Fig 1. DUET PHD is a reader of H3K4me2.

(A) Schematic representation of DUET protein. DUET encodes a 704 amino acid plant specific protein with a nuclear localization sequence (NLS), a region showing homology with the meiotic gene SWITCH1 (SWI1), and a C-terminal Plant Homeo Domain (PHD). (B) Calf thymus histone pull down followed by western blot with the indicated antibodies. BPTF PHD2 is used as positive control. (C) Peptide pull downs followed by western blot with anti-GST antibody. (D) The PHD finger of DUET is conserved across eukaryotes. Sequence alignment of DUET PHD finger with characterized homologues showing highest homology obtained by PSI-BLAST. DUET PHD shares 46% identity with human MLL5 PHD (E = 9e-08), 44% identity with S.cerevisiae Set3 PHD (E = 2e-07), and 41% identity with human BPTF PHD2 (E = 3e-05). Conserved and similar residues are in red and blue respectively, conserved C4HC3 structural residues are in bold green. White circles indicate mutation sites in the triple mutant (C21A/C24A/H29A), and black circles indicate the point mutations S20A and W27A. (E) Histone peptide pull downs with methylated H3K4 peptides and the indicated mutant DUET PHD fingers. (F) Representative Alexander staining of T1 duet plants transformed with a wild-type DUET construct (WT). Left panel: fully viable pollen (purple) indicating full rescue; middle panel: mix of viable and dead pollen (green) indicating partial rescue; right panel: absence of pollen indicating no rescue. (G) Quantification of rescue phenotypes obtained for independent T1 duet plants transformed with the indicated construct. WT, WT full length DUET construct; n, number of independent transformants analyzed per construct.