supplemental figure 1. chemical structures of hydroxamic ......supplemental figure 1. chemical...
TRANSCRIPT
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
1
Supplemental Figure 1. Chemical structures of hydroxamic acid HDAC inhibitors and allelochemicals.
TSA SAHA
DIBOA DIMBOA
BOA MBOA
APO AMPO
Hydroxamic acids
Cyclic hydroxamic acids
Derivatives of cyclic hydroxamic acids
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
2
Supplemental Figure 2. HDAC conservation across different organism. Multiple sequence alignments of HDACs of Homo sapiens (Hs), Aquifex aeolicus (Aa) and A. thaliana (At). Between human HDAC8 and HDAC4 most of the active site is conserved. In HDAC8 the hydroxyl group of Tyr306 forms a hydrogen bond to an oxygen atom of hydroxamic acid inhibitors (Somoza et al., 2004), whereas in HDAC4 the equivalent Tyr is replaced by His and the bond is formed by a water molecule (Bottomley et al., 2008). A tyrosine in A. thaliana HDA1/HDA19, HDA2 and HDA6 makes this part of the binding site more similar to HDAC8. In the central part of the binding site, His142, His143 and Phe152 are identical across species; Phe208 is
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
3
replaced by Tyr in HDA2. For clarity, only the central part of the alignment, as present in the crystal structure of HDAC8 (PDB-ID 1T64), is shown. Blue circles below the alignment: zinc binding site; green boxes below the alignment: residues predicted to bind to APO or AMPO in HDAC8. Figure drawn with ESPript.
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
4
Supplemental Figure 3. Modeled binding of known and putative HDAC inhibitors to human and bacterial HDACs. A-C, TSA (green), SAHA (yellow), APO (red) and AMPO (blue), with crystal structure reference coordinates of TSA (atom colors), in the binding pocket of human class I HDAC8 (A), human class II HDAC4 (B) and bacterial HDLP (C) (all HDACs in surface representation). Residues with predicted interactions with the ligands are shown in stick representation; the zinc ion of the HDAC is shown as a grey sphere. Water molecules are shown in stick representation (black arrows). Figures were rendered with BALLView and POVRay (v3.6).
HDAC8 HDAC8
HDAC4 HDAC4
HDLP HDLP
A
B
C
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
5
Supplemental Figure 4. Homology models of A. thaliana HDACs. Class I HDA6 (A) and class II HDA2 (B) are shown in cartoon representation (red), compared to the template structure of bacterial HDLP (transparent grey). View into the binding site with the zinc ion at the bottom (grey sphere), indicating the high conservation of secondary structure elements of the binding site (center).
A
B
HDA6
HDA2
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
6
Supplemental Figure 5. DIBOA, DIMBOA, BOA and MBOA do not inhibit plant-derived HDAC enzymes. Overall HDAC inhibition in nuclear extracts of A. thaliana by increasing concentrations of DIBOA, DIMBOA, BOA and MBOA (5, 20, 50, 100 and 200 µM); SAHA (50 µM) was used as a reference inhibitor. Data points represent the mean ± SD of 4 individual measurements (two independent experiments, each performed in duplicate).
0 50 100 150 200
-250
255075
100DIBOADIMBOABOAMBOASAHA
DIBOA
Concentration (µM)
HD
AC in
hibi
tion
(%)
BOA APO
DIMBOA MBOA AMPOOxidationHydrolysis
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
7
Supplemental Figure 6. Inhibition of human class I, II and IV HDAC enzymes by APO. Human HDAC profiling was performed for different concentrations of APO (5, 20, 100 and 200 µM). Red horizontal line indicates 50% inhibition of HDAC activity. Data points represent the mean ± SD of 4 individual measurements (two independent experiments, each performed in duplicate).
HDAC1
0 50 100 150 200
0
25
50
75
100
Concentration (µM)
HD
AC
inhib
itio
n (
%) HDAC2
0
25
50
75
100
HDAC3
0
25
50
75
100
HDAC4 HDAC5 HDAC6
HDAC7 HDAC8 HDAC9
HDAC10 HDAC11
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0
25
50
75
100
HD
AC
inhib
itio
n (
%)
25
50
75
100
25
50
75
100
0
25
50
75
100
HD
AC
inhib
itio
n (
%)
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
HD
AC
inhib
itio
n (
%)
0
25
50
75
100
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
8
Supplemental Figure 7. Inhibition of human class I, II and IV HDAC enzymes by AMPO. Human HDAC profiling was performed for different concentrations of AMPO (5, 20, 100 and 200 µM). Red horizontal line highlights 50% inhibition of HDAC activity. Data points indicate the mean ± SD of 4 individual measurements (two independent experiments, each performed in duplicate).
0
25
50
75
100
HD
AC
inhib
itio
n (
%)
0
25
50
75
100
HD
AC
inhib
itio
n (
%)
0
25
50
75
100
HD
AC
inhib
itio
n (
%)
0
25
50
75
100
HD
AC
inhib
itio
n (
%)
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
0 50 100 150 200
Concentration (µM)
HDAC1 HDAC2 HDAC3
HDAC4 HDAC5 HDAC6
HDAC7 HDAC8 HDAC9
HDAC10 HDAC11
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
9
Supplemental Figure 8. Exposure to hydroxamic acids and their derivatives inhibits plant growth in lettuce. Concentration-response assays for APO and AMPO on root growth of L. sativa seedlings. The commercially available herbicide PEN was used as a positive control. Root length was measured 5 days after germination (≥ 1 mm minimum root length) and concentration-response curves were calculated using a logistic regression model. The quality of curve fitting was verified by F test for lack-of-fit based on an analysis of variances (α=0.05).
0.0
0.2
0.4
0.6
0.8
1.0
1.2
101 102 103
Rela
tive r
oot le
ngth
Concentration (mM)
APO
AMPO
PEN
100 10-1 10-2 10-3 0
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
10
Supplemental Figure 9. Growth defects in HDAC loss-of-function mutants. Seedlings were grown for 7 days on half-strength MS medium after stratification at 4°C for 4 days; the exact alleles used are indicated in the Methods section.
Ws-2
Col-0
Wildtype hda19
Wildtype hda6-6 hda6-710
mm
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
11
Supplemental Figure 10. Validation of ChIP enrichment for H3ac by quantitative real-time PCR. Relative enrichment of acetylated histone H3 was determined by qRT-PCR using specific primers for loci previously identified as acetylated (TUB2) or not acetylated (AT1TE46405) locus (Supplemental Table 2) (Wang et al., 2015). For each chromatin extract, ChIPs were performed sequentially using anti-acetylated histone H3 and anti-H3 antibodies. Levels of H3ac are given as percentages of IP/input relative to histone H3 occupancy.
0
0.04
0.08
0.12
0.16
Control APO TSARel
ativ
e H
3 ac
etyl
atio
n AT5G62690(TUB2), Replicate 1 AT5G62690 (TUB2), Replicate 2 AT1TE46405, Replicate 1 AT1TE46405, Replicate 2
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
12
Supplemental Figure 11. Schematic representation of the ChIP peak calling and filtering. Peaks were called independently from two biological replicates using MACS2, and subjected to IDR (irreproducibility discovery rate) (Li et al., 2011) analysis to filter for reliably detected peaks. Moreover, alignments of each replicate were split into two self-pseudoreplicates and each pair of self-pseudoreplicates was subjected to IDR filtering. In parallel, reads of the two biological replicates were pooled and peaks were called on the pooled dataset. Finally, the merged alignments were split into two pseudoreplicates and subjected to the same peak calling and filtering process. Only peaks called in all three sets were used for differential analysis. N1, N2, Np and Nt represent the number of peaks retained after the respective filtering and were used for quality assessment (Supplemental Table 3). Scheme modified from Landt et al. (2012).
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
13
Supplemental Figure 12. Differential ChIP-seq analysis. A, Principal component analyses (PCA) of histone acetylation. The left panel is based on acetylation at all analyzed peaks; the right panel shows PCA on peaks identified as differentially acetylated between APO- and non-APO-treated samples using the edgeR implementation of the DiffBind package. Percentages in square brackets represent the amount of variation explained by the respective principal component. B-C, Manhattan plots of differential ChIP-seq analysis of APO- (B) and TSA-treated (C) samples compared to the untreated control using the DiffBind package. Red dots indicate statistically significantly differentially acetylated peaks (FDR <0.1). Left and right panels show results using the edgeR and DESeq2 implementations in DiffBind, respectively. D, Venn diagrams showing the overlap of differentially acetylated peaks upon APO and TSA treatment using the edgeR or DESeq2 implementations in DiffBind.
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
14
Supplemental Figure 13. Correlation between TSA-dependent gene expression and H3 acetylation. H3 acetylation in control-, TSA- and APO-treated samples at peaks overlapping with TSA-upregulated (left panel) and TSA-downregulated genes (right panel). (RPKM: Reads Per Kilobase per Million; * p<0.05, unpaired two-tailed Student’s t-test; ns: not significant).
Control TSA APO0
20
40
60
80
Control TSA APO0
20
40
60
TSA-upregulated genes TSA-downregulated genes
nsns
nsns
*
ns
His
tone
H3
acet
ylat
ion
(RP
KM
)
His
tone
H3
acet
ylat
ion
(RP
KM
) 80
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
15
Supplemental Figure 14. Annotation of acetylated and differentially acetylated regions. A, All positions in the respective set of regions were assigned to one annotation element in the following order: CDS > intron > 5’UTR > 3’UTR > 2 kb upstream > 2 kb downstream > transposon (TE) > intergenic. Annotation was based on the TAIR10 model of A. thaliana gene annotation, for TEs we used the annotation of Slotte et al. (2013). B, Histograms of χ2 test statistics. Left panel: from the unified set of H3ac peaks, n1 randomly drawn peaks were compared against n2 randomly drawn peaks, n1 and n2 corresponding to the number of APO- and TSA-hyper-acetylated regions, respectively. The red line indicates the test statistic of the real-data comparison of APO-hyper-acetylated regions to TSA-hyper-acetylated regions. Right panel: same as left panel, but for hypo-acetylated regions.
Co
ntr
ol
TS
A
AP
O
TS
A h\SHUí$
F
TS
A h\SRí$F
AP
O h\SHUí$
F
AP
O h\SRí$F
Re
lativH�Fo
ve
rag
e
0.0
0.2
0.4
0.6
0.8
1.02kb upstream
5'UTR
CDS
Intron
3'UTR
2kb downstream
Transposon
,QWHUJHQLF
ȋ2 THVW�VWDWLVWLF
)UHTXHQF\
0 5 10 15 20 25 30
01
00
20
03
00
40
0
ȋ2 THVW�VWDWLVWLF0 5 10 15 20 25 30
$32�YV��76$�K\SHUDFHW\ODWLRQ $32�YV��76$�K\SRDFHW\ODWLRQ
Random peaks
$32�YV��76$�K\SR�$FRandom peaks
APO vs. TSA
K\SHU�$F
A
B
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
16
Supplemental Figure 15. Summary of gene ontology (GO) term analysis of differentially expressed genes. Heatmap showing p-values of ‘molecular function’ and ‘cellular component’ GO terms that were over-represented in at least one treatment. Black color indicates that the respective GO term was not significantly over-representated.
iron ion bindingWKUHRQLQHíW\SH�HQGRSHSWLGDVH�DFWLYLW\trDQVferDVH�DFWLYLW\, trDQVferring he[RV\O�JURXSVnXFOHRVLGHíWrLSKRVSKDWDVH�DFWLYLW\HOHFWURQ�FDUrLHU�DFWLYLW\JOXWDWKLRQH�ELQGLQJdrXJ�WrDQVPHPErDQH�WrDQVSRrWHU�DFWLYLW\HQGRSHSWLGDVH�DFWLYLW\DQWLSRrWHU�DFWLYLW\DGHn\O\OíVXOfDWH�UHGXFWDVH�DFWLYLW\ATPDVH�DFWLYLW\ATPDVH�DFWLYLW\��FRXSOHG�WR�WrDQVPHPErDQH�PoYHPHQW�RI�VXEVWDQFHVo[\JHQ�ELQGLQJ8'3íJO\FRV\OWrDQVferDVH�DFWLYLW\8'3íJOXFRV\OWrDQVferDVH�DFWLYLW\o[LGRUHGXFWDVH�DFWLYLW\JOXWDWKLRQH�WrDQVferDVH�DFWLYLW\LQGROHí�íDFHWRQLWrLOH�QLWrLODVH�DFWLYLW\nitrLODVH�DFWLYLW\KHPH�ELQGLQJSHUo[LGDVH�DFWLYLW\o[LGRUHGXFWDVH�DFWLYLW\��DFWLQJ�RQ�SDLUHG�GRQRUV, with
Molecular function
� � �ORJ���S�YDOXH�
F\WRVROSURWHDVRPH�FRUH�FRPSOexFHOO�wDOOYDFXRODU�PHPErDQHF\WRSODVPSURWHDVRPH�FRPSOexDSRSODVWFKORURSODVWextrDFHOOXODU�UHJLRQ
6$+$BXS
SA
HA
_dow
n76
$BXS
TSA
_dow
n$32BXS
AP
O_d
own
Cellular component
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
17
Supplemental Figure 16. Validation of RNAseq by quantitative real-time PCR. From genes classified as differentially expressed upon APO treatment, 6 genes were randomly chosen for validation by reverse transcription followed by quantitative PCR (qRT-PCR). Bars represent the mean of RNA-seq read counts; the four individual replicates are shown by black circles. The expression fold-change, determined by qRT-PCR relative to the control sample, is shown by the red line. Filled triangles represent the mean, empty triangles represent the three biological replicates. RNA-seq and qRT-PCR data are represented to scale; the RNA-seq control level was set to 1 for qRT-PCR fold changes.
AT1G11190
Norm
alized r
ead c
ount (R
NA
seq)
Control TSA APO
050
100
0.2
0.4
0.6
0.8
1.0
1.2
AT2G23680
040
80
1.0
1.5
2.0
2.5
3.0
3.5
AT4G33070
0200
400
600
800
2
4
6
8
10
12
AT2G34300
Norm
alized r
ead c
ount (R
NA
seq)
0100
200
300
400
500
0.2
0.4
0.6
0.8
1.0
AT4G19880
02000
4000
5
10
15
AT3G14420
02000
4000
6000
0.4
0.6
0.8
1.0
1.2
Fold
change (q
PC
R)
Fold
change (q
PC
R)
Control TSA APO Control TSA APO
Control TSA APO Control TSA APO Control TSA APO
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
18
Compound EC10 (µM) EC50 (µM) EC70 (µM) EC90 (µM)APO 7 75 192 856AMPO 1491 4724 7369 14966PEN 51 664 1782 8597
Lactuca sativaCompound EC10 (µM) EC50 (µM) EC70 (µM) EC90 (µM)
APO 2896 6868 9582 16289AMPO 3623 12291 19685 41694PEN 9 14 16 20
Arabidopsis thaliana
Supplemental Table 1. Effective concentrations for different HDAC inhibitors. EC-concentrations causing a 10, 50, 70 and 90% inhibition in root length of A. thaliana Col-0 and L. sativa cv. Maikönig for APO and AMPO, as calculated using a logistic concentration-response model. For comparison the EC50-values of the commercial herbicide PEN are also presented.
TAIR10 Gene ID Gene name Direction Sequence (5'->3')AT5G62690 TUB2 forward AAGAACCATGCACTCATCAGCAT5G62690 TUB2 reverse ATCCGTGAAGAGTACCCAGATAT1G37110 AT1TE46405 forward CTGCGTGGAAGTCTGTCAAAAT1G37110 AT1TE46405 reverse CTATGCCACAGGGCAGTTTT
Supplemental Table 2. Primer sequences for chromatin immunoprecipitation analyses.
Sample N1/N2 Np/NtReplicate1 Replicate2
APO 39686 48141 1.81 2.85TSA 28325 32629 1.21 1.80Control 23913 41660 1.58 1.58
Number of peaks
Supplemental Table 3. Quality control analysis of ChIP peak calling. Quality control parameters were calculated as indicated in Supplemental Figure 10. Nt/Np and N1/N2 reference values as used in the ENCODE project, details are described in Landt et al. (2012).
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
19
TAIR10 Gene ID Gene name Direction Sequence (5'->3')
AT1G11190 BFN1 forward GGGATACAAAGGCGTCAAGTCTGAT1G11190 BFN1 reverse GTGGCAGCAACACCAGCAATAGAT2G23680 n/a forward CGCTCTCCTCACCGTTATTCTCAGAT2G23680 n/a reverse TCCCGTATATCTCAGGTCGTCTCTGAT4G33070 PDC1 forward GGTCTCGTTGACGCCATTCATAACAT4G33070 PDC1 reverse CTTTCTTCTCCGTCGTCGCTGTCAT2G34300 n/a forward CCATGCCGATCATCTTTTCTCTACCAT2G34300 n/a reverse TTCTCCTAATGTTTCCATGTCATCCCAT4G19880 n/a forward TGCAAAGAAACAAGGACCTTATGAGAT4G19880 n/a reverse TTTCAGTCAAAGTGTTACCACAGATGAT3G14420 GOX1 forward TGTTCGACGTGGCACTGATGTCAT3G14420 GOX1 reverse CCTTTCTAACTCCAGCCTCTCCTTC
Supplemental Table 4. Primer sequences for RNA-seq validation by qRT-PCR.
Supplemental Data. Venturelli et al. (2015). Plant Cell 10.1105/tpc.15.00585
20
SUPPLEMENTAL REFERENCES
Bottomley, M.J., Lo Surdo, P., Di Giovine, P., Cirillo, A., Scarpelli, R., Ferrigno, F., Jones, P., Neddermann, P., De Francesco, R., Steinkuhler, C., Gallinari, P., and Carfi, A. (2008). Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain. J Biol Chem 283, 26694-26704.
Landt, S.G., Marinov, G.K., Kundaje, A., Kheradpour, P., Pauli, F., Batzoglou, S., Bernstein, B.E., Bickel, P., Brown, J.B., Cayting, P., Chen, Y., DeSalvo, G., Epstein, C., Fisher-Aylor, K.I., Euskirchen, G., Gerstein, M., Gertz, J., Hartemink, A.J., Hoffman, M.M., Iyer, V.R., Jung, Y.L., Karmakar, S., Kellis, M., Kharchenko, P.V., Li, Q., Liu, T., Liu, X.S., Ma, L., Milosavljevic, A., Myers, R.M., Park, P.J., Pazin, M.J., Perry, M.D., Raha, D., Reddy, T.E., Rozowsky, J., Shoresh, N., Sidow, A., Slattery, M., Stamatoyannopoulos, J.A., Tolstorukov, M.Y., White, K.P., Xi, S., Farnham, P.J., Lieb, J.D., Wold, B.J., and Snyder, M. (2012). ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res 22, 1813-1831.
Li, Q., Brown, J., Huang, H., and Bickel, P. (2011). Measuring reproducibility of high-throughput experiments. Ann Appl Stat 5, 1752–1779.
Slotte, T., Hazzouri, K.M., Agren, J.A., Koenig, D., Maumus, F., Guo, Y.L., Steige, K., Platts, A.E., Escobar, J.S., Newman, L.K., Wang, W., Mandakova, T., Vello, E., Smith, L.M., Henz, S.R., Steffen, J., Takuno, S., Brandvain, Y., Coop, G., Andolfatto, P., Hu, T.T., Blanchette, M., Clark, R.M., Quesneville, H., Nordborg, M., Gaut, B.S., Lysak, M.A., Jenkins, J., Grimwood, J., Chapman, J., Prochnik, S., Shu, S., Rokhsar, D., Schmutz, J., Weigel, D., and Wright, S.I. (2013). The Capsella rubella genome and the genomic consequences of rapid mating system evolution. Nat Genet 45, 831-835.
Somoza, J.R., Skene, R.J., Katz, B.A., Mol, C., Ho, J.D., Jennings, A.J., Luong, C., Arvai, A., Buggy, J.J., Chi, E., Tang, J., Sang, B.C., Verner, E., Wynands, R., Leahy, E.M., Dougan, D.R., Snell, G., Navre, M., Knuth, M.W., Swanson, R.V., McRee, D.E., and Tari, L.W. (2004). Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases. Structure 12, 1325-1334.
Wang, C., Liu, C., Roqueiro, D., Grimm, D., Schwab, R., Becker, C., Lanz, C., and Weigel, D. (2015). Genome-wide analysis of local chromatin packing in Arabidopsis thaliana. Genome Res 25, 246-256.