Appendix 1.
Supplementary Figure Legends
Figure S1: Characterization of transgenic line 68lf-3. (a) Map of 68lf construct
indicating the position of BamHI sites and the probes used for Southern analysis. (b-d)
Southern analysis of Col-0 (WT) and 68lf-3 genomic DNA digested with BamHI and
sequentially probed with probes A, B, and C. The endogenous PHYA gene is marked
with an arrowhead. (e) Detection of siRNA complementary to PHYA exon 1 and exon 2
in transgenic line 68lf-3. U6-8 served as a control.
Figure S2: (a) Map of PHYA locus displaying EcoRI (E), BamHI (B), and HindIII (H)
sites. (b-d) Southern blot of BamHI, EcoRI or HindIII cut genomic DNA of phyA-7,
phyA-17, 68lf-3 or Col-0 (WT) indicating the absence of transgene fragments in phyA-7
and phyA-17. The blots were probed with full-length PHYA promoter (probe A) or
coding region (probe B). Endogenous PHYA gene on the BamHI blot is indicated by
arrowhead.
Figure S3: Map of primers used for sequencing the PHYA gene.
Figure S4. HaeIII, DdeI, and Sau3A I Southern Analysis. (a). Schematic map (not to
scale) of the PHYA gene displaying HaeIII, DdeI, and Sau3A I sites.
Dotted line
represents the PHYA fragment missing in 68lf construct (see Fig. 1a). Fragment sizes
(bp) between the sites are shown. (b-d). Southern blots of genomic DNA from phyA-7,
phyA-17, and Col-0 (WT) lines was restricted with EcoRI (E) and a methylationsensitive enzyme, and hybridized with various probes. DNA size markers (M) are given
in kb with each blot.
Figure S5. BglII, PstI, and PvuII Southern Analysis. (a). Schematic map of the PHYA
gene displaying BglII, PstI, and PvuII sites . Dotted line represents the PHYA fragment
missing in 68lf construct (see Fig. 1a). Fragment sizes (bp) between the sites are shown.
(b-d). Southern blots of genomic DNA from phyA-7, phyA-17, and Col-0 (WT) lines
was restricted with EcoRI (E) and a methylation-sensitive enzyme, and hybridized with
various probes. DNA size markers (M) are given in kb.
Figure S6. Exact methylation pattern of the promoter, 5’ UTR, and exon fragments. (a)
PHYA map displaying promoter, 5’ UTR, exons and introns along with the transcription
start site (+1) and start codon (ATG). Boxes below the map (P1-2, U1-3, E1-3) represent
regions subjected to bisulfite sequencing. Open boxes represented unmethylated regions
and shaded boxes represent methylated regions of phyA’. Number of nucleotides in each
fragment is given below. The amplicons for P1, P2, U1, U2, E1, and E2 derived from both
Col-0 (WT) and phyA’ genotypes were sequenced directly, whereas the amplicons for P2,
U3, and E3 derived from phyA’ were cloned into the TOPO vector and 13-19 individual
clones (detailed in Figure S6) were sequenced. (b) Lack of cytosine methylation
(unmarked) within P1 and U1 fragments of phyA’ and WT gene was determined by direct
sequencing of PCR fragment.
Methylation pattern within P2, U3 and E3 fragments
derived from the phyA’ gene was obtained by sequencing 13-19 independent clones of
each fragment. Frequency of methylation (number of 5mC/ total number of clones) for
each cytosine is indicated by solid boxes (100%), half-filled boxes (88%), and a hatched
box (45%). TATA box and transcription start site (+1) are shown in P2, and the ATG
codon in U3.
Figure S7. Characterization of the T-DNA insertion line SALK_076522 (met1-7). (a-c),
Genomic DNA from Col-0, SALK_076522 (met1), SALK_000590 (ddm1), and the F2
and F3 progeny of phyA-17 x met1 cross was digested with HpaII and hybridized with a
180 bp centromeric repeat fragment. The ddm1 genotype serves as a positive control for
centromeric hypomethylation. (d) Northern hybridization of total RNA of Col-0, phyA-7,
phyA-17, and SALK_076522 with a MET1 probe. Hybridization with actin serves as a
loading control.
Figure S8. DNase I accessibility assay. (a). Map of PHYA gene displaying the location
of primers used in the assay. (b). Nuclei were isolated from Col-0 (WT) and phyA’
seedlings, and subjected to DNase I digestion for 0, 5, 10, 15 and 20 min. DNA was
isolated from the treated nuclei, and subjected PCR amplification of the phyA locus. The
amplified products were fractionated on a 0.8% agarose gel, and hybridized with the 32P
labeled complementary DNA fragment. Actin and a 180 bp single repeat served as
controls for euchromatin and heterochromatin regions, respectively.
Figure S9. Exact fragment sizes of Southern blots shown in Figure 2.
Supplementary Experimental Procedures
siRNA analysis
Fifty micrograms of total RNA was denatured at 65oC for 10 min and fractionated on
TBE-urea 15% polyacrylamide gel (Bio-Rad Inc.), and transferred to Hybond N+
membrane by electroblotting for 1.5 hours at 300 mA in 1xTBE. The blot was UV
crosslinked and hybridized in Church and Gilbert solution with 5’ end labeled oligo
complementary to PHYA exon 1 (5’- GTT ATC GAG TTC CAC CTC CTT ATT GAC
GTG TAT GGC AAA CAC TT- 3’), exon 2 (5’-TGC TAG AGC CTT TAG TCT CTT
CAC TGC GGT TCG CTC AGC TAA ACG TTG AA-3’) or snRNA U6-8 (5’ -TTC
TTT GTA TCG TTT CAA TTT TAT CGG ATG - 3’). Hybridizations with oligos
complementary to exon 3, 4, introns 1-3, 5’ UTR, PHYA promoter, hpt, and 35S promoter
were also performed. The sequences of these oligos are available upon request. Each
oligo was labeled with 32P using T4 Polynucleotide kinase (Ambion Inc.), and purified
through Sephadex G25 spin column (Roche Inc.), denatured in boiling water before
hybridization at 42 oC overnight. Blots were washed at 42 oC twice with 2X SSC + 0.2%
SDS for 10 minutes each, and twice with 0.5X SSC + 0.2% SDS for 5 minutes each.
Washed blots were viewed on Phosphorimager. Blots were stripped in boiling 0.1% SDS
before subsequent hybridizations.
DNase I Accessibility Assay
Five grams of fresh dark grown seedlings were ground in 20 ml of Hamilton buffer (10
mM TRIS-HCl pH 7.6, 1.14 M sucrose, 5 mM MgCl2, 5mM β-mercaptoethanol) using
pestle and mortar kept on ice. The slurry was gently stirred for 1 h and filtered through a
Miracloth (Calbiochem, CA) followed by centrifugation for 10 min at 1000g. The pellet
was resuspended in Hamilton buffer containing 0.15% Triton X-100, incubated for 30
min, and centrifuged for 10 min at 1000g. The pellet was washed again with Hamilton
buffer containing 0.15% Triton X-100, resuspended in nuclease digestion buffer (50 mM
TRIS-HCl pH 8.0, 0.3 M sucrose, 5 mM MgCl2, 1.5 mM NaCl, 0.1 mM CaCl2, 5 mM βmercaptoethanol) and centrifuged for 10 min at 1000g. The crude nuclear pellet was then
resuspended in 1.2 ml of nuclease digestion buffer. All manipulations were performed at
4oC. Before addition of DNase I, 200 μl aliquots were removed for the 0 time point. 0.5
U of DNase I (Qiagen) was added to the remaining 1 ml. At various time points, 200 μl
aliquots were removed and added to 40 μl of stop buffer (0.25 M EDTA). Aliquots of 10
μl of naked DNA were digested with 0.01 - 0.02 U of DNase I in 160 μl of nuclease
digestion buffer. The 40 μl aliquots were taken at different time points and added to 8 μl
of stop buffer. To extract DNA from the nuclei, 120 μl of H2O and 40 μl of 10% SDS
was added, followed by 15 min incubation at 50oC. Then 132 μl of 5 M potassium
acetate was added and mixed well; vials were then incubated for 15 min on ice and
centrifuged for 15 min at 13000 rpm at 4oC. The supernatant was extracted once with
phenol/chloroform and DNA was precipitated by addition of 10 μg of yeast tRNA and 1
ml of 96% ethanol, incubation for 1 h at -20oC and centrifugation at 13000 rpm at 4oC.
The pellet was washed with 70% ethanol and finally dissolved in 50 μl of water. Naked
DNA was isolated as described above. Digested DNA was extracted with
phenol/chloroform, and precipitated by addition of 10 μg yeast tRNA, 20 μl of 3M
sodium acetate and 500 μl of 96% ethanol, incubation for 1 h at -20 oC and centrifugation
at 13000 rpm at 4oC. The pellet was washed with 70% ethanol, dried and resuspended in
50 μl H2O. DNA isolated from nuclei was diluted 1:5 and 2 μl was taken for each PCR,
blotted on Hybond N+ membrane, and hybridized with DNA probes.