Dynamic regulatory interactions of Polycomb group genes: MEDEA autoregulation is required for imprinted gene expression in Arabidopsis

Abstract

The imprinted Arabidopsis Polycomb group (PcG) gene MEDEA (MEA), which is homologous to Enhancer of Zeste [E(Z)], is maternally required for normal seed development. Here we show that, unlike known mammalian imprinted genes, MEA regulates its own imprinted expression: It down-regulates the maternal allele around fertilization and maintains the paternal allele silent later during seed development. Autorepression of the maternal MEA allele is direct and independent of the MEA-FIE (FERTILIZATION-INDEPENDENT ENDOSPERM) PcG complex, which is similar to the E(Z)-ESC (Extra sex combs) complex of animals, suggesting a novel mechanism. A complex network of cross-regulatory interactions among the other known members of the MEA-FIE PcG complex implies distinct functions that are dynamically regulated during reproduction.

Figure 1.

MEA regulates its own imprinted expression. (A,B) Quantification of maternal and paternal transcripts in ovules and developing seeds derived from wild-type (A) or mea-2 mutant (B) embryo sacs. Transcripts were quantified before fertilization in entire gynoecia containing unfertilized ovules (BF) and after fertilization in developing fruits following hand pollination (1–4 dap) (Supplementary Fig. S1). Transcript levels were normalized to ACTIN11; average and standard deviation of triplicate quantification are shown. In B, the fold increase of maternal transcripts in mea ovules compared with wild-type is indicated for each time point below the graph. (C–E) Expression of a pMEA∷GUS reporter gene in unfertilized ovules of MEA/MEA wild-type plant (C) or mea-2/MEA mutant plants (D). (E) The percentage of ovules showing a weak staining as in C or a strong staining as in D was quantified in wild-type and mea-2/MEA mutant segregants. (P) Level of significance relative to the difference between the two segregants; (n) number of ovules; (cc) central cell; (ec) egg cell. Bar, 10 μm. (F–H) Expression of a paternally inherited pMEA∷GUS reporter gene 5–6 d after crossing to wild-type (F) or mea-2/mea-2 mutant (G) plants. In H, the percentage showing derepression of the usually silent paternal allele in crosses with wild-type, heterozygous, and homozygous mea-2 plants is given. (n) Number of seeds analyzed. Bar, 20 μm.

Figure 2.

ChIP experiments show an interaction of MEDEA with its own promoter (A) Schematic representation of the MEA locus indicating the regions analyzed by PCR after ChIP. The numbers indicate a genomic position relative to the transcriptional start site (arrow) upstream the of the ORF of MEA (dark shaded gray). The dashed-line box indicates the promoter region where fragments were enriched in the immunoprecipitated fractions in the presence of the αMEA antibody. (B) ChIP experiment showing MEA interaction with its own promoter. Chromatin was prepared from 1 g of tissue typically consisting of ∼1000 wild-type flowers. The gels show PCR products from immunoprecipitated fractions treated with no antibody (lane a), with preimmune serum (lane b), and with αMEA antibody (lane c). The complete set of ChIP experiments is shown in Supplementary Figure S2.

Figure 3.

Expression profiles of MEA, FIS2, FIE, and MSI1. (A) Relative transcript levels in wild-type gynoecia (BF) or developing siliques (1, 2, and 3 dap). The levels were normalized to 1 before fertilization. The average of three biological replicates is plotted on the graph, and the standard deviation is shown.(B) Relative transcript abundance at the BF time point. The ratios are expressed relative to the level of FIS2, which was normalized to 1.

Figure 4.

Complex regulatory interactions among the FIS class PcG genes. (A) MEA regulates its own imprinted gene expression. Schematic representation of the known regulators of imprinted MEA expression. Direct interactions are shown as solid lines, and interactions that are either direct or indirect are shown as stippled lines. Before fertilization, the maternal MEA^m allele is activated through antagonistic interactions of DME and MET1 (Xiao et al. 2003) and kept at a low expression level by direct autorepression. After fertilization, the paternal MEA^p allele is maintained silent through MEA-mediated repression and activity of the chromatin remodeling factor DDM1, based on genetic evidence (Vielle-Calzada et al. 1999). (B) Genetic regulation of the FIS class PcG genes involves positive auto- and cross-regulations. Only MEA and FIE autoregulate. MEA autorepression is direct but independent of the other components of the MEA–FIE complex. The mechanism of FIE autoregulation is unknown but is also independent of the MEA–FIE complex. A comparison of regulatory interactions before and after fertilization illustrates the highly dynamic nature of PcG regulation.