Nature - USA (2020-01-02)

Nature | Vol 577 | 2 January 2020 | 125

mCherry

eYFP–LacI

WT

WT T1 T2 T3

T1 T2 T3 WT

LacI alone

T1 T2 T3

d

e

j

f

g

hi

a

c

b

WT T1 T2 T3

eYFP–WT–LacI

mCherry–WT

Merged Merged Merged Merged

mCherry–T1 mCherry–T2 mCherry–T3

eYFP–T1–LacI eYFP–T2–LacI eYFP–T3–LacI

eYFP ENL LacI

mCherry ENL

LacO

ENL

LacI

WT T1 T2 T3

T1(FL)

T1(YEATS)

T1(ΔIDR)

SEC

Ac Pol II

ENLmut

SEC

Pol II

Ac

ENLwt

Normal development Tumorigenesis

T1(ΔAHD)

YEATS IDR AHDWT(FL) Chromatin occupancy Gene expression

0.0 102 103 104

0.2

0.4

0.6

Mean nuclear intensity (AU)

In-punct

a intensity fraction

Self-association

2 3

1

0

2

4

mCherry enrichment of

the LacO array

P = 0.004

P = 0.026

P = 0.031

0

1

2

3

4

5

Rela

tive expres

sion

HOXA1 1

0

2

4

6

8

10

Rela

tive expres

sion

HOXA 13

0.0

0.1

0.2

0.3

0.4

0.5

Input (%)

HOXA 11 TSS + 1 kb TSS + 3 kb

0.0

0.1

0.2

0.3

Input (%)

HOXA1 3 TSS + 1 kb TSS + 4 kb

0.0 1 × 10 (^2) 1 × 10 (^3) 1 × 10 4
0.2
0.4
0.6
0.8
In-puncta intensity fractioMean nuclear intensity (AU)
n
WTT1
T2
T3
T1(FL)
T1(YEATS)
T1(ΔIDR)
T1(ΔAHD)
WT(FL)
T1(FL)
T1(YEATS)
T1(ΔIDR)
T1(ΔAHD)
WT(FL)
Fig. 4 | Tumour mutations enhance ENL self-association to drive reinforced
recruitment and gene activation. 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 self-
association. 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 f luorescence 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 (ChIP–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.