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. Author manuscript; available in PMC: 2020 Mar 9.
Published in final edited form as: Nature. 2019 Dec 18;577(7788):121–126. doi: 10.1038/s41586-019-1842-7

Fig. 4 |. Tumour mutations enhance ENL self-association to drive reinforced recruitment and gene activation.

Fig. 4 |

a, Testing of ENL self-mediated recruitment to the LacO array. mCherry–ENL can be recruited to the array only through self-association with eYFP-ENL-LacI proteins that have already been recruited. b, Fluorescence images of LacO-containing U2OS cells that have co-expressed various combinations of mCherry–ENL and eYFP-ENL-LacI. White dashed circles indicate the LacO array. Scale bar, 5 μm. c, Quantification of mCherry–ENL enrichment at the LacO array bound by various eYFP-ENL-LacI proteins. Enrichment of mCherry above an expression level of 1 suggests ENL-ENL sel-fassociation. Shown are means ± s.e.m.; n = 24, 9, 13, 14, 51, 62, 39, 38 cells from left to right; one-tailed unpaired t-test. d, Fluorescence images of HEK293 cells that express similar levels of WT or mutant mCherry–ENL. Scale bar, 5 μm. e, 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 intensity. Each dot represents one cell. f, Schematics of full-length (FL) or different deletion forms of ENL. AU, arbitrary units. h, ChIP with quantitative PCR (ChlP-qPCR) analysis of the indicated Flag–ENL constructs at HOXA genes in HEK293 cells. TSS, transcription start site. i, mRNA expression analysis (normalized to GAPDH) of HOXA genes in HEK293 cells expressing equal levels of the indicated ENL constructs. The data in panels h, i represent means ± s.e.m. from n = 3 technical replicates; independent experiments were repeated three times with similar results. j, During normal kidney development, wild-type ENL (ENLwt), a component of the SEC, binds to acetylated histone proteins in chromatin. The CDK9 component of the SEC phosphorylates RNA polymerase II (yellow circle on pol II), resulting in transcription appropriate to normal development. By contrast, mutant ENL (ENLmut) shows increased self-association and increased phosphorylation of pol II, resulting in aberrant gene activation that contributes to the development of Wilms tumour. Potential strategies to inhibit the oncogenic effects of ENL mutations are indicated by numbers 1–3.