1. Volume 11, Issue 5, Pages 659-677 (May 2018)
Trithorax Group Proteins Act Together with a Polycomb Group Protein to Maintain Chromatin Integrity for Epigenetic Silencing during Seed Germination in Arabidopsis 
Fan Xu, Tony Kuo, Yenny Rosli, Mao-Sen Liu, Limin Wu, Long-Fang Oliver Chen, Jennifer C. Fletcher, Zinmay Renee Sung, Li Pu 
Molecular Plant 
Volume 11, Issue 5, Pages (May 2018)
DOI: /j.molp
Copyright © 2018 The Author Terms and Conditions

2. Figure 1
EMF1, ATX1, and ULT1 Together Prevent Excessive Seed Storage Reserve Accumulation Following Germination.
(A and B) Wild-type (WT) seedling (A) and root (B). Scale bar, 1 mm.
(C) Fat red-stained WT seedlings. Scale bar, 0.5 mm.
(D–F) atx1 (D), ult1 (E), and atx1 ult1 (F) seedlings. Scale bars, 1 mm.
(G) emf1 mutant seedling. Scale bar, 0.5 mm.
(H) Fat red-stained emf1 seedling. Scale bar, 0.5 mm.
(I) emf1 atx1 ult1 triple mutant (triple) with emf1-like shoot and swollen primary root tip (arrow). Scale bar, 0.5 mm.
(J) Fat red-stained emf1 atx1 ult1 seedlings. Arrow indicates a Fat red-positive swollen root. Scale bar, 0.5 mm.
(K–M) emf1 atx1 ult1 triple mutants. Arrows indicate the swollen primary root (K), embryo-like structures arising from hypocotyl (L), and callus-like tissues arising from cotyledon (M). Scale bar, 0.5 mm.
(N) emf1 ult1 double mutant with swollen primary root tip (arrow). The inset shows Fat red-stained emf1 ult1 root.
(O) emf1 atx1 double mutant with swollen primary root tip (arrow). The inset shows Fat red-stained emf1 atx1 root. Scale bar, 0.5 mm.
(P and Q) pkl mutant (P) and Fat red-stained pkl mutant (Q). Arrows indicate the pkl root tip. Scale bar, 1mm.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

3. Figure 2
EMF1, ATX1, and ULT1 Together Prevent Derepression of Selected Gene Expression Programs Following Germination.
(A) mRNA expression levels of the seed genes ABI3, LEC1, LEC2, FUS3, LTP3, and AT2S3, the flower MADS box genes AG and AP3, the GA-related genes RGL1, GA2OX2, and GA3OX1, and PKL in WT, emf1, atx1, ult1, atx1 ult1, emf1 ult1, and emf1 atx1 ult1 seedlings at 14 DAG. Graphs show the relative expression levels measured by qPCR, normalized to a UBIQUITIN (UBQ) reference gene. Error bars represent SD.
(B) Correlation between qPCR and RNA-seq/microarray data for selected genes. Comparison of expression levels from qPCR and RNA-seq (WT, emf1, atx1, atx1 ult1, and emf1 atx1 ult1 mutants) or microarray (ult1 mutants) data. R (Pearson's correlation coefficient) was calculated using six fold changes of qPCR and RNA-seq/microarray for selected genes. Star indicates that similar fold change values overlapped in the scatter chart.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

4. Figure 3
EMF1, ATX1, and ULT1 Together Repress Gene Transcription and Promote H3K27me3 Enrichment.
(A) Chromosomal distribution of H3K27me3 on the five Arabidopsis chromosomes in WT (black), emf1 (green), atx1 (red), and emf1 atx1 ult1 (blue) plants.
(B) The H3K27me3 patterns of 5988 trimethylated genes in WT, emf1, atx1, and emf1 atx1 ult1 plants. The gene sequences were aligned at the transcription start site (TSS) and average signals of the H3K27me3 enrichment 1 kb upstream and 3 kb downstream of the TSS were plotted.
(C) Number and percentage of genes with a 2-fold or greater reduction in H3K27me3 enrichment in atx1, emf1, and emf1 atx1 ult1 plants compared with WT plants.
(D) Correlation between percentage of genes upregulated and percentage of genes with decreased H3K27me3 enrichment among the atx1, emf1, and emf1 atx1 ult1 mutants.
(E and F) Change on a log2 scale in H3K27me3 enrichment (E) and mRNA expression levels (F) of selected genes in emf1 and emf1 atx1 ult1 mutants compared with WT.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

5. Figure 4
EMF1, ATX1, and ULT1 Together Affect Histone Methylation at Target Gene Loci.
(A) ChIP-seq analysis of H3K27me3 distribution at the ABI3, LEC2, LTP3, and AT2S3 seed gene loci in WT, emf1, atx1, and emf1 atx1 ult1 plants. ACT3 (AT3G53750) was used as a negative control. Gene models shown at the bottom include 5′ UTR (medium black line), exons (black boxes), introns (thin black line), and 3′ UTR (medium black line). The white arrow indicates transcriptional direction. The black line below each gene model indicates 500 bp.
(B) ChIP–qPCR analysis of H3K27me3 levels at the ABI3, LEC2, LTP3, AT2S3, and ACT3 loci in WT, emf1, atx1, ult1, and emf1 atx1 ult1 seedlings at 14 DAG. Primers (double arrowheads) correspond to the gene regions shown in (A). ChIP–qPCR results are expressed as a percentage of input DNA, with error bars representing SD.
(C) ChIP–qPCR analysis of H3K4me3 levels at the ABI3, LEC2, LTP3, AT2S3, and ACT3 loci in WT, emf1, atx1, ult1, and emf1 atx1 ult1 seedlings at 14 DAG. Primers (double arrowheads) correspond to the gene regions shown in (A). ChIP–qPCR results are expressed as a percentage of input DNA, with error bars representing SD.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

6. Figure 5
ATX1 and ULT1 Affect the Expression of Genes Normally Bound by EMF1 When EMF1 Is Absent.
(A) Number and percentage of misregulated genes in emf1, atx1, and ult1 single, double and triple mutants.
(B–D) Pie charts of the effect of atx1 ult1 mutations on the expression of EMF1-bound genes that are 2-fold or greater upregulated (B), unchanged (C), and downregulated (D) in emf1 mutants.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

7. Figure 6
EMF1, ATX1, and ULT1 Together Affect Global Chromatin Organization.
(A–C) DAPI-stained WT (A), emf1 (B), and emf1 atx1 ult1 triple mutant (C) primary roots (200×). Insets show a single WT (A), emf1 (B), or triple mutant (C) nucleus. Scale bars, 50 μm.
(D and E) Quantification of nuclear diameter (D) and size (E) in primary root tips. *p < 0.01, **p < 0.001 by t-test. Error bars indicate SD; n = 60 nuclei.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

8. Figure 7
ULT1 and ATX1 Directly Bind Seed Gene Loci but Not PRC1 Gene Loci.
(A) ChIP-seq analysis of ATX1 or ULT1 binding at the seed genes ABI3, AT2S3, LEC2, and LTP3 and at the PRC1 genes AtBMI1A, AtBMI1B, AtRING1A, and AtRING1B in 35S::HA-ULT1 ult1 35S::ATX1-HA atx1 seedlings at 14 DAG. ACT3 was used as a negative control. Gene models shown at the bottom include 5′ UTR (medium black line), exons (black boxes), introns (thin black line), and 3′ UTR (medium black line). The white arrowheads indicate transcriptional direction. The double arrowheads above each gene model indicate the regions amplified for the ChIP–qPCR analysis in (B) and (C). The black bar above each gene model indicates 500 bp.
(B) ChIP–qPCR analysis of ATX1 binding at the seed genes ABI3, AT2S3, LEC2, and LTP3, at the PRC1 genes AtBMI1A, AtBMI1B, AtRING1A, and AtRING1B, and at ACT3 in 35S::ATX1-HA atx1 seedlings at 14 DAG. The graphs show ChIP–qPCR using an anti-HA antibody (IP) or no antibody (mock control) as a percentage of input DNA, with error bars indicating SD.
(C) ChIP–qPCR analysis of ULT1 binding at the seed genes ABI3, AT2S3, LEC2, and LTP3, at the PRC1 genes AtBMI1A, AtBMI1B, AtRING1A, and AtRING1B, and at ACT3 in 35S::HA-ULT1 ult1 seedlings at 14 DAG. The graphs show ChIP–qPCR using an anti-HA antibody (IP) or no antibody (mock control) as a percentage of input DNA, with error bars indicating SD.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

9. Figure 8
EMF1 and ULT1 Physically Associate to Maintain Chromatin Condensation and Silencing of Seed Genes.
(A) Yeast two-hybrid analysis showing an interaction between EMF1 and ULT1 and between ATX1 and ULT1. AD-ULT1, BD-EMF1, AD-ATX1, BD-ATX1, and BD-EMF1 constructs were co-transformed into Golden Yeast cells in pairwise combinations. The AD/BD, AD-ULT1/BD, AD/BD-EMF1, AD/BD-ATX1, and AD-ATX1/BD combinations were employed as negative controls. Transformed yeast cells were selected on synthetic dextrose medium lacking Leu and Trp (−Trp/−Leu), then transferred to synthetic dextrose medium lacking Leu, Trp, and His (−Trp/−Leu/−His) to assay for protein–protein interactions. The interactions were confirmed on synthetic dextrose medium lacking Leu, Trp, His, and Ade (−Trp/−Leu/−His/−Ade).
(B) Luciferase (LUC) complementation imaging (LCI) assays showing that EMF1 interacts with ULT1 and ATX1 interacts with ULT1 in N. benthamiana. The full-length EMF1, ATX1, and ULT1 coding regions were fused with the N- or C-terminal domain of the luciferase reporter gene LUC (-nLUC or cLUC-). N. benthamiana leaves were infiltrated with A. tumefaciens strain GV3101 containing the indicated constructs. The EMF1-nLUC/cLUC combination was employed as a negative control. The pseudocolor bar shows the range of luminescence intensity in the image.
(C) A proposed model for PRC1 (EMF1, AtRING1, AtBMI1), PRC2 (EMF2, CLF, FIE, MSI1), and trxG (ATX1 and ULT1) proteins in maintaining seed gene repression during germination in Arabidopsis. In WT, EMF1 is required for chromatin compaction and physically interacts with the trxG protein ULT1 as well as with PRC1 and PRC2 proteins, thus maintaining chromatin in a condensed and stable repressive state. The seed genes are enriched with H3K27me3 and their expression is “OFF” at the specific developmental phase of seed germination. The trxG proteins may also promote the expression of an unknown gene (?) that negatively regulates seed gene expression (dashed line). In atx1 ult1 double mutants, seed genes are more enriched in H3K27me3 relative to WT and are kept silent (OFF). In emf1 mutants, H3K27me3 levels are reduced and seed genes are derepressed (ON) although the mRNA levels of some PcG components are similar to those of WT. Alternatively, trxG activity may promote the expression of an unknown seed repressor gene (?), which allows seed gene upregulation in triple mutants. In emf1 atx1 ult1 triple mutants, loss of EMF1, ATX1, and ULT1 significantly reduces H3K27me3 levels, loosens chromatin, and consequently greatly enhances seed gene expression (ON). These seedlings develop swollen roots, shown as the green rectangle at the root end of the seedling. The “+” indicates relative transcript level. Red triangles represent H3K27me3 marks; green rectangles represent PRC2 proteins; orange ovals represent PRC1 proteins; blue clouds represent trxG proteins; pink hexagons represent putative unknown factors.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions