Nature - USA (2019-07-18)

Letter reSeArCH

7.5mins 10mins

Pre 0mins 2.5mins

5.0mins

FOXA1-P375fs

7.5mins 10mins

Pre 0mins 2.5mins

5.0mins

FOXA1-A287fs

C-terminal domain-disrupted Class2 mutants

TD Forkhead RD

50 100 150 200 250 300 350 400 450

0

2

4

6

8

10

missense inframe frameshift

Class1 FOXA1

mutations

Wing1Winggggggggggggggggggggggggggggggggggg 1

H1

H2

H3

Wing2

M253

I176

D226

C227

V229

H247

S250

FOXA3 FKHD

Crystal struture:

3D-mutation hotspot

residues (Gao et al. 2017)

a

Wing2 (H247-E269aa)

FOXA1 WT

(n=2)

FOXA1 Class1

(I176M and R261G)(n=4)

Union FOXA1 cistromes

10420 4701

0% 20% 40% 60%

% peaks with core FOX motif

29572

FOXA3 FKHD: DNA-contacting residues -

Major Groove (Clark et al. 1993 & Li et al. 2017)

DNA-contacting residuesin FOXA2 & FOXA3

(Clark et al. 1993 & Li et al.2017)

DNA-contacting residues only in FOXA3

(Clark et al. 1993)

R213

N216

S217

H220

S223

R219

W244

K230

S242

S217

Major

Groove

Mobility

Fraction of molecules

log [D(μm^2 /s)]

Slow Fast Unbound

0.0

0.1

0.2

0.0

0.1

0.2

0.0

0.1

0.2

FOXA1-WT

FOXA1-R261G

FOXA1-I176M

-4 -3 -2 -1 01

FOXA1AR

544605632676

(22RV1 + FOXA1-WT )

FOXA1AR

477486547975

(22RV1 + Class1 mutant)

Wing2-disrupted Class1 mutants

FOXA1-R265-71del

1.0mins 2.0mins

Pre 0mins 0.25mins

0.50mins

FOXA1 WT cistrome

1234567891011121314151617

0

0.5

1

1.5

2

Info

rmation content

(^123456789) Position 1011121314151617
0
0.5
1
1.5
2
Position
FOXA1 class1 mutant cistrome
WT
0255075 100
Percentage(%)
Promoter(<=1kb)
Promoter(1−2kb)
Promoter(2−3kb)
5' UTR
3' UTR
1st Exon
Other Exon
1st Intron
Other Intron
Downstream(<=3kb)
Distal Intergenic
Genomic feature distribution
Class1
010203040 50
BORIS
TATA−Box
SP1
NF1:FOXA1
AP−2gamma
CTCF
NKX3.1
GATA 3
GATA2
HOXB13
GRE
AR−halfsite
ARE
FOXA1:AR
% of peaks with motif matches
FOXA1
FOXA1:AR
ARE
AR−halfsite
GRE
HOXB13
Gata2
GATA 3
Nkx3.1
CTCF
AP−2gamma
NF1:FOXA1
Sp1
TATA−Box
BORIS
Odd−ratio−1024
Mutant
Wildtype
WT
Class1
1 2 3 4 5 6 7 8
FOXA
ARID5
FOS/JUN
HOXC
NFI
HOXB
1e-7361
1e-646
1e-520
1e-324
1e-321
1e-222
67.77%
3.21%
6.77%
8.12%
26.67%
14.10%
CTCF
ARE(NR)
1e-214
1e-152
2.47%
FOXA1-Class1 cistrome 3.44%
Motif Best match P-value with motif%peaks
De novo
motif analyses (HOMER)
Rank
1 2 3 4 5 6 7 8
FOXA
ARID5
FOS/JUN
CTCF
NFI
HOXB
1e-10729
1e-1788
1e-666
1e-443
1e-431
1e-372
67.90%
12.46%
5.29%
1.97%
23.83%
17.31%
SP1
HNF4a(NR)
1e-153
1e-138
1.60%
1.08%
FOXA1-WT
cistrome
FOXA1-WT
FOXA1-R261G
Unbound
Fast
Slow
58.5%37.7%
3.8%
42%55.2%
2.8%
45.9%41.6%
12.5%
FOXA1-I176M
FOXA1-WT
Sample
FOXA1-R261G
FOXA1-I176M
Fast (s)Slow (s)
1.06 ± 0.08
0.78 ± 0.09
0.65 ± 0.07
8.32 ± 0.84
7.63 ± 0.64
3.85 ± 0.56
Class1
0
2
4
6
8
10
(^12) Vector
FL
I176M
R261G
HPRT1 FOXA1
Relativ
e expression
b
c
FOXA1-R261GFOXA1-R265-71del
FOXA1-A287fs
FOXA1-WT
FOXA1-I176M
FOXA1-P375fs
PhalloidinDAPI
PhalloidinDAPI Merged
FOXA1-WT-GFP
PhalloidinDAPI
FOXA1-P375fs-GFP (Class 2)
FOXA1-I176M-GFP (Class 1)
GFP
GFP
GFP
Representative FRAP nuclei
d
Merged
Merged
0
20
40
(^60) eGFP
WT
I176M
R261G
Top de novo motif
29335
FOXA1-WT 10217
FOXA1-I176M
FOXA1-R261G
14873
3157
951
835
WT cistrome
I176M cistrome
R261G cistrome
1e-4367
1e-2962
1e-2021
p-value %sites with motif
64.95%
67.79%
58.05%
HPRT1 FOXA1
Relati
ve expression
j
n
0.0 012345678910
0.2
0.4
0.6
0.8
1.0
Time (mins)
WT
P375fs
I176M
R261G
R265-71del
t1/2
Relative fluorescence post-bleaching
Class1
WT
e
f
ghi
klm
o
p q
rs
1.0mins
0mins
2.0mins
Pre 0.25mins
0.50mins
FOXA1-I176M
6207
0.0
0.5
1.0 UnBleachbleacheded
Normalized uorescent intensity
Time (mins)
0.0
0.5
1.0
0.0 4.9 9.6
0.0
0.5
1.0
Normalized uorescent intensity
0.0
0.5
1.0
UnBleachbleacheded
0.0 Time (m4.9ins) 9.6
Extended Data Fig. 3 | See next page for caption.