Gal4 plasmid pm

To construct the Gal4 DBD-NINJA plasmid, the Arabidopsis NINJA full-length coding sequence was polymerase chain reaction (PCR)–amplified, digested, and ligated into the Gal4 plasmid pM (Clontech). To generate VP16 fusion constructs, full-length and truncated coding sequences of rice TPR2, TPR1, and TPL were PCR-amplified from pLexA-N fusion plasmids (13 (link)), digested, and inserted into the pVP16 plasmid (Clontech). The reporter plasmid pG5-Luc contains a luciferase gene under the control of a Gal4 upstream activating sequence element. Gal4 fusion constructs (30 ng) were cotransfected with VP16 fusion constructs (30 ng), together with 100 ng of pG5-Luc and 1 to 3 ng of phRG-TK/Renilla (Promega), into AD293 cells in 24-well plates by Lipofectamine (Invitrogen) method according to the manufacturer’s manual. Cells were harvested 17 hours after transfection with 1× Passive Lysis Buffer (Promega). Luciferase/Renilla activities were measured with the Dual-Luciferase Kit (Promega), and data were plotted as firefly luciferase activity relative to Renilla luciferase activity.

The TPL/TPR expression clones were described previously (25 (link)). For the M2H assay, the full-length D53 open reading frame was cloned into Gal4 plasmid pM (Clontech). Cloning of full-length rice TPL, TPR1, and TPR2 open reading frames as fusions with an N-terminal VP16 activation domain was described previously (25 (link)). Site-directed mutagenesis was performed using the QuikChange method (Stratagene). All expression constructs and mutations were confirmed by DNA sequencing. Nonhistone peptides were synthesized by Peptide 2.0 Inc. Histone peptides were synthesized by the High-Throughput Peptide Synthesis and Array Core Facility at the University of North Carolina at Chapel Hill. Biotinylated recombinant human nucleosomes were produced by EpiCypher Inc.

To construct the Gal4 DBD-D53 plasmid, full-length, codon-optimized D53 coding sequence was synthesized by GeneWiz with flanking restriction sites. The restriction fragment was cloned into the Gal4 plasmid pM (Clontech). The full-length TPL/TPR1/TPR2-VP16 activation domain constructs were described previously (25 (link)). Gal4 fusion constructs (25 ng) were cotransfected with VP16 fusion constructs (25 ng), together with 100 ng of pG5-Luc reporter and 5 ng of phRG-TK/Renilla (Promega) control into AD293 cells using FuGENE 6 (Promega) according to the manufacturer’s instructions. Cells were harvested 24 hours after transfection and lysed in 1× passive lysis buffer (Promega). Luciferase/Renilla activities were measured with the Dual Luciferase Kit (Promega), and data were plotted as relative activities (Luciferase activity:Renilla activity).