FIG 2.

Mit1 requires its conserved chromatin tethering domains for silencing. (A) Sequence alignment of Mit1's chromodomain. Sequence alignment of Mit1 with chromodomains of CHD family remodeling enzymes and Suvar39h1. Conserved aromatic (red), acidic (blue), basic (green), and hydrophobic (yellow) residues are indicated. (B) Mit1 has conserved PHD and CD domains. Schematic representation of SpMit1 and Mi-2 conserved motifs including the plant homeodomain (PHD), chromodomain (CD), and SWI/SNF-like ATPase domains. (C) PHD and CD contribute to silencing activity of Mit1. Serial dilution spotting assay on medium lacking leucine (to maintain expression plasmids) with or without 5′-fluoroorotic acid (FOA) of strains containing a centromeric ura4⁺ reporter gene (otr1::ura4⁺), indicated in the schematic. Lines underneath the schematic represent the locations of primers used for transcript analyses. Plates were incubated at 32°C. EV, empty vector. (D) Purification of recombinant GST fusions of domains of Mit1. SDS-PAGE and Coomassie staining of purified recombinant GST, GST-PHD(205–273)^Mit1, and GST-CD(447–499)^Mit1 fusion proteins is shown. (E) Mit1's CD domain can bind DNA in vitro. Biotinylated double-stranded DNA or an equivalent amount of biotin or unlabeled DNA was incubated with recombinant GST or GST fused to the Mit1 chromodomain. The DNA and associated proteins were captured by streptavidin beads and analyzed by Western blotting. (F) Mit1 can bind nucleosomal DNA. EMSA to compare the binding of Mit1 chromodomain to free DNA and nucleosomal DNA. Radiolabeled 147-bp Widom 601 nucleosome positioning sequence (50 ng; lanes 1 to 5) or mononucleosomes without free DNA ends reconstituted by salt dilution on the same sequence (50 ng DNA equivalent; lanes 6 to 10) were incubated with GST (2 μg and 4 μg) or GST-CD^Mit1 (2 μg and 4 μg) fusion protein. (G) Mit1's PHD domain can bind histone H3 in vitro. Calf thymus histone pulldown experiment comparing the abilities of the GST, GST-PHD^Mit1, and GST-PHD^Ing2 proteins to bind to histones. Histone association was monitored by immunoblotting using antibodies specific for different histones or histone modifications. (H) Mit1 PHD and CD are not required to maintain association with Chp2. Immunoprecipitation of wild-type 3×V5-tagged Mit1, Mit1^PHDΔ, Mit1^CDΔ, and Mit1^K587A in strains expressing 6×FLAG-Chp2 and blotting against the V5 and FLAG epitopes. Note that the SDS-PAGE gels used in these experiments do not effectively resolve wild-type Mit1 from Mit1^PHDΔ and Mit1^CDΔ. The asterisk indicates a nonspecific band. (I) Centromeric association of Chp2 is partially dependent on Mit1. ChIP of Chp2 association with centromeres relative to a euchromatic locus in the indicated strain backgrounds is shown. Error bars reflect the SEM (n = 4).