ear1 ear2 879 264 tassel1+tassel2 - genes &...
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5632
879
264
4506 18226
Ear2 Ear1
Tassel1+Tassel2
Figure S1: Number of peaks identified by KN1 ChIP-seq in the four
biological replicates. A total of 6511 peaks, referred as high
confidence-bound loci, overlap between the two ears samples.
C
KN1 - + - + - + - + - +
0
2
4
6
8
10
1 2 3 4 5 6 7 8 9 10En
ric
hm
en
t re
lati
ve
to
tu
b
500 bp
A
0
20
40
60
80
100
+/- -/- +/- -/- a b c d e f
a b c d e f
lg3
Ear1
Ear2
Primers *
B73 Ears SAM Tassels
B
EMSA
ChIP-seq
ChIP-qPCR
Figure S2: ChIP-seq results and validation. (A) ChIP-seq identified KN1 binding in liguleless3 (lg3). Genic exon-intron
structures is shown with red arrow indicating gene orientation. The blue bars with letters a-f mark primer positions used for
qPCR in (B). Longer bars mark positions of probes used for EMSA in (C), while the star (*) shows the position of a short probe
used for EMSA (Supplemental Fig. 6). (B) ChIP-qPCR validation of KN1 binding at the lg3 locus. Primers in position “c” were
used for ChIP-qPCR in SAM and tassels (right). Enrichment is not detected in kn1-e1 (-/-). Error bars correspond to the
standard variation of two (SAM and tassels) or three (ears) biological replicates. (C) EMSA using a 1 kb probe (black bar) or
shorter overlapping probes (colored bars) from lg3 (A). Of the short probes, the light blue and pink probes generated the
strongest binding (colored arrowheads), consistent with the binding to a motif present in the overlap of the two probes [star (*)
in (A), “lg3” probe in Supplemental Fig. 6C)]. The weak binding detected with the red and green probes is occasionally
observed in regions not bound in vivo and considered as non-specific.
0
10
20
30
40
50
60
70
80
90
NB
149
_N
B15
0
NB
502
_N
B50
3
NB
434
_N
B43
5
NB
793
_N
B79
4
NB
498
_N
B49
9
NB
735
_N
B73
6
NB
781
_N
B78
2
KM
10
2_K
M1
03
KM
10
4_K
M1
05
KM
10
6_K
M1
07
KM
10
8_K
M1
09
KM
11
0_K
M1
11
KM
11
2_K
M1
13
KM
11
4_K
M1
15
KM
11
6_K
M1
17
KM
86
_K
M87
KM
88
_K
M89
KM
90
_K
M91
KM
92
_K
M93
KM
94
_K
M95
KM
96
_K
M97
KM
98
_K
M99
NB
366
_N
B36
7
NB
370
_N
B37
1
NB
376
_N
B37
7
NB
382
_N
B38
3
NB
396
_N
B39
7
NB
406
_N
B40
7
NB
414
_N
B41
5
NB
432
_N
B43
3
NB
442
_N
B44
3
NB
448
_N
B44
9
NB
454
_N
B45
5
NB
472
_N
B47
3
NB
504
_N
B50
5
NB
506
_N
B50
7
NB
747
_N
B74
8
NB
769
_N
B77
0
NB
787
_N
B78
8
NB
795
_N
B79
6
En
ric
hm
en
t re
lati
ve
to
tu
bu
lin
Ears WT
negatives
Figure S3: ChIP-qPCR validation in wild-type ears. Primer pairs labeled “negatives” are those called negative in all three wild-
type tissues tested in Supplemental Table 3. Errors bars are the standard variation of two biological replicates.
0
50
100
150
200
250
NB
149
_N
B15
0
NB
502
_N
B50
3
NB
434
_N
B43
5
NB
793
_N
B79
4
NB
498
_N
B49
9
KM
10
2_K
M1
03
KM
10
4_K
M1
05
KM
10
6_K
M1
07
KM
10
8_K
M1
09
KM
11
0_K
M1
11
KM
11
2_K
M1
13
KM
11
4_K
M1
15
KM
11
6_K
M1
17
KM
86
_K
M87
KM
88
_K
M89
KM
90
_K
M91
KM
92
_K
M93
KM
94
_K
M95
KM
96
_K
M97
KM
98
_K
M99
NB
366
_N
B36
7
NB
370
_N
B37
1
NB
376
_N
B37
7
NB
382
_N
B38
3
NB
396
_N
B39
7
NB
406
_N
B40
7
NB
414
_N
B41
5
NB
432
_N
B43
3
NB
442
_N
B44
3
NB
448
_N
B44
9
NB
454
_N
B45
5
NB
472
_N
B47
3
NB
504
_N
B50
5
NB
506
_N
B50
7
NB
747
_N
B74
8
NB
769
_N
B77
0
NB
795
_N
B79
6
En
ric
hm
en
t re
lati
ve
to
tu
bu
lin
Tassels WT
Tassels kn1-e1
negatives
Figure S4: ChIP-qPCR validation in wild-type (WT) and kn1-e1 tassels. Primer pairs labeled “negatives” are those called negative
in all three wild-type tissues tested in Supplemental Table 3. Errors bars are the standard variation of two biological replicates.
0
20
40
60
80
100
120
140
160
180
200
NB
149
_N
B15
0
NB
502
_N
B50
3
NB
434
_N
B43
5
NB
793
_N
B79
4
NB
498
_N
B49
9
KM
10
2_K
M1
03
KM
10
4_K
M1
05
KM
10
6_K
M1
07
KM
10
8_K
M1
09
KM
11
0_K
M1
11
KM
11
2_K
M1
13
KM
11
4_K
M1
15
KM
11
6_K
M1
17
KM
86
_K
M87
KM
88
_K
M89
KM
90
_K
M91
KM
92
_K
M93
KM
94
_K
M95
KM
96
_K
M97
KM
98
_K
M99
NB
366
_N
B36
7
NB
370
_N
B37
1
NB
376
_N
B37
7
NB
382
_N
B38
3
NB
396
_N
B39
7
NB
406
_N
B40
7
NB
414
_N
B41
5
NB
432
_N
B43
3
NB
442
_N
B44
3
NB
448
_N
B44
9
NB
454
_N
B45
5
NB
472
_N
B47
3
NB
504
_N
B50
5
NB
506
_N
B50
7
NB
747
_N
B74
8
NB
769
_N
B77
0
NB
795
_N
B79
6
KM
10
0_K
M1
01
En
ric
hm
en
t re
lati
ve
to
tu
bu
lin
SAM WT
SAM kn1-e1
negatives
Figure S5: ChIP-qPCR validation in wild-type (WT) and kn1-e1 SAM. Primer pairs labeled “negatives” are those called negative in
all three wild-type tissues tested in Supplemental Table 3. Errors bars are the standard variation of two biological replicates.
GA
P0
G
AP
1
GA
P2
G
AP
2b
G
AP
3
GA
P3
b
W
T
GA
P3
G
AP
3b
G
AP
3c
WT
WT
V
1
V2
V
3
V4
B
Bound
probe
Free
probe
MD1
MD4 MD3
MD2
A
WT
C
1/2
M
D1a
M
D1b
M
D2a
M
D2
b
MD
3a
M
D3b
M
D4
Free
probe
Bound
probe
C
ga2o
x1
lg3
ga-c
1
ga-c
1/2
kn
1-1
tb1
lg3
Bound
probe
Free
probe
ga2ox1 TGACTGATTGAC
lg3 TGATGATATGAT
ga-c1 TCGCTGATTGAC
ga-c1/2 TCGCTGATTCGC
kn1-1 TGACAGATAGAC
tb1 TGATGGATGGAC
Figure S6: see next page for legend
Figure S6: KN1 binds in vitro to motifs identified in the in vivo KN1-bound sequences. (A) We carried out motif
discovery starting from the high confidence KN1-bound regions. Motifs were identified that appear as two NGAC
sequences spaced by one or two residues, instead of the four residues found in the ga2ox1 binding site
(Supplemental Table 4). However, no KN1 binding was detected through EMSA assay using oligos derived from
these motifs (right). (B) EMSA comparing KN1 binding to the ga2ox1 binding site (labeled as “WT”) with the binding
to variants of the WT motif. (C) EMSA comparing KN1 in vitro binding to motifs derived from ga2ox1, lg3, kn1 and
tb1 (see Supplemental Table 4 for full probes sequences). The weak ga2ox1 variant ga-c1 could recruit KN1 in
planta as efficiently as the original ga2ox1 motif, suggesting that weaker motifs such as lg3 are sufficient to promote
KN1 binding in vivo.
0.E+00
1.E-05
2.E-05
3.E-05
4.E-05
5.E-05
up 10 kb up 1 kb 5'UTR exon intron 3'UTR down 1 kb down 10 kb
Pe
ak
de
ns
ity (
pe
ak
nu
mb
er/
bp
)
Figure S7: Detailed distribution of KN1 peaks around transcribed genes. Because exons, introns and UTRs vary in size,
the number of peaks was normalized relative to the size (bp) of each gene structure. The up/down 1 and 10 kb represent
all the peaks located from the UTR to 1 or 10 kb away, respectively. Thus, the “10 kb” categories also include the peaks in
the “1 kb” categories.
- + KN1 C
3000 bp
64 kb
tb1
Ear1
Ear2
*
A
*
B
-66 kb -65 kb -64 kb -63 kb -62 kb
Tourist Hopscotch
408 bp 4,885 bp
* TGATGGATGGAC
ChIP-seq
Figure S8: KN1 binding at the tb1 locus. (A) A binding site is detected 64 kb upstream of the tb1 transcription start, within
the region previously identified as responsible for morphological differences between maize and teosinte (58-69 kb
upstream of the transcription start; Clark et al 2006). The red bar marks the region used for the EMSA in (C). The star (*)
shows the position of the oligo probe used for the EMSA in Fig. S6C. Another peak is located 10 kb downstream of the
transcription end, in addition to a peak detected only in Ear1 in the 5’ of the gene. (B) KN1 binding site (red star) similar to
that found in ga2ox1 was identified immediately upstream of the Hopscotch transposon found to be important for tb1
expression (Studer et al 2011). Positions are relative to the transcription start site of tb1. (B) EMSA identifying KN1
binding in vitro (arrow) to the same region bound in vivo.
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
kn1 ga2ox1 lg3
Rela
tive
ex
pre
ss
ion
+/+
Kn1-N/+
Kn1-N/Kn1-N
Figure S9: Quantification of kn1, ga2ox1 and lg3 mRNA accumulation in leaf primordia from wild-type
(+/+) and Kn1-N seedlings. ga2ox1 and lg3 mRNA levels in Kn1-N leaf primordia follow a gradient of
expression proportional to kn1 overexpression. Error bars are the standard deviation of two biological
replicates.
+/+ Kn1-N/+ Kn1-N/Kn1-N
A B
2G087741
2G094241
2G452178
2G135447
2G102161
2G126018
2G074543
2G167824
2G005353
2G529859
2G116646
2G079080
2G021406
lg3
lg4a
gn1
knox8
zmm8
tsh4
yab9
yab10
yab14
yab15
yab5
ago
log
FDR < 5E-2 *
< 1E-2 **
< 1E-4 ***
< 1E-8 ****
-2.0
-1.75
-1.5
-1.1
1.0
1.1
1.25
1.5
2.0
20
40
♀ ♂ K/+ K/K GRMZM
0
0.01
0.02
0.03
0.04
0.05
Flu
ore
sc
en
ce
/ug
to
tal
pro
tein
+/+
Kn
1-N
/+
+/+
; D
R5
Kn
1-N
/+;
DR
5
*
C
argonaute GRMZM2G079080
E1
E2
E1
E2
1 kb
2 kb
lonely guy GRMZM2G021406 D
Figure S10: (A) Kn1-N phenotype. Shown are 10 days old seedlings, the stage used for sequencing. (B) Changes in
gene expression for selected direct targets in ears (♀), tassels (♂) or leaves [Kn1-N heterozygotes (K/+) and
homozygotes (K/K)]. (C) Quantification of fluorescence in protein extracts from immature leaves of Kn1-N/+ plants and
wild-type siblings segregating the DR5rev::mRFPer reporter (DR5). The difference between +/+;DR5 and Kn1-N/+;DR5
is statistically significant (*; P-value = 0.0003). Error bars correspond to the standard variation of 9 (non-DR5 siblings)
or 18 (DR5) biological replicates. (D) ChIP-seq identified KN1 binding in selected targets. Exon-intron structure is
shown with red arrows indicating gene orientation. Peak traces are shown for the two biological replicates of ears (E1-
E2).
bHLH GRMZM2G080054
Ear1
Ear2
5 kb
Figure S11: Peaks detected around gene GRMZM2G080054 (bHLH), which is up-regulated in ears (fold change
3.07, P value = 1.8E-06). Other genes in the vicinity are either not expressed, or expressed at a low level and
are not modulated.
MEME version 4
ALPHABET= ACGT
strands: + -
Background letter frequencies
A 0.275 C 0.225 G 0.225 T 0.275
MOTIF ga2ox1 like
letter-probability matrix: alength= 4 w= 12
0.000000 0.000000 0.000000 1.000000
0.000000 0.000000 1.000000 0.000000
1.000000 0.000000 0.000000 0.000000
0.000000 0.820000 0.000000 0.180000
0.100000 0.080000 0.320000 0.500000
0.000000 0.000000 1.000000 0.000000
1.000000 0.000000 0.000000 0.000000
0.000000 0.380000 0.000000 0.620000
0.250000 0.000000 0.250000 0.500000
0.000000 0.000000 1.000000 0.000000
1.000000 0.000000 0.000000 0.000000
0.000000 0.820000 0.000000 0.180000
Figure S12: Probability matrix used to identify KN1 binding sites in the KN1-bound loci and B73
genome.
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