Control of PHERES1 Imprinting in Arabidopsis by Direct Tandem Repeats Villar Corina Belle R. , Erilova Aleksandra , Makarevich Grigory , Trösch Raphael , Köhler Claudia   Molecular Plant  Volume 2, Issue 4, Pages 654-660 (July 2009) DOI: 10.1093/mp/ssp014 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Structure of the PHE1 and PHE2 Loci. Numbers indicate position relative to the translational start sites. Molecular Plant 2009 2, 654-660DOI: (10.1093/mp/ssp014) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 PHE2 Is a Direct Target Gene of the FIS PcG Complex. (A) Schematic diagram of the PHE1 and PHE2 loci indicating the regions probed by PCR after ChIP. The numbers indicate the position relative to the translational start sites. (B) ChIP analysis of MEA and H3K27me3 enrichment at the PHE1 and PHE2 (corresponding to the faster migrating band) loci in closed flowers. ChIP PCR was performed in triplicate, indicated by the numbers on top of the panel. Quantification of the results as relative enrichment over ACTIN is shown in the right panel. I, input; ACT, ACTIN. Molecular Plant 2009 2, 654-660DOI: (10.1093/mp/ssp014) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 PHE2 Is Biallelically Expressed. (A) Allelic expression analysis of PHE1 after crosses of the Col-0 and C24 accessions. Analysis was performed at 1–4 d after pollination (DAP). (B) Allelic expression analysis of PHE2 in wild-type and mea mutant plants after crosses of Ler and mea plants with the Col-0 accession. Analysis was performed at 1–4 d after pollination. (C) Expression analyses of PHE2::GUS transgene in seeds at 3 DAP. Scale bar, 100 μm. (D) Relative PHE2 messenger RNA levels in wild-type and mea flowers before fertilization (0 DAP) and siliques harvested at different DAP. Numbers in all panels correspond to DAP. wt, wild type; g, genomic DNA; ACT, ACTIN. Molecular Plant 2009 2, 654-660DOI: (10.1093/mp/ssp014) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Two Copies of Repeat Sequences Are Sufficient for DNA-Methylation. Cytosine methylation profiles of the PHE1 downstream region in the Col-0, Br-0, Bur-0, and Var2-1 accessions analyzed by bisulfite sequencing. Cytosine positions within CG contexts relative to the translational stop codon are indicated on the x-axis. Numbers refer to the Col-0 accession. For corresponding locations in other accessions, see Supplemental Figure 1. Asterisks indicate the lack of CG sites within a particular accession. Molecular Plant 2009 2, 654-660DOI: (10.1093/mp/ssp014) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Deletion of the PHE1 Repeats Abolishes PHE1 Imprinting. (A) Schematic diagram of PHE1 reporter constructs containing parts of the 3’ region with or without the repeat region. (B–E) Percent of GUS-positive seeds after reciprocal crosses of wild-type plants with plants heterozygous for the full-length PHE1::GUS_3’ construct (B), for the PHE1::GUS_3’ construct including the tandem repeats but lacking the region after the repeats (construct 1) (C), for the PHE1::GUS_3’ construct lacking only the repeats (construct 2) (D), and for the PHE1::GUS_3’ construct lacking the repeats and the 3’ region after the repeats (construct 3) (E). Numbers on the x-axis refer to different transgenic lines. n, number of seeds analyzed. Molecular Plant 2009 2, 654-660DOI: (10.1093/mp/ssp014) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions