Figure 2.

AtMBD6-GFP Is Tightly Bound to Chromatin and Is Associated with Centromeric Repeats and the 18S rDNA.

(A) Nuclei prepared from leaves of transgenic Arabidopsis expressing AtMBD6-GFP or of wild-type (ecotype Wassilewskija) plants were extracted with increasing concentrations of NaCl. Soluble (S) and insoluble pellet (P) fractions were analyzed for the presence of AtMBD6-GFP or histone H3 methylated at Lys 4 by immunoblotting using anti-GFP (αGFP) and anti-dimethylated K4 histone H3 (αK4m2H3), respectively. M indicates molecular weight markers. Note that AtMBD6-GFP appears as multiple protein bands that are absent from wild-type plants. Arrow indicates a breakdown product of the AtMBD6-GFP, which is loosely bound to chromatin.

(B) ChIP assay demonstrating the association of AtMBD6-GFP with CEN180 but not with actin-encoding sequence. ChIP was performed on nuclei prepared from AtMBD6-GFP–expressing plants. Chromatin was immunoprecipitated using αGFP, αK4m2H3, or antidimethylated K9 histone H3 (αK9m2H3). DNA was extracted and subjected to PCR using sets of primers to amplify the centromeric 180-bp repeats (CEN180), the 18S rDNA, or actin-encoding sequence as a control.

(C) Immunolabeling/FISH assay showing that AtMBD6-GFP is associated with CEN180 and has preference for perinucleolar chromocenters adjacent to the 18S rRNA genes. DAPI was used as a counterstain. Bars = 5 μm.