repeated more than 20 times with similar results. Panel e) shows the recovery of interactors between two independent replicates while f) shows the amounts of reproducible interactors between two independent replicates stratified as indicated in the panel (see also Supplementary Table 1). g) Accumulated reproducibility between two independent experiments. h) Map of the MYC locus with arrows indicating the position of interactors identified by using MYC as initial bait. See reference31 for further information. Also shown are the NUP153 DamID-seq peaks of U2OS cells, publicly available from GEO (accession number GSE87831)24. i) Comparison between qPCR analysis of 3 C DNA products and the resulting normalised reads from the same sample. Data are represented as mean + SEM from two independent replicates. Dots indicate the actual values. The numbers indicate the positions of the interactors identified from the Nodewalk analysis shown in h).
a) Schematic visualisation of sequential “Nodewalking”31. The iterative nature of the Nodewalk assay enables the detection of a network of interacting loci (nodes representing distinct genomic regions) and edges (lines) representing their interactions. The identity of the numbered network nodes (pin-pointing new baits) is indicated on the linear genome map in panel c. b) The actual network generated from the MYC locus in HCT116 cells, using the above strategy. c) The position of each new bait highlighted in b). All baits were selected from the interactors of MYC that were included in the flanking TADs on chromosome 8, except for bait nr 10 that originated from chromosome 5 (Supplementary Table 2). Vertical lines indicate the interactors and their ligation events (LE) impinging on MYC. d) The network structure from HCT116 cells stratified by its k-core values. The red and green nodes identify regions overlapping with H3K27ac and H3K4me1 peaks, respectively. The size of each node reflects the number of detected interactions. e) Distribution of interactors generated from enhancer baits from within TAD 1 and 2, respectively. f) The interactions of enhancer hubs largely follow the TAD boundaries - with the exception for the MYC bait (nr 5), which interacts with regions equally distributed within both flanking TADs in both HCEC and HCT116 cells.
a) Schematic view of the genomic position of the Nodewalk baits (arrow heads). Nodewalk is a 3C-based technique that is based on the conversion of ligated chromatin DNA fragments into chimeric RNA sequences followed by cDNA priming using strategically positioned primers (baits) close to either end of key Hind III fragments listed in Supplementary Table 231. The ligation events (LEs) in A) indicate the frequency of interactions between MYC and its neighbouring regions. With the exception of its most immediate neighbourhood, by far the most prominent region to contact MYC is represented by the distal super-enhancer depicted by the b and c baits. b) Enhancer hubs with or without NUP153 binding sites (NUP153-positive or negative, respectively) were numbered and colour-coded as indicated in (a) and in the images. The larger circles represent baits (indicated by letters in (a)), while the smaller circles represent interactors detected by these baits and which are connected to 3 or more nodes in the network (K core > 3). c) The extent to which the enhancer baits are connected to one another positively correlates with NUP153 binding sites positioned within 5 kb from the point of interaction (left image; p = 4.68E-06), but not with location within constitutive lamina-associated domains (cLADs) (right image; p = 1.2E-05). The Y axes show the % of interactors with or without NUP153 binding sites or a genomic position inside or outside constitutive LADs (cLADs), while the X axes show the number of connections an enhancer bait has to other enhancer baits. The data is based on 9 independent Nodewalk analyses (See Supplementary Table 1 for additional information). P values: two-sided Fisher’s exact test. d) Interpretation (viewed from the nuclear side) of data in panel c.
Extended Data Fig. 4 Schematic illustration of the ChrISP technique using the fluorescently labelled splinter approach to score for chromatin fibre proximities.
The method is based on aptamer-conjugated secondary antibodies against primary rabbit or mouse antibodies, targeting either biotin- and digoxygenin-labelled DNA FISH probes, or against a protein epitope and a biotin-labelled DNA FISH probe. The fluorescently labelled splinter (in green) will anneal to the aptamers of the secondary antibodies only if the epitopes they recognise are within 162 Å from each other. The annealing step is subsequently stabilised by the ligation of a backbone DNA (in black)35,36.
a) Comparison of the sub-nuclear distribution of MYC and its OSE in relation to the nuclear periphery in HCECs using the “c” value strategy. The data is based on two independent experiments counting in total 300 alleles (p value: two-sided Fisher’s exact test). b) ChrISP analysis showing the proximity between the OSE and MYC. Each dot represents a ChrISP signal indicating proximity between one allele of the OSE and MYC in relation to the position of the OSE to the nuclear periphery/pore (a total of 310 alleles were counted in two independent experiments) (p value: two-sided Fisher’s exact test). c) ChrISP analyses of proximities between EnhD and MYC in relation to the position of the EnhD to the nuclear periphery. The number of counted alleles (155) was derived from three independent experiments (p value: two-sided Fisher’s exact test). Source Data
Extended Data Fig. 6 TCF4 and AHCTF1 are proximal to each other in control, but not in BC21-treated HCT-116 cells in extended view images.
The in situ proximity ligation assays were performed using mouse anti-ß-catenin and rabbit anti-AHCTF1 antibodies followed by staining with oligo-DNA modified secondary antibodies and ligation of splinter oligo DNA12. The physical proximity between TCF4 and AHCTF1 is subsequently detected by rolling circle amplifications. The amplification products were detected using a labelled oligo detector DNA to generate bright yellow dots. The nuclei were counterstained with DAPI. The experiments were repeated on two independent occasions with similar results. Bar = 10 μm. See Fig. 5a for a quantitation of the ISPLA signals in control (DMSO ctrl) and BC21-treated cells.
Extended Data Fig. 7 The colorectal super-enhancer is flanked by domains enriched in the repressive H3K9me2 mark, while the MYC gene and its associated enhancer, which reside close to an inter-TAD boundary, are devoid of this feature.
The major CTCF binding site within the OSE is indicated. Data for ChIP-seq peaks of the H3K9me2 mark were retrieved from GEO (accession number GSE58534). The CTCF peaks were called using the HCT-116 data set from GEO (accession number GSM749690). See Methods for additional information.
Primary cultures of colon epithelial cells were treated with WNT3a at different concentrations and for different lengths of time, as indicated in the image. Total RNA was extracted from two independent experiments and MYC mRNA expression levels were examined by qRT-PCR using primers for exon2, as described in the Methods section. The results were normalised to total cDNA input. Ctrl = control cells. Source Data
Extended Data Fig. 9 BRD4 and TCF4 binding to chromatin in the super-enhancer region (black rectangle).
Data for ChIP-seq peaks of BRD4 binding was retrieved from GEO (accession number GSM2058664). The TCF4 ChIP-seq peaks were called using the HCT-116 TCF7L2 UC Davis ChIP-seq Signal from ENCODE/SYDH (GSM782123).
Extended Data Fig. 10 The percentage of contaminations into the nuclear and cytosolic compartments with mitochondrial cytochrome B mRNA and unspliced nuclear MYC transcripts.
The bars show the mean value ± SD resulting from 11 (CYTB) and 8 (MYC intron) independent experiments. Source Data
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Scholz, B.A., Sumida, N., de Lima, C.D.M. et al. WNT signaling and AHCTF1 promote oncogenic MYC expression through super-enhancer-mediated gene gating. Nat Genet 51, 1723–1731 (2019). https://doi.org/10.1038/s41588-019-0535-3
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