Figure 7.
MLK4 Is Required for H2A.Z Deposition and H4 Acetylation.
(A) A yeast two-hybrid assay revealed interaction between CCA1 and YAF9a. The growth of two dilutions (2 × 10−2 and 2 × 10−3) of the yeast culture on SD medium lacking Trp, Leu, His, and adenine is shown.
(B) Beads containing a His tag (His) or His-fused CCA1 were assayed for their ability to bind a soluble GST-fused YAF9a. The input and bound protein was detected with an antibody to GST (anti-GST).
(C) Beads containing a GST tag or a GST-fused YAF9a were assessed for their ability to bind a soluble His-fused CCA1 and detected with an antibody to His (anti-His).
(D) YAF9a fused to the N terminus of YFP or the N terminus of YFP alone were tested for the ability to bind to the C terminus of YFP fused to LHY or the C terminus of YFP fused to CCA1. Twenty-five cells were examined for each transformation. Bar = 10 µm.
(E) Coimmunoprecipitation of CCA1 and YAF9a. FLAG-CCA1 and GFP-YAF9a were cotransformed into Arabidopsis protoplasts, immunoprecipitated using an anti-FLAG antibody, and detected with anti-FLAG and anti-GFP.
(F) Coimmunoprecipitation of MLK4 and YAF9a, FLAG-MLK4 and GFP-YAF9a, were cotransformed into Arabidopsis protoplasts, immunoprecipitated using an anti-FLAG antibody, and detected with anti-FLAG and anti-GFP.
In (A) to (F), experiments were repeated at least three times, and representative experiments are shown. Molecular mass markers in kilodaltons are indicated on the left, and the bands agree with expectation.
(G) and (H) The amounts of H2A.Z and H4 acetylation at different regions of GI were determined using ChIP-PCR. The y axis denotes enrichment relative to input or UBIQUITIN. Experiments were repeated at least three times, and the data from the representative experiments shown are presented as means ± se, n = 3 replicates.