Epigenetic regulation of gene programs by EMF1 and EMF2 in Arabidopsis

Abstract

The EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative development in Arabidopsis (Arabidopsis thaliana). Loss-of-function emf mutants skip the vegetative phase, flower upon germination, and display pleiotropic phenotypes. EMF1 encodes a putative transcriptional regulator, while EMF2 encodes a Polycomb group (PcG) protein. PcG proteins form protein complexes that maintain gene silencing via histone modification. They are known to function as master regulators repressing multiple gene programs. Both EMF1 and EMF2 participate in PcG-mediated silencing of the flower homeotic genes AGAMOUS, PISTILLATA, and APETALA3. Full-genome expression pattern analysis of emf mutants showed that both EMF proteins regulate additional gene programs, including photosynthesis, seed development, hormone, stress, and cold signaling. Chromatin immunoprecipitation was carried out to investigate whether EMF regulates these genes directly. It was determined that EMF1 and EMF2 interact with genes encoding the transcription factors ABSCISIC ACID INSENSITIVE3, LONG VEGETATIVE PHASE1, and FLOWERING LOCUS C, which control seed development, stress and cold signaling, and flowering, respectively. Our results suggest that the two EMFs repress the regulatory genes of individual gene programs to effectively silence the genetic pathways necessary for vegetative development and stress response. A model of the regulatory network mediated by EMF is proposed.

Figure 1.

Genes up-regulated or down-regulated in three emf mutants. A total of 11,447 and 10,204 genes with a hybridization signal greater than 50 in 7- and 15-d-old plants, respectively, were analyzed for overlapping expression patterns among the three mutants. Number (percentage) of genes up-regulated (A and C) and down-regulated (B and D) greater than 2-fold difference in the mutants are shown.

Figure 2.

RT-PCR analysis of flowering time genes in emf mutants. RNA samples extracted from 7- and 14-d-old wild-type (WT) and emf mutant plants were subjected to RT-PCR analysis. ACTIN8 was used as a loading control. Numbers presented below the gels represent relative expression levels and were calculated using the ACTIN8 signal as a reference.

Figure 3.

Overlapping seed gene expression patterns in the three emf mutants. Sixty-one and 56 of the 120 seed maturation genes up-regulated in one of the three emf mutants at 7 and 15 DAG, respectively, were analyzed for overlapping expression in two or all three emf mutants. Number (percentage) of genes up-regulated in mutants at 7 (A) and 15 (B) DAG are shown.

Figure 4.

RT-PCR analysis of seed and stress genes in emf mutants. RNA samples extracted from 7- and 14-d-old wild-type (WT) and emf mutant plants were subjected to RT-PCR analysis. ACTIN8 was used as a loading control. Numbers presented below the gels represent relative expression levels and were calculated using the ACTIN8 signal as a reference.

Figure 5.

EMF2promoter∷EMF2∷3XFLAG construct and the protein expression pattern. A, The construct that harbors tagged EMF2 used in plant transformation is depicted. EMF2 cDNA was tagged with the 3XFLAG sequence and expressed under the EMF2 promoter. This construct was introduced into emf2-1, and it rescued the mutant phenotype. B, emf2-1 mutant and rescued emf2-1 (E2-3F) harboring EMF2promoter∷EMF2∷3XFLAG and E2-3F grown on agar plates (left) as well as E2-3F and the wild type (WT) grown in soil (right). C, Western-blot analysis of nuclear protein from various tissues of wild-type and E2-3F seedlings at 7 and 14 DAG, rosette leaves, inflorescence apex, and siliques of plants grown in soil. Anti-FLAG antibody was used to detect the EMF2-3XFLAG fusion protein. A portion of the Coomassie Brilliant Blue-stained gels in the range of 50 to 100 kD is shown as a loading control for western-blot analysis.

Figure 6.

EMF1 and EMF2 proteins interaction with target genes. A, Schematic representation of gene structures of PI, ABI3, LOV1, FLC, FT, and PHE1. Black boxes depict the first exon including the 5′ untranslated region, and arrows indicate the region of the gene amplified in ChIP analysis, −320 to −74 bp for PI, −746 to −586 bp for ABI3, +267 to +393 bp for LOV1, −94 to +232 bp for FLC, −330 to −70 bp for FT-P, +1,050 to +1,325 bp for FT-I, and −59 to +141 bp for PHE1. P and I denote promoter and intron regions in FT, respectively. B, ChIP results showing EMF1 and EMF2 interaction with target genes. Anti-FLAG antibody was used to immunoprecipitate nuclear proteins from 14-d-old transgenic plants, and primers from A were used to amplify the DNA in the immunoprecipitated chromatin (IP). Input is preimmunoprecipitated DNA after sonication. Thirty cycles of PCR were performed using the primers covering the DNA regions shown in A. PHE1 was used as an internal negative control. E1-3F represents emf1-2 rescued by EMF1∷EMF1∷3XFLAG (Calonje et al., 2008).

Figure 7.

A model of EMF repression of developmental and stress pathways in Arabidopsis. Arrows and blocking bars indicate transcriptional activation and repression, respectively. Thick blocking bars show EMF repression of target genes (LOV1, ABI3, PI, AG, and FLC) through direct EMF-target gene interaction. The dotted arrow indicates potential EMF interaction with TEM.