a maize gene regulatory network for phenolic metabolism · a maize gene regulatory network for...
TRANSCRIPT
A Maize Gene Regulatory Network For Phenolic Metabolism
Fan Yanga,b, Wei Lib,c, Nan Jianga,b, Haidong Yua,b, Kengo Morohashia,b, Wilberforce
Zachary Oumaa,b,d, Daniel E Morales-Mantillab,c,e, Fabio Gomez-Canoa,b, Eric Mukundia,b,
Luis Daniel Prada-Salcedob,c, Roberto Alers Velazquezb,c,e, Jasmin Valentinb,c,e, Maria
Katherine Mejía-Guerraa,b, John Grayf, Andrea I Doseffb,c, Erich Grotewolda,b
aCenter for Applied Sciences (CAPS), The Ohio State University, Columbus, OH 43210; bDepartment of Molecular Genetics, The Ohio State University, Columbus, OH 43210; cDepartment of Physiology and Cell Biology, Heart and Lung Research Institute, The
Ohio State University, Columbus, OH 43210; dMolecular, Cellular, and Developmental Biology (MCDB) Graduate Program, The Ohio
State University, Columbus, OH 43210. eSuccess in Graduate Education (SiGuE) Program, The Ohio State University, Columbus,
OH 43210. fDepartment of Biological Sciences, University of Toledo, Toledo, OH 43560.
A gene-centered approach based on the yeast one-hybrid assay revealed ~1,100
transcription factor-DNA interactions associated with the regulation of maize phenolic
compound biosynthesis. Results were validated by a combination of chromatin
immunoprecipitation and transient regulation. Several transcription factors recognize the
promoters of 10 or more genes, suggesting a role in coordinating phenolic metabolism.
1To whom correspondence should be addressed. E-mail, [email protected]; FAX :
(614) 292-8037; TEL (614) 292-2483
918 bps
E1 PAL3: GRMZM2G160541
-801 +116 +119 -144
-855 +122 +134
978 bps
E1 PAL4: GRMZM2G063917
-415 -792
+123 958 bps
E1 PAL7: GRMZM2G170692
-841 +116
903 bps
E1
PAL5: GRMZM2G081582
-763 +139 +178 +587 +2177
E2 ?
Cloned region
CAGE tags
TSS CAGE
TSS Annotation
LINEs
Dust
MuDR
Copia
hAT
TRF
ADN/EnSpm
Unknown
ADN/hAT
Gypsy
Helitron
RTE
DNA-TA
DNA/Harbinger
919 bps
E1 PAL9: GRMZM2G029048
-816 +102 +167 -549
-889 +93
C4H1: GRMZM2G147245
+111 982 bps
E1 -237 -472 -747
+906
904 bps
E1 PAL2: GRMZM2G441347
-870 +33 +100 -42 -187 -459 -645
941 bps
E1 C4H2: GRMZM2G139874
-938 +2 -39 +61 -174 -501 +187 -588
P1 P2
-402 -519
-886 +34 921 bps
E1 PAL1: GRMZM2G074604
-151 +109 -449 -683
P1 P2
-568 -648
P1 P2
-456 -535
P1 P2
-324 -414
Supplemental Figure 1
953 bps
E1 PAL6: GRMZM2G118345
-794 +158 -552 -6 -53 +159
P1 P2
-289 -398
951 bps
E1 PAL8: GRMZM2G334660
-874 +76 -345 +113 -392 -618 -298
P1 P2
-374
+126 961 bps
E1 4CL2: GRMZM2G174574
-934 +126
-517
500 bps
E1 4CL4: GRMZM2G122787
-490 +9 +206 -101 -262
902 bps
E1 HCT5: GRMZM2G158083
-679 +221 +247 -164 -410
942 bps
E1 HCT6: GRMZM2G035584
-787 +156 -199 -275 -504 +213
+84 957 bps
E1 HCT10: GRMZM2G034360
-875 +84
ATG
+208 -431 -516 -656 -716
921 bps
HCT11: GRMZM2G156296
-932 -12
+82 -56 -217 -408 E2 E1
+512 +9124 +7682
500 bps
E1 HCT12: GRMZM2G179703
-794 -296 +59
+43 1811 bps
E1 +480
+566
HCT13: GRMZM2G129266
-1244
922 bps
E1 CCoAOMT1: GRMZM2G033952
-890 +31 +82 -234
922 bps
E1 4CL1:GRMZM2G055320
-996 -76 +143 -339
ATG ATG
+243 +906 -407
500 bps
E1 4CL3: GRMZM2G096020
-550 -51 +145 -396
P1 P2
-338 -216
P1 P2
-352 -492
P1 P2
-220 -308
P1 P2
-245 -343
Supplemental Figure 1 (cont.)
910 bps
E1 CCR1: GRMZM2G099420
-827 +82 +113 -585
910 bps
E1 CCR4: GRMZM2G131836
-735 +173 +196
937 bps
E1 CAD1: GRMZM2G046070
-827 +109 -133 +123 -63
907 bps
E1 CAD3 (bm1): GRMZM5G844562
-838 +68 +142 -154 -417 -628 -683 -765
924 bps
E1 CCoAOMT2: GRMZM2G099363
-844 +79 +104 -614
941 bps
ALDH5: GRMZM2G097706
-846 +94
-391 +111 -712 E2 E1
901 bps
E1 F5H1: AC210173.4_FG005
-840 +60 +103 -164 -511
953 bps
E1 COMT2: GRMZM2G082007
-936 +13 +359 -419 -263 -340 +15
P1 P2
-377 -452
P1 P2
-230 -329
Supplemental Figure 1 (cont.)
921 bps
E1 CCR3: GRMZM2G131205
-654 +266 +100 -237 -19 +151
E2 +324
ATG ATG
-324 -420 -566
P1 P2
-128 -216
901 bps
E1
ALDH1 (rf2): GRMZM2G058675
-735 +165 +190 -163 +2770
E2 +290
P1 P2
-242 -344
812 bps
E1
COMT1 (bm3): AC196475.3_FG004
-810 +1 +114 -83 -392 -725
P1 P2
-460 -558
WHP1: GRMZM2G151227
941 bps
E1
-787 +51 -472 +119 -498 -667
E2
+63 1095 bps
E1 -358 -775 -1004
CHI1: GRMZM2G155329
-1080
929 bps
E1 F3H1 (fht1): GRMZM2G062396
-866 +62 +115 -788
869 bps
E1 FNS1 (fnsi1): GRMZM2G099467
-770 +98 +110 -164
927 bps
E1 F3’H (pr1): GRMZM2G025832
-933 -4 +59
982 bps -982 -1
E1 F3’H3: GRMZM2G313750
-184 -283 +37
FLS2: GRMZM2G069298
+34 1500 bps
E1 -491 -709 -1326
-1464 +33
+36 969 bps
E1
FLS1: GRMZM2G152801
-934 +35
-530 -872
P1 P2
-532 -652
915 bps
E1 F3’H2: GRMZM2G160763
-851 +63 +66 -189 -327 -488
P1 P2
-97 -197
-256 -380 794 bps
E1 C2: GRMZM2G422750
-781 +12 +725 -673
P1 P2
Supplemental Figure 1 (cont.)
+23 988 bps
E1 F3H2: GRMZM2G146234
-976
A1: GRMZM2G026930
+87 1866 bps -776
E1 E2 +220 +315 +497
-1370
-135 -763
+496
A2: GRMZM2G345717
-1186 +4
1122 bps
E1 -167 -554
438 bps
E1 A4: GRMZM2G013726
-329 +106 +43 -101 -262
911 bps
E1 ANR1: GRMZM2G097841
-738 +173 +241 -699
500 bps
E1 Bz1: GRMZM2G165390
-491 +8 +81 -344
500 bps
E1 Bz2: GRMZM2G016241
-582 -83 +7 -136 -337
+1134 992 bps
E1
-28 -537
RHM1 (sm1): GRMZM2G031311
-1018 -217 -293
937 bps
E1 RHM2: GRMZM2G072911
-887 +49 -233 +112 -63 -360
+890 997 bps
E1
-48 -967
RHM4: GRMZM2G166767
-1039
-245 -161
P1 P2
P1 P2
-46 -558
Supplemental Figure 1 (cont.)
+953 1000 bps
E1 +1 +502 +88
RHM3: GRMZM2G044281
ATG
+825 -46
+551 +599 +843
ATG
+952
Supplemental Figure 1. Graphic representation of the gene promoters used as baits in the Y1H screens. Representation of the promoters of the 54 genes in the phenolic biosynthetic pathway used in this study. Gene name and model are shown in the top. Transcription start sites (TSSs; indicated by +1) were obtained from CAGE data (red arrows) (Mejia-Guerra et al., 2015), or from the latest genome annotation (V3.0, http://www.maizegdb.org/) (black arrows), respectively. Repetitive elements (RE) present in the cloned regions used in the Y1H assays (represented by a blue line) are in accordance with the annotation available in MaizeGDB and are represented by the colored boxes following the criteria shown in the legend box. Exons (E; gray rectangles) are linked with UTRs (thinner gray rectangles). P1 and P2 represent the primers used for ChIP-qPCR experiments.
Num
ber o
f pla
smid
s pe
r ng
1.0E+00
1.0E+02
1.0E+06
1.0E+04
ALF
18O
FP41
MY
B11
8H
AG
15M
YB
19
GR
AS
70
YH
AB
5bH
LH14
5LB
D9
HS
F3W
RK
Y82
OR
PH
AN
314
ZIP
84
GB
P17
ZIM
32
bHLH
10M
YB
108
C3H
21
Supplemental Figure 2. Representation of TFs in the AD-TF library.Number of plasmid molecules present per ng of AD-TF library, as determined by qPCR using specific standards, are shown in blue. Number of promoters to which each TF was found to bind are shown in red. Values represent means±SE of three replicates.
1.0E+01
1.0E+05
1.0E+03
0
20
15
10
5
25
30
Num
ber o
f pro
mot
ers
in
whi
ch T
F w
as id
entif
ied
bHLH
1
LBD
1LI
M6
LBD
20LB
D35
BA
F60.
21bH
LH17
7C
PP
8M
YB
31M
YB
40
OR
PH
AN
249
HB
112
MY
B3
Number of plasmids per ng
Random selected TFs TFs of interest
Number of promoters in which TF was identified
PAL5
PAL1
CO
MT2
F5H
1
CC
R4
CC
R3
CAD
3 (b
m1)
ALD
H5
CC
R1
CO
MT1
(bm
3)
ALD
H1
(rf2)
CAD
1
CC
oAO
MT2
CC
oAO
MT1
HC
T13
PAL2
4CL3
HC
T12
HC
T11
HC
T10
HC
T6H
CT5
4CL4
4CL2
4CL1
C4H
1PA
L9PA
L8PA
L7PA
L6
PAL4
PAL3
F3’H
2F3
’H (p
r1)
FNS1
(fns
i1)
F3H
2 F3
H1
(fht1
)C
HI1
WH
P1C
2
A4A1FLS2
FLS1
F3’H
3
RH
M3
RH
M4
RH
M2
RH
M1
(sm
1)Bz
2Bz
1AN
R1
A2C4H
2
Promoter TestedGeneral
Lignins Flavonoids Transcription Factors
Phenylpropanoids
Frequency
Direct Interactions
0
0.4
0.2
0.1
0.3
Supplemental Figure 3. Heat map representing PDIs identified by Y1H. Genes encoding phenolic pathway enzymes are shown on top. The 568 TFs identified as recognizing one or more of the promoters indicated in the columns are listed on the right. The brown-yellow gradient represents PDIs identified from the Y1H screenings, with the color corresponding to the frequency by which a particular TF was identified. Blue represent PDIs identified from Y1H directed assays. TFs are ordered from most (top) to least (bottom) interactions. All the data used to generate this heat map is available as Supplemental Table 2.
*
*
*
0 2015105
*
*
0 252015105
*
0
*
54321A B
SD-His-Ura-Leu+ 3AT (mM)
Fold enrichment over input
p35S::GFP-JMJ13p35S::GFP
p35S::GFP-MBF1.3p35S::GFP
p35S::GFP-GBP20p35S::GFP
Copia
ALDH1
4CL3
C2
Copia
4CL3
PAL5
Bz2
Copia
FLS1
CCoAOMT2
C2
*
*
*
0 12963 15
p35S::GFP-DOF28p35S::GFPCopia
4Cl3
CCR3
HCT10
C2
2
567
8
43
1
HCT10 CCR3
C2 4CL3
1,3,5,7: EV2,4,6,8: AD-DOF28
21
56 3
4
1,3,5: EV2,4,6: AD-GBP20
FLS1CCoAOMT2
C2
21
5
Bz2
4CL3 PAL5
21
5
C2
ALDH1 4CL3
1,3,5: EV2,4,6: AD-JMJ13
1,3,5: EV2,4,6: AD-MBF1.3
6 34
6 3
4
60
80 60
60 20
100 60
40
40
60 60
SD-His-Ura-Leu
60
120
Supplemental Figure 4. Comparison of ChIP and Y1H validation of select PDIs(A) Plates show the growth of various promoter bait yeast strains transformed with AD-JMJ13, AD-MBF1.3, AD-GBP20 and AD-DOF28 in non-selective (SD-His-Ura-Leu) and selective (SD-His-Ura-Leu +3-AT) media. AD empty vector (EV) was used as a negative control. (B) Validation of Y1H-identified PDIs in maize protoplasts transformed with p35S::GFP-TF by ChIP-qPCR using αGFP antibodies. Fold enrichment was calculated using the Copia genomic regions as control, and normalizing to the respective input controls (see Methods). Values represent the mean and SE of three independent biological replicates (represented by transformation of three different protoplast preparations from seedlings grown at different times). Statistically (two-sided Student t-test) significant values are indicated with an asterisk (p<0.05).
LBD35 LBD24 LBD20
DOF28 MYB65CPP8
NLP14 bZIP63 MYB19
C3H42 MBF1.3
General Phenylpropanoids
Flavonoids
Lignins
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
RHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl linginp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
PAL C4H
4CL
CHS
CHI
F3’H
DFR
FNSF3HFLS
FLS FNSF3H
DFR
F3’H
LDOX/ANS
UGT
F2H
Phlobaphene Maysin
CGTUGTRHM
Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl
C3H CSE
HCT
CAD CADCADCAD
4CLCCoAOMT
CCRF5H COMT
F5H COMT
CCR
GST
HCT
lignin
ANR
Stablization and vacuolar sequestration
ALDHALDH
Supplemental Figure 5. Distribution of candidate target genes for those TFs binding the largest number of promoters.Representation of the phenolic pathway shown in Figure 1 indicating in red which of the biosynthesis genes are targeted by each of the indicated TFs.
Supplemental Table 1. Yeast bait strains representing genes encoding main enzymes of the phenolic pathway.
Bait strain name
Promoter gene name
Promoter gene identifier
[3-AT] used in
Y1H screen (mM)
Number of transformants
screened
Number of TFs
validated
Y346 PAL1 GRMZM2G074604 100 94 10 Y062 PAL2 GRMZM2G441347 20 94 1 Y171 PAL3 GRMZM2G160541 100 94 2 Y153 PAL4 GRMZM2G063917 80 94 3 Y347 PAL5 GRMZM2G081582 80 94 3 Y349 PAL6 GRMZM2G118345 40 94 0 Y096 PAL7 GRMZM2G170692 40 94 2 Y185 PAL8 GRMZM2G334660 80 94 1 Y108 PAL9 GRMZM2G029048 60 94 4 Y258 C4H1 GRMZM2G147245 80 144 4 Y221 C4H2 GRMZM2G139874 80 113 10 Y143 4CL1 GRMZM2G055320 40 96 17 Y092 4CL2 GRMZM2G174574 20 96 10 Y050 4CL3 GRMZM2G096020 60 96 10 Y028 4CL4 GRMZM2G122787 40 96 7 Y114 HCT5 GRMZM2G158083 60 96 17 Y285 HCT6 GRMZM2G035584 80 96 31 Y149 HCT10 GRMZM2G034360 80 96 34 Y140 HCT11 GRMZM2G156296 80 192 22 Y033 HCT12 GRMZM2G179703 40 96 14 Y269 HCT13 GRMZM2G129266 100 96 11 Y330 CCoAOMT1 GRMZM2G033952 100 192 2 Y335 CCoAOMT2 GRMZM2G099363 100 96 21 Y159 CCR1 GRMZM2G099420 40 96 2 Y279 CCR3 GRMZM2G131205 40 306 36 Y214 CCR4 GRMZM2G131836 20 144 8 Y112 CAD1 GRMZM2G046070 100 96 13 Y178 CAD3 (bm1) GRMZM5G844562 40 96 7 Y188 ALDH1 (rf2) GRMZM2G058675 60 96 28 Y319 ALDH5 GRMZM2G097706 80 96 26 Y239 F5H1 AC210173.4_FG005 80 408 22 Y332 COMT1 (bm3) AC196475.3_FG004 80 1670 159 Y302 COMT2 GRMZM2G082007 100 288 8 Y203 C2 GRMZM2G422750 100 290 50 Y255 WHP1 GRMZM2G151227 80 138 2 Y316 CHI1 GRMZM2G155329 40 94 10 Y081 F3H1 (fht1) GRMZM2G062396 40 94 2 Y183 F3H2 GRMZM2G146234 40 92 4 Y161 FNS1 (fnsi1) GRMZM2G009467 80 94 13 Y296 F3'H (pr1) GRMZM2G025832 80 94 5 Y206 F3'H2 GRMZM2G160763 40 138 5
Y150 F3'H3 GRMZM2G313750 100 92 11 Y323 FLS1 GRMZM2G152801 100 94 5 Y271 FLS2 GRMZM2G069298 40 94 12 Y230 A1 GRMZM2G026930 80 559 83 Y098 A4 GRMZM2G013726 80 192 7 Y341 A2 GRMZM2G345717 40 94 2 Y326 ANR GRMZM2G097841 60 94 2 Y339 Bz1 GRMZM2G165390 100 461 129 Y350 Bz2 GRMZM2G016241 80 94 27 Y077 RHM1 (sm1) GRMZM2G031311 60 164 11 Y227 RHM2 GRMZM2G072911 80 96 14 Y344 RHM3 GRMZM2G044281 100 94 2 Y337 RHM4 GRMZM2G166767 40 94 3
TOTAL 8999 944