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MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch

Nature Genetics volume 56, pages 663–674 (2024)Cite this article

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A Publisher Correction to this article was published on 09 July 2024

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Abstract

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

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Fig. 1: MYC binds enhancers defined by cancer type-specific transcription factors.
Fig. 2: Expression of MYC enhancer target genes predicts patient outcome.
Fig. 3: MYC directly modulates activity of cancer type-specific enhancers.
Fig. 4: Glucocorticoid signaling reprograms MYC enhancer activity.
Fig. 5: MYC induces RNAPII recruitment and enhancer transcription.
Fig. 6: MYC recruits BRD4 to facilitate RNAPII loading at enhancers.
Fig. 7: MYC-mediated GCN5 recruitment induces an epigenetic switch promoting BRD4 binding at enhancers.
Fig. 8: Model for MYC-mediated regulation of enhancer activity in cancer.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD039586. Raw and processed next-generation sequencing data have been deposited to the GEO database with the dataset identifier GSE224613. Preproccesed data to re-create the figures are available on Zenodo (https://zenodo.org/records/8323614). In addition to the data generated for this study, publicly available raw sequencing data were downloaded from GEO using the following accession codes: GSE86504, GSE86511, GSE78064, GSE44672, GSE36354, GSE23893, GSE15220, GSE96867, GSE44672, GSE65201, GSE167154, GSE197062 and GSE32222. Preproccessed RNA-seq data from cell lines were downloaded from DepMap (https://depmap.org/portal/). Clinical and expression data from TCGA and METABRIC were downloaded from cBioPortal (https://www.cbioportal.org/) and Rueda et al.71. DNA binding motifs were downloaded from the JASPAR database (https://jaspar2020.genereg.net/). Source data are provided with this paper.

Code availability

The authors confirm that data processing and analysis used existing code/software packages whose details are provided in Methods. The code used for figure generation is available on GitHub (https://github.com/SimonToftholm/Jakobsen_et_al_2024).

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Acknowledgements

Sequencing was carried out at the Center for Functional Genomics and Tissue Plasticity, in the Functional Genomics and Metabolism research unit at SDU. The authors thank T. P. Mortensen, M. Wishoff, G. Jørgensen, and R. Nielsen for sequencing assistance. Work in the R.S. laboratory was supported by the Novo Nordisk Foundation (NNF18OC0053276 and NNF21OC0071373 to R.S.), the Danish Cancer Society (R231-A13786 to R.S.), and the VILLUM Foundation (project no. 42066 to R.S.). The mass spectrometry and proteomics platform at SDU, used to perform qPLEX-RIME, was financed by grants from the VILLUM Foundation (VILLUM Center for Bioanalytical Sciences, grant no. 7292 to O.N.J.), the Novo Nordisk Foundation (INTEGRA grant no. NNF20OC0061575 to O.N.J.), and by PRO-MS: Danish National Mass Spectrometry Platform for Functional Proteomics (grant no. 5072-00007B to O.N.J.). The work in the R.A. laboratory was supported by the Novo Nordisk Foundation (NNF20OC0059796 and NNF21SA0072102 to R.A.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Simon T. Jakobsen, Rikke A. M. Jensen, Maria S. Madsen, Tina Ravnsborg, Majken S. Siersbæk, Ole N. Jensen & Rasmus Siersbæk

  2. Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Christian S. Vaagenso, Hjorleifur Einarsson & Robin Andersson

  3. Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Hjorleifur Einarsson & Robin Andersson

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  1. Simon T. Jakobsen
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Contributions

S.T.J. and R.S. conceived and designed the study. S.T.J., R.A.M.J., M.S.M., T.R., C.S.V. and M.S.S. performed the experiments. S.T.J., T.R. and H.E. performed the computational analysis. R.S., O.N.J. and R.A. supervised the study. S.T.J. and R.S. wrote the manuscript with input from the other authors.

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Correspondence to Rasmus Siersbæk.

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Extended data

Extended Data Fig. 1 MYC expression and chromatin binding.

a, MYC expression across cancer cell lines summarized for tumor types from depmap.org. Boxplot: Center line, median; box limits; upper and lower quartiles; whiskers, 1,5x interquartile range; points, outliers. b, MYC expression in four breast cancer cell lines from depmap.org. c, Occurrence of strong/weak E-box motifs within MYC-bound promoters (−1 kb to +100 bp around the TSS) and enhancers (intronic and intergenic regions) from BT549, MDA-MB-231, MCF7, and T47D cell lines. d, Average MYC ChIP-seq signal at MYC-bound promoters and enhancers across four breast cancer cell lines. e, Spearman correlation of MYC binding across four breast cancer cell lines in MYC-bound enhancers (n = 89,631) and promoters (n = 10,049) called in at least one of the cell lines. Circle size and color indicate the correlation values (range: 0 to 1). f, Genome browser screenshot of MYC breast cancer subtype-specific enhancers defined in Fig. 1d. g, Accessibility of MYC breast cancer subtype-specific enhancers from Fig. 1d is shown for TNBC (n = 28) and ER+ (n = 109) breast cancer patients based on the TCGA ATAC-seq data13. Boxplots show the average accessibility of the given regions for patients in each group. P values were calculated using Welch’s two-sided t-test. Boxplot: Center line, median; box limits; upper and lower quartiles; whiskers, 1,5x interquartile range; outliers are not shown. h, Expression of genes located within 50 kb from any of the breast cancer subtype-specific enhancers defined in Fig. 1d in TNBC and ER+ breast cancers from TCGA14. Average expression of the gene groups for each patient (TNBC n = 292, ER+ n = 867) is summarized. P values were calculated using Welch’s two-sided t-test. Boxplot: Center line, median; box limits; upper and lower quartiles; whiskers, 1,5x interquartile range; outliers are not shown. i, Differential binding of MYC in response to 1 h fulvestrant treatment (100 nM) in MCF7 cells as determined by ChIP-seq. Red dots indicate significantly regulated binding sites (Padj < 0.05, two sided, FDR corrected using the Benjamini–Hochberg method in DiffBind). j, Protein expression of ER and MYC during 1 h fulvestrant treatment (100 nM) in MCF7 cells. β-tubulin is included as a control. The blot is representative of three independent biological replicates.

Source data

Extended Data Fig. 2 MYC enhancer and promoter binding target different gene programs.

a, Same as in Fig. 2a for MCF7 cells considering all genes within 50 kb of MYC binding sites. Lines represent the means and gray ranges represent 95% CI. b, Density of enhancer–promoter pairs involving MYC-bound enhancers over the promoter/enhancer ranking of putative MYC target genes in BT549 cells from Fig. 2a. Enhancer–promoter pairs were extracted from17. Line represents the mean and gray range represents 95% CI. Statistical testing was performed using one-way analysis of variance (ANOVA). c, Western blot validation of 72 h siRNA-mediated MYC knockdown and/or 2 h Dexamethasone (Dex) treatment in BT549 cells. β-tubulin is included as a control. Results from three independent biological replicates (Rep) are shown.

Source data

Extended Data Fig. 3 Validation of KJ-PYR-9.

a, Genome browser screenshot showing MYC ChIP-seq in response to KJ-PYR-9 treatment (3 h) for part of chromosome 8. The y-axis shows normalized FPKM counts. b, Differentially expressed genes (Padj < 0.05, two-sided, FDR corrected using the Benjamini–Hochberg method in DEseq2) upon KJ-PYR-9 treatment (3 h) in BT549 cells were identified and their response to KJ-PYR-9 was compared with the response to siRNA-mediated knockdown of MYC in a scatterplot. The color intensity indicates the density of genes in the scatterplot. c, Enrichment of three MYC target gene sets over genes ranked by Log2 fold change (Log2FC) in response to KJ-PYR-9 treatment (3 h) (part of the data from Fig. 4f) or siRNA-mediated MYC knockdown in BT549 cells. d, Percentage of ERCC spike-ins in RNA-seq libraries from BT549 cells upon siRNA-mediated knockdown of MYC or KJ-PYR-9 treatment (3 h) (part of the data from Fig. 4f) in three independent biological replicates. Bars represent mean and error bars represent s.e.m. P values were determined by paired Student’s t-test (two-sided).

Extended Data Fig. 4 Crosstalk between MYC and cancer type-specific transcription factors at enhancers.

a, Enrichment of a weak E-box motif (Extended Data Fig. 1c) in MYC-bound enhancers significantly activated or constitutively active as determined by MED1 ChIP-seq in response to KJ-PYR-9 treatment (3 h) in BT549 cells (see Fig. 3c). b, Average MYC ChIP-seq signal in BT549 cells at MYC-activated, MYC-repressed, and constitutively active enhancers as determined by MED1 ChIP-seq (see Fig. 3c). c, Percentage overlap of MYC enhancers and promoters with differential MED1 binding upon KJ-PYR-9 treatment (3 h) defined in Fig. 3b,c between BT549 and MCF7 cells. d, Giggle analysis22 on MYC-activated enhancers from MCF7 and BT549 cells (Fig. 3c). Bars indicate the mean of the observed Giggle scores and dots represent individual datasets. A high Giggle score indicates a high overlap with chromatin binding of a given transcription factor as determined by ChIP-seq. Only ChIP-seq experiments performed in TNBC or ER+ breast cancer cell lines were used for this analysis. e, Change in STAT3 footprint scores calculated from corrected cut counts within MYC-bound promoters (n = 3811) and enhancers (n = 6723) upon KJ-PYR-9 treatment (3 h) in BT549 cells. Boxplot: Center line, median; box limits; upper and lower quartiles; whiskers, 1,5x interquartile range; outliers are not shown. P value was calculated with Welch’s t-test, two-sided. f, MED1 binding in MCF7 cells upon KJ-PYR-9 treatment (3 h) at ER-bound MYC enhancers (see Fig. 1f).

Extended Data Fig. 5 Co-binding of MYC and GR on chromatin.

ChIP-seq heatmaps (left) and average line plots (right) for all common GR and MYC enhancer binding sites upon Dex treatment (2 h) in BT549 cells. GR Tag counts were normalized to a spike-in control.

Extended Data Fig. 6 MYC-mediated regulation of enhancer features.

a, Same as Fig. 5a for genes that are not regulated upon 3 h KJ-PYR-9 treatment (Padj > 0.2, two-sided, FDR corrected using the Benjamini–Hochberg method in DEseq2) without strong MYC binding at the promoter (Tag counts < 25) in BT549 cells. P value was calculated using Welch’s two-sided t-test. b, Average spike-in normalized MYC ChIP-seq signal in response to MYCMI-6 and MYCi975 treatment for 3 h in BT549 cells. The average signal for all MYC-bound enhancers and promoters is shown. c, Average ChIP-seq signal for total RNAPII at MYC-activated enhancers located within introns or intergenic regions in response to KJ-PYR-9 treatment (3 h) of BT549 cells. d, Changes in CAGE-seq signal in response to 3 h KJ-PYR-9 treatment in BT549 cells are plotted for MYC-activated, constitutive, and MYC-repressed enhancers as determined by changes in MED1 binding during KJ-PYR-9 treatment (see Fig. 3c). * P < 0.05 calculated using Welch’s two-sided t-test. e, eRNA within MYC-activated enhancers not located within genes (n = 1899) measured using rRNA-depleted (Ribo-Zero) RNA-seq in BT549 cells (part of the data from Fig. 4f). Boxplot: Center line, median; box limits; upper and lower quartiles; whiskers, 1,5x interquartile range; outliers are not shown. P value was calculated using Welch’s two-sided t-test. f, Heatmaps showing ATAC-seq and MED1 ChIP-seq signal at MYC-activated enhancers (see Fig. 3c) in response to KJ-PYR-9 treatment (3 h) in BT549 cells.

Extended Data Fig. 7 Loss of BRD4 binding at enhancers upon MYC inhibition.

a, Average BRD4 ChIP-seq signal at MYC-activated enhancers and gene promoters (see Figs. 3c and 5a) in response to MYCMI-6 and MYCi975 treatment (3 h) in BT549 cells. b, Western blot for BRD4 in response to KJ-PYR-9 treatment (3 h) in BT549 cells. β-tubulin is included as a control. Representative of two independent biological replicates. c, Western blot of BRD4 in response to MYCMI-6 and MYCi975 treatment (3 h) in BT549 cells. β-tubulin is included as a control. Representative of three independent biological replicates. d, Western blot showing BRD4, GCN5, MYC, and H3K9ac levels upon JQ1 treatment (1 h) in BT549 cells. β-tubulin is included as a control. Representative of two independent biological replicates.

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Extended Data Fig. 8 Loss of GCN5, H3K9ac, and KDM3A at enhancers upon MYC inhibition.

a, Venn diagram showing overlap between GCN5 (veh) and MYC (from Fig. 1a) binding as determined by ChIP-seq in BT549 cells (top). Genomic annotation of shared GCN5-MYC binding sites (bottom). b, Average GCN5 ChIP-seq signal at MYC-bound promoters and enhancers in BT549 cells. c, Western blot showing KDM3A, GCN5, MYC, H3K9me1/2, and H3K9ac protein levels upon KJ-PYR-9 treatment (3 h) in BT549 cells. β-Tubulin and H3 are included as controls. Representative of three independent biological replicates. d, Western blot showing GCN5, MYC, and H3K9ac upon MYCi975 treatment (3 h) in BT549 cells. β-Tubulin and H3 are included as controls. Representative of three independent biological replicates. Vertical black line indicates that lanes between the two conditions were cropped. e and f, Change in GCN5 binding at all MYC-bound promoters and enhancers in response to 3 h KJ-PYR-9 (e) or MYCi975 (f) treatment of BT549 cells. Black lines in the horizontal violin plots represent the median. P values were calculated using Welch’s two-sided t-test. g, Average ChIP-seq signal for GCN5 and H3K9ac at MYC-activated enhancers and gene promoters (defined in Figs. 3c and 5a) in response to MYCi975 treatment (3 h) of BT549 cells. h, Boxplot showing the change in KDM3A binding with KJ-PYR-9 treatment (3 h) as determined by ChIP-seq in BT549 cells at shared (KDM3A and MYC) (enhancers n = 10327 and promoters n = 7094) or KDM3A-only (enhancers n = 6320 and promoters n = 2138) enhancers and promoters. Boxplot: Center line, median; box limits; upper and lower quartiles; whiskers, 1,5x interquartile range; outliers are not shown. P values were calculated using Welch’s t-test, two-sided. i, Venn diagram showing overlap between KDM3A and MYC binding sites as determined by ChIP-seq in BT549 cells. The percentages of shared MYC-KDM3A binding sites in relevant sub-groups of the Venn are indicated. j, Western blot showing BRD4, GCN5, MYC, and H3K9ac levels in response to siRNA-mediated GCN5 knockdown in BT549 cells. β-tubulin is included as a control. Representative of two independent biological replicates. k, BT549 cell growth upon siRNA-mediated knockdown of GCN5 as determined by Incucyte live-cell imaging. The lines show the average confluency of 15 technical replicates and the error bars represent s.e.m. The data shown is representative of two independent biological replicates.

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Extended Data Table 1 Cancer type information for cell lines used in Fig. 3d
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Jakobsen, S.T., Jensen, R.A.M., Madsen, M.S. et al. MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch. Nat Genet 56, 663–674 (2024). https://doi.org/10.1038/s41588-024-01676-z

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