WNT signaling and AHCTF1 promote oncogenic MYC expression through super-enhancer-mediated gene gating

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The OSE and MYC interact with the nuclear pore.
Fig. 2: The colorectal OSE recruits the transcriptionally active MYC gene to the nuclear pores.
Fig. 3: The gating of MYC increases the level of MYC transcripts in the cytoplasm of cancer cells.
Fig. 4: The gating of MYC to the nuclear pore or periphery is regulated by AHCTF1.
Fig. 5: The OSE-mediated gating of MYC is regulated by β-catenin in HCT-116 cells.
Fig. 6: Model summarizing the enhancer-mediated gating of MYC and its regulation.

Data availability

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. 15 and Extended Data Figs. 13, 5, 8, 10.

Code availability

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.

References

  1. 1.

    Göndör, A. & Ohlsson, R. Enhancer functions in three dimensions: beyond the flat world perspective. F1000Res. https://doi.org/10.12688/f1000research.13842.1 (2018).

    Google Scholar 

  2. 2.

    Bickmore, W. A. & van Steensel, B. Genome architecture: domain organization of interphase chromosomes. Cell 152, 1270–1284 (2013).

    CAS  Article  Google Scholar 

  3. 3.

    Misteli, T. Beyond the sequence: cellular organization of genome function. Cell 128, 787–800 (2007).

    CAS  PubMed  Google Scholar 

  4. 4.

    Shachar, S. & Misteli, T. Causes and consequences of nuclear gene positioning. J. Cell Sci. 130, 1501–1508 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Feinberg, A. P., Koldobskiy, M. A. & Gondor, A. Epigenetic modulators, modifiers and mediators in cancer aetiology and progression. Nat. Rev. Genet. 17, 284–299 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Burns, L. T. & Wente, S. R. From hypothesis to mechanism: uncovering nuclear pore complex links to gene expression. Mol. Cell Biol. 34, 2114–2120 (2014).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Capelson, M. et al. Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes. Cell 140, 372–383 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Fraser, P. & Bickmore, W. Nuclear organization of the genome and the potential for gene regulation. Nature 447, 413–417 (2007).

    CAS  PubMed  Google Scholar 

  9. 9.

    Deng, B., Melnik, S. & Cook, P. R. Transcription factories, chromatin loops, and the dysregulation of gene expression in malignancy. Semin Cancer Biol. 23, 65–71 (2013).

    CAS  PubMed  Google Scholar 

  10. 10.

    Maharana, S., Sharma, D., Shi, X. & Shivashankar, G. V. Dynamic organization of transcription compartments is dependent on functional nuclear architecture. Biophys. J. 103, 851–859 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Ohlsson, R., Lobanenkov, V. & Klenova, E. Does CTCF mediate between nuclear organization and gene expression? Bioessays 32, 37–50 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Zhao, H. et al. PARP1- and CTCF-mediated interactions between active and repressed chromatin at the lamina promote oscillating transcription. Mol. Cell 59, 984–997 (2015).

    CAS  PubMed  Google Scholar 

  13. 13.

    Hansen, A., Cattoglio, C., Darxacq, X. & Tjian, R. Recent evidence that TADs and chromatin loops are dynamic structures. Nucleus 9, 20–32 (2018).

    CAS  PubMed  Google Scholar 

  14. 14.

    Reddy, K. L. & Feinberg, A. P. Higher order chromatin organization in cancer. Semin. Cancer Biol. 23, 109–115 (2012).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Wen, B., Wu, H., Shinkai, Y., Irizarry, R. A. & Feinberg, A. P. Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. Nat. Genet. 41, 246–250 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Guelen, L. et al. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453, 948–951 (2008).

    CAS  PubMed  Google Scholar 

  17. 17.

    McDonald, O. G., Wu, H., Timp, W., Doi, A. & Feinberg, A. P. Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition. Nat. Struct. Mol. Biol. 18, 867–874 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Schneider, R. & Grosschedl, R. Dynamics and interplay of nuclear architecture, genome organization, and gene expression. Genes Dev. 21, 3027–3043 (2007).

    CAS  PubMed  Google Scholar 

  19. 19.

    Blobel, G. Gene gating: a hypothesis. Proc. Natl Acad. Sci. USA 82, 8527–8529 (1985).

    CAS  PubMed  Google Scholar 

  20. 20.

    Cremer, T. et al. The 4D nucleome: evidence for a dynamic nuclear landscape based on co-aligned active and inactive nuclear compartments. FEBS Lett. 589, 2931–2943 (2015).

    CAS  PubMed  Google Scholar 

  21. 21.

    Pascual-Garcia, P. & Capelson, M. Nuclear pores as versatile platforms for gene regulation. Curr. Opin. Genet. Dev. 25, 110–117 (2014).

    CAS  PubMed  Google Scholar 

  22. 22.

    Pascual-Garcia, P. et al. Metazoan nuclear pores provide a scaffold for poised genes and mediate induced enhancer-promoter contacts. Mol. Cell 66, 63–76 e66 (2017).

    CAS  PubMed  Google Scholar 

  23. 23.

    Liang, Y., Franks, T. M., Marchetto, M. C., Gage, F. H. & Hetzer, M. W. Dynamic association of NUP98 with the human genome. PLoS Genet. 9, e1003308 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Ibarra, A., Benner, C., Tyagi, S., Cool, J. & Hetzer, M. W. Nucleoporin-mediated regulation of cell identity genes. Genes Dev. 30, 2253–2258 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Grosschedl, R. (eds Brenner, S. & Miller, J. H.) Brenner’s Encyclopedia of Genetics 624–625 (Elsevier, 2001).

  26. 26.

    Seeber, A. & Gasser, S. M. Chromatin organization and dynamics in double-strand break repair. Curr. Opin. Genet. Dev. 43, 9–16 (2017).

    CAS  PubMed  Google Scholar 

  27. 27.

    Ohlsson, R. I., Schwarze, P., Ruud, E. & Pfeifer-Ohlsson, S. Potential multiple functions of the v-myc oncogene within a single cell clone of OK10 retrovirus-transformed quail fibroblasts. Oncogene 3, 457–461 (1988).

    CAS  PubMed  Google Scholar 

  28. 28.

    Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Hnisz, D. et al. Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers. Mol. Cell 58, 362–370 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kahn, M. Can we safely target the WNT pathway? Nat. Rev. Drug Discov. 13, 513–532 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Sumida, N. et al. The ultra-sensitive Nodewalk technique identifies stochastic from virtual, population-based enhancer hubs regulating MYC in 3D: implications for the fitness of cancer cells. Preprint at bioRxiv https://doi.org/10.1101/286583 (2018).

  32. 32.

    Gondor, A. et al. Window into the complexities of chromosome interactomes. Cold Spring Harb. Symp. Quant. Biol. 75, 493–500 (2010).

    CAS  PubMed  Google Scholar 

  33. 33.

    Seidman, S. Network structure and minimum degree. Soc. Netw. 5, 269–287 (1983).

    Google Scholar 

  34. 34.

    Maeshima, K. et al. Nuclear pore formation but not nuclear growth is governed by cyclin-dependent kinases (Cdks) during interphase. Nat. Struct. Mol. Biol. 17, 1065–1071 (2010).

    CAS  PubMed  Google Scholar 

  35. 35.

    Chen, X. et al. Chromatin in situ proximity (ChrISP): single-cell analysis of chromatin proximities at a high resolution. Biotechniques 56, 117–124 (2014).

    PubMed  Google Scholar 

  36. 36.

    Chen, X. et al. The visualization of large organized chromatin domains enriched in the H3K9me2 mark within a single chromosome in a single cell. Epigenetics 9, 1439–1445 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Schwartz, M., Travesa, A., Martell, S. W. & Forbes, D. J. Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin. Nucleus 6, 40–54 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Sur, I. K. et al. Mice lacking a Myc enhancer that includes human SNP rs6983267 are resistant to intestinal tumors. Science 338, 1360–1363 (2012).

    CAS  PubMed  Google Scholar 

  39. 39.

    Jackson, D., Pombo, A. & Iborra, F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells. FASEB J. 14, 242–254 (2000).

    CAS  PubMed  Google Scholar 

  40. 40.

    Kudo, N. et al. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242, 540–547 (1998).

    CAS  PubMed  Google Scholar 

  41. 41.

    Hansen, M., Desai, R., Simpson, M. & Weinberger, L. Cytoplasmic amplification of transcriptional noise generates substantial cell-to-cell variability. Cell Syst. 7, 384–397 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Gillespie, P. J., Khoudoli, G. A., Stewart, G., Swedlow, J. R. & Blow, J. J. ELYS/MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing. Curr. Biol. 17, 1657–1662 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Rennoll, S. & Yochum, G. Regulation of MYC gene expression by aberrant Wnt/β-catenin signaling in colorectal cancer. World J. Biol. Chem. 6, 290–300 (2015).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Togel, L. et al. Dual targeting of bromodomain and extraterminal domain proteins, and WNT or MAPK signaling, inhibits c-MYC expression and proliferation of colorectal cancer cells. Mol. Cancer Ther. 15, 1217–1226 (2016).

    CAS  PubMed  Google Scholar 

  45. 45.

    Bafico, A., Liu, G., Goldin, L., Harris, V. & Aaronson, S. A. An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell 6, 497–506 (2004).

    CAS  PubMed  Google Scholar 

  46. 46.

    Voloshanenko, O. et al. β-catenin-independent regulation of Wnt target genes by RoR2 and ATF2/ATF4 in colon cancer cells. Sci. Rep. 8, 3178 (2018).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Schuijers, J. et al. Transcriptional dysregulation of MYC reveals common enhancer-docking mechanism. Cell Rep. 23, 349–360 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Kurukuti, S. et al. CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc. Natl Acad. Sci. USA 103, 10684–10689 (2006).

    CAS  PubMed  Google Scholar 

  49. 49.

    Sandhu, K. S. et al. Nonallelic transvection of multiple imprinted loci is organized by the H19 imprinting control region during germline development. Genes Dev. 23, 2598–2603 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Nemeth, A., Guibert, S., Tiwari, V. K., Ohlsson, R. & Langst, G. Epigenetic regulation of TTF-I-mediated promoter-terminator interactions of rRNA genes. EMBO J. 27, 1255–1265 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Delmore, J. E. et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904–917 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Rathert, P. et al. Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature 525, 543–547 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Cowling, V. H. & Cole, M. D. Turning the tables: Myc activates Wnt in breast cancer. Cell Cycle 6, 2625–2627 (2007).

    CAS  PubMed  Google Scholar 

  54. 54.

    Narendra Talabattula, V. A. et al. Non-canonical pathway induced by Wnt3a regulates β-catenin via Pyk2 in differentiating human neural progenitor cells. Biochem Biophys. Res. Commun. 491, 40–46 (2017).

    CAS  PubMed  Google Scholar 

  55. 55.

    Yang, Q., Riblet, R. & Schildkraut, C. L. Sites that direct nuclear compartmentalization are near the 5′ end of the mouse immunoglobulin heavy-chain locus. Mol. Cell Biol. 25, 6021–6030 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Pai, S. G. et al. Wnt/beta-catenin pathway: modulating anticancer immune response. J. Hematol. Oncol. 10, 101 (2017).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Posternak, V., Ung, M. H., Cheng, C. & Cole, M. D. MYC mediates mRNA cap methylation of canonical Wnt/β-catenin signaling transcripts by recruiting CDK7 and RNA methyltransferase. Mol. Cancer Res. 15, 213–224 (2017).

    CAS  PubMed  Google Scholar 

  58. 58.

    Fagnocchi, L. et al. A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity. Nat. Commun. 7, 11903 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Su, Y. et al. Post-translational modification localizes MYC to the nuclear pore basket to regulate a subset of target genes involved in cellular responses to environmental signals. Genes Dev. 32, 1398–1419 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Liu, X. et al. Deregulated Wnt/β-catenin program in high-risk neuroblastomas without MYCN amplification. Oncogene 27, 1478–1488 (2008).

    CAS  PubMed  Google Scholar 

  61. 61.

    Niederriter, A. R., Varshney, A., Parker, S. C. & Martin, D. M. Super enhancers in cancers, complex disease, and developmental disorders. Genes (Basel) 6, 1183–1200 (2015).

    CAS  Google Scholar 

  62. 62.

    Khan, A. & Zhang, X. dbSUPER: a database of super-enhancers in mouse and human genome. Nucleic Acids Res. 44, D164–D171 (2016).

    CAS  PubMed  Google Scholar 

  63. 63.

    Gondor, A., Rougier, C. & Ohlsson, R. High-resolution circular chromosome conformation capture assay. Nat. Protoc. 3, 303–313 (2008).

    PubMed  Google Scholar 

  64. 64.

    Pant, V. et al. The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains. Genes Dev. 17, 586–590 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Ramirez, F. et al. High-resolution TADs reveal DNA sequences underlying genome organization in flies. Nat. Commun. 9, 189 (2018).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

B.A.S. did most of the ChrISP and RNA and DNA FISH analyses and contributed to the export and mRNA decay analyses. N.S. did most of the ChIP and qRT–PCR analyses, and contributed to the kinetic and RNA stability analyses. C.D.M.L. performed some of the ChIP and qRT–PCR analyses. I.C. contributed to some of the ChrISP analyses. M.M. and I.T. performed ISPLAs and contributed to the qRT–PCR analyses. A.N. contributed to the co-immunoprecipitation experiments. H.Z. contributed to the 3D DNA FISH analyses. R.M. performed the simulation experiments. D.B. and E.G.S. performed the bioinformatic analyses. A.G. and R.O. conceived, supervised and planned the experiments and wrote the manuscript.

Corresponding authors

Correspondence to Anita Göndör or Rolf Ohlsson.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The Nodewalk principle and associated quality controls.

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).

Extended Data Fig. 2 The generation of Nodewalk networks and their link to enhancers.

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.

Extended Data Fig. 3 The link between chromatin networks, enhancers and NUP153 in HCT-116 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.

Extended Data Fig. 5 The sub-nuclear localisation of enhancer-MYC regions.

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.

Extended Data Fig. 8 WNT3a represses MYC expression in HCEC cells.

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

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading