Impaired cell fate through gain-of-function mutations in a chromatin reader

Abstract

Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by ‘reader’ proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia1. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour2,3, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.

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Fig. 1: ENL mutations drive aberrant developmental programs and impair nephron differentiation.
Fig. 2: ENL mutations enhance chromatin occupancy by ENL and associated SEC complex to enforce active transcription.
Fig. 3: Acylation-reading activity is required for enhanced chromatin occupancy by ENL T mutants.
Fig. 4: Tumour mutations enhance ENL self-association to drive reinforced recruitment and gene activation.

Data availability

The ChIP–seq and RNA-seq data have been deposited in the Gene Expression Omnibus database under accession number GSE125186. All other raw data generated or analysed during this study are included in this paper, the Extended Data figures, and the Supplementary Information.

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Acknowledgements

We thank R. Nishinakamura, A. Taguchi and Z. Li for providing reagents and discussions related to kidney differentiation assays; M. Leboeuf for technical assistance; A. Soshnev for help with figure preparation; and members of the Allis and Wen laboratories for scientific input throughout the study. We thank the Rockefeller University Genomic Resource Center, the Bio-Imaging Resource Center, the Flow Cytometry Resource Center and the MD Anderson Science Park Next-Generation Sequencing Facility (Cancer Prevention and Research Institute of Texas (CPRIT), grant RP120348). The research was supported by funds from the National Cancer Institute (grant 5R01CA204639-03), the Leukaemia and Lymphoma Society (LLS-SCOR 7006-13), and the Rockefeller University and St Jude Children’s Research Hospital Collaborative on Chromatin Regulation in Pediatric Cancer (to C.D.A.); from CPRIT (grant RP160237) and the Van Andel Institute (to H.W.); and from the National Institutes of Health (NIH grants R01HG007538, R01CA193466 and R01CA228140, to W.L.). L.W. is supported by the Jane Coffin Childs Memorial Fund and an NIH Pathway to Independence Award (1K99CA226399-01). M.V.O. is supported by a K12 Award from the National Cancer Institute (K12CA184746).

Author information

L.W. and H.W. conceived and designed the study. L.W., H.W., A.L. and F.X. performed most of the molecular and genomic studies. L.W. and A.L. performed kidney differentiation studies. S.C. and L.W. performed imaging studies with assistance from L.F. X.C., T.S.C., H.X. and W.L. performed bioinformatics analyses. Y.L., G.C. and H.L. performed the calorimetric studies. X.W. provided technical assistance. L.G., S.-P.W., S.K.D., R.G.R. and T.W.M. generated unpublished data. M.V.O. and A.K. provided discussion and assistance with pathology analysis. L.W. wrote the paper with critical input and edits from C.D.A., H.W., R.T., L.G., X.C., A.L. and S.C. C.D.A. and H.W. jointly supervised the research.

Correspondence to Liling Wan or Hong Wen or C. David Allis.

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Competing interests

C.D.A. is a co-founder of Chroma Therapeutics and Constellation Pharmaceuticals and a Scientific Advisory Board member of EpiCypher.

Additional information

Peer review information Nature thanks Tanja Mittag and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data figures and tables

Extended Data Fig. 1 ENL mutations induce transcriptional changes that are implicated in developmental programs and in Wilms tumour.

a, Bottom, ENL protein structure, with the region that is mutated in cancer shown in red. Above, amino-acid sequences of the T1 to T8 tumour-associated mutations and the corresponding WT region. b, c, Western blots showing the levels of ectopically expressed WT or mutant Flag–ENL proteins in HEK293 (b) and HK-2 (c) cells. Independent experiments were repeated four times with similar results. β-Tubulin is used as a loading control. d, e, Venn diagrams showing the number and overlap of genes for which expression is significantly upregulated upon expression of mutant ENL as compared with WT ENL in HEK293 (d) and HK-2 (e) cells. Genes with a fold change of 1.5 or more and a false discovery rate (FDR) of 0.01 or less are considered to be significantly upregulated. f, g, GSEA plots evaluating the changes in the indicated gene signatures upon expression of the indicated ENL mutants compared with WT in HEK293 (f) and HK-2 (g) cells. h, i, Volcano plots of RNA-sequencing data demonstrating the −log10 P-values versus log2 fold changes in HEK293 (h) and HK-2 (i) cells. HOXA genes are highlighted in red. P-values were determined by two-tailed exact test, adjusted by FDR. j, Western blot showing the close-to-endogenous levels of ectopically expressed WT or mutant Flag–ENL in HEK293 cells. Independent experiments were repeated three times with similar results. k, mRNA expression analysis (normalized to GAPDH) of selected ENL target genes in HEK293 cells (from panel j) expressing the indicated constructs. vec, vector control. Data represent mean ± s.e.m., n = 3 technical replicates, independent experiments were repeated three times with similar results. l, western blot showing the protein levels of ectopically expressed wildtype or indicated mutants (as illustrated in a) Flag-ENL in HEK293 cells. Experiment repeated three times independently with similar results. m, mRNA expression analysis (normalized to GAPDH) of selected ENL target genes in HEK293 cells (from panel l) expressing the indicated constructs. Vec, vector control. Data represent means from n = 2 technical replicates; results are representative of three independent experiments. For gel source data (b, c, j, l), see Supplementary Fig. 1. Source data

Extended Data Fig. 2 Three-dimensional nephron structures derived from mESCs.

a, Diagram showing the in vitro directed differentiation assay. Signature genes expressed at each step are shown at the bottom. T, the Brachyury gene. b, mRNA expression analysis (normalized to Gapdh) of the indicated genes at different time points during the assay. Data shown are representative of two independent experiments. c, Haematoxylin and eosin staining shows the induced embryoid body co-cultured with the spinal cord (sp). Green and red arrowheads point to nephric tubule and glomerulus, respectively. Scale bars: left, 500 μm; middle, 100 μm; right, 50 μm. d, Representative immunofluorescence staining of induced kidney structures for the nephric distal-tubule marker E-cadherin (green) and the glomerular marker WT1 (red). DNA is stained with DAPI (blue). Scale bar, 25 μm. e, Representative immunofluorescence staining of induced kidney structures for E-cadherin (green) and the proximal-tubule marker lotus tetragonolobus lectin (LTL, red). DNA was stained with DAPI (blue). Scale bar, 25 μm. f, Western blot showing the protein levels of ectopically expressed WT or mutant Flag-tagged ENL in mESCs. For gel source data, see Supplementary Fig. 1. For panels cf, independent experiments were repeated three times with similar results.

Extended Data Fig. 3 Characterization of ENL-mutant kidney structures.

a, d, g, Representative haematoxylin and eosin staining of the indicated kidney structures. b, e, h, Representative immunohistochemistry staining of the indicated kidney structures for the proliferation marker Ki-67. c, f, i, Representative immunohistochemistry staining of the indicated kidney structures for the mesenchymal marker vimentin. In panels c, f, the vimentin-positive cells shown are stroma cells. In panel i, the vimentin-positive cells shown are mostly blastema components. ac, WT epithelium; df, mutant epithelium; gi, mutant blastema. All experiments were repeated twice with similar results. Scale bars, 50 μm.

Extended Data Fig. 4 ENL mutants occupy largely the same genomic loci as the wild-type protein.

a, b, Bar graphs showing the genomic distribution of Flag–ENL-bound peaks in HEK293 (a) and HK-2 (b) cells. c, d, Heat maps of normalized WT or mutant Flag–ENL ChIP–seq signals in HEK293 (c) and HK-2 (d) cells, centred on ENL-bound peaks across a ±5-kb window. The colour key represents the signal density, with darker colour representing more signal. More details are in Supplementary Tables 4, 5. e, f, Venn diagrams showing the overlap of WT or mutant ENL-bound peaks in HEK293 (e) and HK-2 (f) cells. Source data

Extended Data Fig. 5 Enhanced occupancy of ENL mutants at a shared subset of target genes correlates with gene activation.

a, Venn diagram showing the number and the overlap of peaks with enhanced binding of individual mutant ENLs as compared with WT ENL in HEK293 cells. b, Heat maps of normalized WT or mutant Flag–ENL ChIP–seq signals in HK-2 cells, centred on mutant-enhanced peaks (fold change greater than 1.5) across a ± 5-kb window. More details are in Supplementary Table 7. c, Genome browser view of Flag–ENL ChIP–seq signals at selected target genes in HK-2 cells expressing indicated Flag–ENL transgenes. d, e, ChIP–qPCR of Flag–ENL at selected ENL target genes in two batches of HEK293 cells that are expressing the indicated ENL transgenes at levels higher than those of the endogenous ENL protein (d; see Extended Data Fig. 1b) or close to endogenous levels (e; see Extended Data Fig. 1j). Data in d represent means from n = 2 technical replicates, and are representative of three independent experiments. Data in e represent means ± s.e.m. from n = 3 technical replicates; independent experiments were repeated twice with similar results. f, GSEA plots showing that genes (n = 91; Supplementary Table 10) with enhanced occupancy of ENL mutants are upregulated in mutant-expressing HK-2 cells. Source data

Extended Data Fig. 6 Enhanced binding of ENL mutants at target genes leads to increased SEC recruitment and activity.

a, Western blot analysis of co-immunoprecipitation (IP) using the M2 anti-Flag antibody with lysates from HEK293T cells that are expressing the indicated Flag–ENL constructs. The experiment was repeated twice with similar results. For source data, see Supplementary Fig. 1. b, c, GSEA plots of genes ranked by their fold-change (mutant over WT) of CDK9 (b) or Pol II S2P (c) ChIP–seq signals in HEK293 cells, annotated against the set of genes (n = 87) that show increased occupancy of ENL mutants compared with WT. df, ChIP–qPCR analysis of CDK9 (d), Pol II S2P (e) and H3K79me2 (f; dimethylation of lysine 79 of histone H3) at selected ENL target genes in HEK293 cells that are expressing the indicated Flag–ENL constructs. Data represent means ± s.e.m.; n = 3 technical replicates. Experiments were repeated twice with similar results. g, mRNA expression analysis (normalized to GAPDH) of selected ENL target genes in HK-2 cells expressing the indicated constructs upon treatment with flavopiridol for 3 h. Increasing dosages (0, 125 nM, 250 nM and 1,000 nM) are depicted by grey wedges. Data represent means from n = 2 technical replicates. Experiments were repeated twice with similar results. Source data

Extended Data Fig. 7 Loss of PAF1 has minimal effect on the functionality of cancer-associated ENL mutants.

a, Western blot analysis of co-immunoprecipitation using the M2 anti-Flag antibody in lysates from HEK293T cells expressing the indicated Flag–ENL constructs. Results are representative of three independent experiments. b, c, mRNA expression (b) and western blot (c) analysis showing the knockdown efficiency of short interfering (si)RNAs that target PAF1 in HEK293 cells. Independent experiments were repeated twice with similar results. d, e, ChIP–qPCR analysis of Flag–ENL at selected ENL target genes in control (siCtrl) or PAF1 knockdown (siPAF1) HEK293 cells. f, mRNA expression analysis (normalized to GAPDH) of selected ENL target genes in control (siCtrl) or PAF1 knockdown (siPAF1) HEK293 cells. Data in df represent means ± s.e.m.; n = 3 technical replicates. For gel source data in panels a, c, see Supplementary Fig. 1. Source data

Extended Data Fig. 8 Interaction with histone acylation is essential but not sufficient for chromatin occupancy by cancer-associated ENL mutants.

a, Western blot showing the protein levels of ectopically expressed WT or mutant Flag–ENL in HEK293 cells. Experiments were repeated twice with similar results. For source data, see Supplementary Fig. 1. bd, Heat maps of normalized WT or mutant Flag–ENL ChIP–seq signals in HEK293 cells, centred on peaks that are enhanced in all three T mutants (as in Fig. 2a; n = 54) across a ± 5-kb window. e, f, ChIP–qPCR analysis of Flag–ENL at selected ENL target genes in HEK293 cells expressing the indicated constructs. Data represent means of n = 2 technical replicates. Independent experiments were repeated twice with similar results. g, Coomassie staining of purified WT ENL YEATS domain, and of YEATS domains with the indicated T mutations or a Y78A mutation. Data represent two independent experiments. h, i, ITC titration fitting curves for the indicated ENL YEATS domains (as in panel g), using a histone H3 peptide that is either acetylated at lysine 27 (H3(17-28)K27ac; h) or crotonylated at lysine 27 (H3(19-30)K27Cro; i). Source data

Extended Data Fig. 9 Characterization of nuclear puncta formed by ectopically expressed ENL tumour mutants.

a, Fraction of in-puncta fluorescence intensity in the nucleus of HEK293 cells that express WT or mutant mCherry–ENL, as a function of mean out-of-puncta nuclear intensity. Each dot represents one cell (the same experiment as in Fig. 4e). b, Dot plots showing the radius of puncta in HEK293 cells that are expressing similar levels of the indicated mCherry–ENL proteins. n = 20 independent puncta, randomly selected from four different cells per group. P-values were obtained using two-tailed unpaired Student’s t-test. Centre lines represent medians; whiskers indicate the minimum to maximum range. c, d, Fraction of in-puncta mCherry–ENL intensity in the nucleus as a function of mean nuclear intensity (c) or mean out-of-puncta nuclear intensity (d) in HEK293 cells that express the indicated mCherry–ENL proteins. Each dot represents one cell. e, Representative images from fluorescence recovery after photobleaching (FRAP) analysis in HEK293 cells expressing T3 mutant mCherry–ENL. The white dashed circles indicate the punctum undergoing targeted bleaching. Images represent 14 FRAP experiments in total with T1/2/3 mCherry–ENL. f, Averaged FRAP curves from areas inside the mCherry–ENL puncta formed by the indicated ENL mutants. Bleaching occurs at t = 0 s. Data represent means ± s.e.m.; n = 6 (T1), 5 (T2) and 3 (T3) distinct puncta from multiple cells. g, Fraction of in-puncta fluorescence intensity in the nucleus of HEK293 cells that express the indicated mCherry–ENL constructs as a function of mean nuclear out-of-puncta intensity. Each dot represents one cell (same experiment as in Fig. 4g). h, Western blot showing the protein levels of ectopically expressed Flag–ENL in HEK293 cells. Experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1. Source data

Supplementary information

Supplementary Information

.This file contains the Supplementary Methods and Supplementary Figure 1. Supplementary Figure 1 shows source gel images, one for each figure panel that contains immunoblot analysis, with the red box indicating the cropped region in the final figures.

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Supplementary Tables

This file contains Supplementary Tables 1-10.

Video 1

This video shows a fusion event of nuclear puncta formed by mutant mCherry-ENL (T3) in HEK293 cells. The video represents six independent events from each of the three T mutants.

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Wan, L., Chong, S., Xuan, F. et al. Impaired cell fate through gain-of-function mutations in a chromatin reader. Nature 577, 121–126 (2020). https://doi.org/10.1038/s41586-019-1842-7

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