Hernandez et al. 10.1073/pnas.0705629104. |
Fig. 5. Micrococcal (MNase) sensitivity assay. (A) PCR of MNase digestion experiments using the nuclear extract from BMS cells bombarded with pA1Luc and the pure (naked) pA1Luc plasmid as a control. PCR was performed by using primers that selectively amplify the pA1Luc or the endogenous A1 promoter, as shown in B. MNase treatments were carried out for 30 sec, 1 min, and 5 min in the presence of MNase (+) and 20 min without MNase (-), followed by agarose gel electrophoresis.
Fig. 6. Enrichment of H3K9/14ac in the proximal A1 promoter region in maize plants expressing the dominant versions of anthocyanin regulators. (A) Semiquantitative PCR of ChIP experiments of b and B-I leaves. PCR was performed on three 4-fold serial dilutions of the chromatin immunoprecipitated material, on the proximal (P) and distal (D) fragments of the A1 gene promoter regions. A fragment corresponding to the coding region of the actin gene was used as a negative control. (B) Quantitative PCR of ChIP experiments of b and B-I leaves. ChIP experiments were performed in biological triplicates, and PCRs were performed by using proximal (P) and distal (D) fragments with [a-32P]dCTP. Amplified products were TCA-precipitated and quantified by scintillation. Asterisks indicates a sample significantly different from the rest (P < 0.05).
Fig. 7. Nucleotide sequence of the coding region of RIF1. The fragment used for the dsRNAi is highlighted in yellow. The GenBank accession of RIF1 is EF647588.
Fig. 8. The ENT domain of RIF1 is sufficient for homodimer formation. Shown are yeast two-hybrid assay interactions between the ENT domain of RIF1 (RIF1ENT) with the full-length protein and itself. Empty pBD-GAL4 or pAD-GAL4 were used as negative controls. For the yeast strain used and the selections applied see Fig. 2.
Fig. 9. RIF1-GFP localizes to nuclear speckles in agroinfiltrated N. benthamiana leaf epidermal cells, and this pattern is not affected by the presence of R (RMYC).
SI Materials and Methods
Protoplast Isolation.
After chopping the third leaves into small pieces, the leaf stripes were digested in 3% cellulase RS, 0.6% macerozyme R10 (both from Yakult Honsha), 0.6 M mannitol, 10 mM Mes (pH 5.7), 5 mM CaCl2, 7.5 mM 2-mercaptoethanol, and 0.1% BSA for 10 min under vacuum followed by 2 h of gentle shaking (40 rpm) at 25°C in the dark. After releasing the protoplasts at 80 rpm, the protoplasts were filtered through a 35-mm nylon mesh and collected by centrifugation at 150 ´ g for 1 min. The protoplasts were washed in ES buffer (0.6 M mannitol/5 mM Mes, pH 5.7/10 mM KCl) and counted with a hemocytometer.Chromatin Immunoprecipitation Analyses.
The cross-linked material was resuspended in 0.1 ml lysis buffer (50 mM Hepes, pH 7.5/150 mM NaCl/1 mM EDTA/1% Triton X-100/0.1% deoxycholate/0.1% SDS/1 mM PMSF/10 mM sodium butyrate) and plant proteinase inhibitor mixture (Sigma). DNA was sheared by sonication to »300- to 1,000-bp fragments with a main peak of 500 bp. Sonication (Sonics & Materials) was performed on ice with an amplitude of 10% using five 15-sec pulses (5 sec between bursts). After preclearing with 40 ml of salmon sperm DNA/Protein A-agarose beads (Upstate) for 120 min at 4°C, immunoprecipitations were performed overnight at 4°C with 1 mg of anti-acetyl-histone H3 (06-599; Upstate), anti-dimethyl-K9-histone H3 (ab7312; abcam), or 1 ml of anti-GFP antibody (ab290; Abcam). After incubation, beads were washed two times with LNDET Buffer (0.25 M LiCl/1% Nonidet P-40/1% deoxycholate/1 mM EDTA) and two times with TE buffer. The washed beads and input fraction were resuspended in elution buffer (1% SDS/0.1 M NaHCO3) with 0.25 mg/ml proteinase K and incubated overnight at 65°C. After cross-link reversal of the immunoprecipitated and input DNA (set aside from the sonication step), the DNA was purified by using the PCR Purification Kit (Qiagen). Semiquantitative PCR was performed under standard PCR conditions (35-38 cycles). DNA was detected by using agarose gel electrophoresis and quantified by ethidium bromide staining.Nuclei Isolation and Micrococcal Nuclease Digestion.
BMS cells (100 mg) were subjected to nuclear isolation with the CellLytic PN plant nuclei isolation kit (Sigma) 40 h after transformation with the pA1Luc plasmid by microprojectile bombardment. Ten micrograms of isolated nuclear protein was resuspended in 300 ml of buffer N (15 mM Hepes, pH 7.5/60 mM KCl/15 mM NaCl/3 mM CaCl2/0.1 mM DTT), and 50-ml aliquots were incubated with 15 units of microccocal nuclease (USB) for various time periods at 37°C. As a control, we used 5 ng of the pA1Luc plasmid. After the reaction was completed, DNA was extracted by using the PCR Purification Kit (Qiagen) into 30 ml of EB buffer. The purified DNA (2 ml) was used in PCRs with primers that recognize the A1 promoter and luciferase (to detect pA1Luc) or the A1 promoter and the A1 5' UTR (to detect the endogenous A1 gene).