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MYC phase separation selectively modulates the transcriptome

Nature Structural & Molecular Biology volume 31, pages 1567–1579 (2024)Cite this article

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An Author Correction to this article was published on 12 June 2024

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Abstract

Dysregulation and enhanced expression of MYC transcription factors (TFs) including MYC and MYCN contribute to the majority of human cancers. For example, MYCN is amplified up to several hundredfold in high-risk neuroblastoma. The resulting overexpression of N-myc aberrantly activates genes that are not activated at low N-myc levels and drives cell proliferation. Whether increasing N-myc levels simply mediates binding to lower-affinity binding sites in the genome or fundamentally changes the activation process remains unclear. One such activation mechanism that could become important above threshold levels of N-myc is the formation of aberrant transcriptional condensates through phase separation. Phase separation has recently been linked to transcriptional regulation, but the extent to which it contributes to gene activation remains an open question. Here we characterized the phase behavior of N-myc and showed that it can form dynamic condensates that have transcriptional hallmarks. We tested the role of phase separation in N-myc-regulated transcription by using a chemogenetic tool that allowed us to compare non-phase-separated and phase-separated conditions at equivalent N-myc levels, both of which showed a strong impact on gene expression compared to no N-myc expression. Interestingly, we discovered that only a small percentage (<3%) of N-myc-regulated genes is further modulated by phase separation but that these events include the activation of key oncogenes and the repression of tumor suppressors. Indeed, phase separation increases cell proliferation, corroborating the biological effects of the transcriptional changes. However, our results also show that >97% of N-myc-regulated genes are not affected by N-myc phase separation, demonstrating that soluble complexes of TFs with the transcriptional machinery are sufficient to activate transcription.

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Fig. 1: N-myc undergoes phase separation, which requires the intrinsically disordered TAD.
Fig. 2: N-myc condensates contain transcriptional markers including DNA-binding and dimerization partner, genomic DNA, transcriptional machinery and nascent RNA.
Fig. 3: Disassembly and assembly of N-myc condensates during mitotic entry and exit, respectively.
Fig. 4: Phase separation of full-length N-myc protein is enhanced by MAX and DNA oligonucleotides containing Myc E-box sequences.
Fig. 5: Both TAD and DNA-binding ___domain are required for transcriptional activity of N-myc condensates.
Fig. 6: Transcriptional role of N-myc phase separation revealed using the chemogenetic tool SparkDrop.
Fig. 7: SparkDrop-induced phase separation of N-myc regulates transcription of a small percentage of genes.

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Data availability

The RNA-seq raw data were uploaded to Gene Expression Omnibus database (accession number GSE259300). All other data are available within the text, Supplementary Information and source data section. Source data are provided with this paper.

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Acknowledgements

We thank H. Madhani, B. Huang, V. Ramani and E. Holland for their critical suggestions. This work was supported by National Institutes of Health (NIH) grants R01CA258237, U01DK127421 and R35GM131766 and a Benioff Initiative for Prostate Cancer Research Award (to X.S.); NIH grants P01CA217959, P30CA082103 and U01CA217864, and grants from the Alex Lemonade Stand, St. Baldrick and Samuel Waxman Cancer Research Foundations (to W.A.W.); U01DA052713 and R21DA056293 (to Y.S.); and ALSAC and the St. Jude Research Collaborative on the Biology and Biophysics of RNP granules (to T.M.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Junjiao Yang, Chan-I Chung.

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA

    Junjiao Yang, Chan-I Chung, Hao He, Zhimin Ma, Qian Zhao, Liang He & Xiaokun Shu

  2. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA

    Junjiao Yang, Chan-I Chung, Hao He, Zhimin Ma, Qian Zhao, Liang He & Xiaokun Shu

  3. Departments of Neurology, Neurological Surgery, Pediatrics, and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA

    Jessica Koach & William A. Weiss

  4. Institute for Human Genetics, Departments of Neurology, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA

    Hongjiang Liu, Xiaoyu Yang & Yin Shen

  5. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Ambuja Navalkar & Tanja Mittag

  6. Helen Diller Family Comprehensive Center, University of California, San Francisco, San Francisco, CA, USA

    Xiaokun Shu

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Contributions

X.S. conceived the project. J.Y., C.-I.C. and L.H. made the constructs. J.Y. performed N-myc phase separation and colocalization with other proteins in cells. C.-I.C. conducted imaging of small-molecule-induced N-myc phase separation and analyzed colocalization with other proteins. C.-I.C. performed and analyzed nascent RNA labeling, RT–qPCR and RNA-seq. J.Y., J.K., X.S. and W.A.W. planned and performed experiments to analyze expression of endogenous N-myc protein in the neuroblastoma cells. H.L. processed RNA-seq data. H.L., H.H., Z.M., C.-I.C, J.Y., Q.Z., X.Y., X.S. and Y.S. analyzed RNA-seq data. A.N. and T.M. designed and analyzed the in vitro experiments. A.N. conducted the in vitro experiments. J.Y., C.-I.C, T.M. and X.S. wrote the paper. All authors contributed to the final draft.

Corresponding author

Correspondence to Xiaokun Shu.

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Competing interests

X.S. and W.A.W. are co-founders of Granule Therapeutics. The other authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Robert Eisenman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Immunofluorescence images of Kelly cells treated with DMSO or MYC/MAX dimerization inhibitor MYCi975.

(Left) Kelly cells were incubated with DMSO or MYCi975 (30 μM) for 20 hours, followed by immunostaining with N-myc antibody. Stars (*) indicate representative cells. Triangle (∆) indicates few (that is un-representative) cells with high MYCN expression. Scale bar, 5 μm. (Right) Normalized average N-myc immunofluorescence intensity in DMSO or MYCi975 treated cells. Data are shown as mean ± SD. N = 3 biological replicates.

Source data

Extended Data Fig. 2 Neuroblastoma cell lines with various degrees of MYC (N-myc, c-MYC) protein expression.

Western blot analysis of MYC proteins in various neuroblastoma cells.

Source data

Extended Data Fig. 3 Estimation of N-myc concentration in the Kelly cells.

The overall procedure is (a) calculation of an mEGFP concentration-brightness standard curve using purified mEGFP protein. (b) Western blot analysis to determine relative amount of N-myc in Kelly vs SH-EP cells. (c) Fluorescence imaging (Hoechst)-based measurement of the nuclear volume of SH-EP and Kelly cells. The N-myc-mEGFP concentration in SH-EP cells is determined by comparing the fluorescence intensity of N-myc-mEGFP versus that of the purified mEGFP. (d) Estimation of the N-myc concentration in the nucleus of Kelly cells. Data are mean + SD (n = 20 cells). Scale bar, 10 μm.

Source data

Extended Data Fig. 4 Relationship of N-myc condensate number and size to the N-myc protein levels.

(a) Number of N-myc condensates was plotted against the N-myc protein level per nucleus. SH-EP cells expressing N-myc-mEGFP were imaged under the spinning disc confocal microscope. (b) Size of N-myc condensates was plotted against the N-myc protein level in single nucleus. (c) SH-EP cells with different expressing levels of N-myc-mEGFP. Images are max-projected to show all condensates. Dim cells are shown with brightness adjustment as listed. Scale bar: 5 μm.

Source data

Extended Data Fig. 5 Endogenous N-myc condensates contain DNA-binding and dimerization partner, transcriptional machinery and nascent RNA but do not co-localize with c-Jun.

Immunostained N-myc condensates colocalized with MED1 (a), Pol II S5p (b), MAX (c), nascent RNA (d) and c-Jun (e). Kelly cells were stained with antibody against N-myc, and antibodies against MED1, Pol II S5p, or c-Jun, or co-expressed with MAX-mKO3, or labeled by 5-ethynyluridine for labeling nascent RNA. The arrows point to representative condensates. The fluorescence intensity profile (right panel) is extracted from the position shown by the dashed line. Co-localization assessment by Pearson’s correlation coefficient is shown in right panels. Center lines show the median values. Green boxes contain the 25th to 75th percentiles of dataset. Black whiskers mark the 10th and 90th percentiles. Outliers are marked with grey dots. NMED1 = 76, NPol II-S5P = 67, NMAX = 61, Nnascent RNA = 82 cells, Nc-Jun = 67 cells. Scale bars, 5 μm.

Source data

Extended Data Fig. 6 Condensate number and size related to protein levels of the truncated N-myc lacking the DNA-binding ___domain.

(a) Number of N-myc1–365 condensates was plotted against the N-myc protein level per nucleus. (b) Size of N-myc1–365 condensates was plotted against the N-myc protein level in single nucleus. (c) SH-EP cells with different expressing levels of N-myc1–365-mEGFP. Images are max-projected to show all condensates. Dim cells are shown with brightness adjustment as listed. Scale bar: 5 μm.

Source data

Extended Data Fig. 7 MAX contributes to N-myc phase separation.

(a) MAX was knocked out by sgRNA in SH-EP cells expressing MYCN-mEGFP. Top left: SPARK value against N-myc concentration in wild type SH-EP cells expressing MYCN-mEGFP. Top middle: SPARK value against N-myc concentration in MAX-KO SH-EP cells expressing MYCN-mEGFP. Lower left: western blot showing MAX protein level in wild type (WT) and MAX-KO (sgMAX) SH-EP cells. Lower middle: SPARK value in MAX-KO SH-EP cells expressing MYCN-mEGFP and MAX-mKO3. Right panel: N-myc saturation concentration in WT, MAX-KO and rescued SH-EP cells. The MAX-KO SH-EP cells were rescued by expressing MAX-mKO3. Center lines show the median values. Boxes contain the 25th to 75th percentiles of dataset. Whiskers mark the minimum and maximum values. NWT = 40, NsgMAX = 49, NsgMAX+MAX-mKO3 = 54. P value, two-sided non-paired t-test. (b) Left: SPARK value against N-myc concentration in SH-EP cells expressing MYCN-mEGFP. Right: SPARK value against N-myc concentration in SH-EP cells expressing MYCN-mEGFP and MAX-mKO3.

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Extended Data Fig. 8 Phase separation of full-length N-myc protein is enhanced by DNA oligonucleotides containing non-canonical Myc E-box sequences.

(a) Fluorescence anisotropy assay showing binding of the N-Myc / MAX heterodimer to non-canonical E-box DNA in 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM DTT, and 0.01% NP-40 containing buffer. The concentration of fluorophore-labeled oligonucleotide DNA containing 1 NCE-box sequence (CATCTG or CATATG) was fixed at 50 nM and a ratio of Myc / Max of 3:1 was used. The dissociation constant (Kd) of the interaction between proteins and DNA was determined by fitting the experimental data in Origin Pro 8.0 {Roehrl, 2004 #1}. MAX/CEbox and N-myc/MAX/Cebox data are the same as in Fig. 4b. (b) Brightfield microscopy images of solutions containing the N-myc / Max complex (10 µM Myc / 3 µM Max) in the presence of 1NCEbox (26-mer), and 7NCEbox (182 mer) DNA in 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM DTT. Purple boxes highlights the images with N-myc phase separation observed. The images show similar phase separation as with canonical E-box sequences. The scale bar is 5 μm.

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Extended Data Fig. 9 Saturation concentration of SparkDrop-tagged N-myc and the stable N-myc/SparkDrop SH-EP cells used for the RNAseq experiments.

(a) Saturation concentration of SparkDrop-tagged N-myc with and without lenalidomide. Each green dot represents data from a single cell (~200 cells for each group). The concentration was calculated based on the green fluorescence of mEGFP in SparkDrop. (b) Representative images of stable SparkDrop-MYCN cells before and 30 minutes after 1 µM lenalidomide (lena) incubation. Scale bar: 20 µm. (c) N-myc concentration is 0.20 ± 0.01 µM (Mean ± SD). N = 200 cells.

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Extended Data Fig. 10 RT-qPCR of SERINC2 mRNA in SH-EP cells expressing N-myc/SparkDrop treated with 1 µM lenalidomide for different duration.

Data are shown as mean ± SD. N = 3 biological independent replicates. P value, two-sided non-paired t-test.

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Supplementary information

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Supplementary Table 2: List of DEGs from RNA-seq.

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Yang, J., Chung, CI., Koach, J. et al. MYC phase separation selectively modulates the transcriptome. Nat Struct Mol Biol 31, 1567–1579 (2024). https://doi.org/10.1038/s41594-024-01322-6

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