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. 2007;35(18):6290-6.
doi: 10.1093/nar/gkm464. Epub 2007 Sep 18.

The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish 'bivalent chromatin marks' at the silent AGAMOUS locus

Abdelaty Saleh  1 , Ayed Al-Abdallat, Ivan Ndamukong, Raul Alvarez-Venegas, Zoya Avramova
Affiliations

The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish 'bivalent chromatin marks' at the silent AGAMOUS locus

Abdelaty Saleh et al. Nucleic Acids Res. 2007.

Erratum in

Abstract

Tightly balanced antagonism between the Polycomb group (PcG) and the Trithorax group (TrxG) complexes maintain Hox expression patterns in Drosophila and murine model systems. Factors belonging to the PcG/TrxG complexes control various processes in plants as well but whether they participate in mechanisms that antagonize, balance or maintain each other's effects at a particular gene locus is unknown. CURLY LEAF (CLF), an Arabidopsis homolog of enhancer of zeste (EZ) and the ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) control the expression of the flower homeotic gene AGAMOUS (AG). Disrupted ATX1 or CLF function results in misexpression of AG, recognizable phenotypes and loss of H3K4me3 or H3K27me3 histone H3-tail marks, respectively. A novel idea suggested by our results here, is that PcG and TrxG complexes function as a specific pair generating bivalent chromatin marks at the silent AG locus. Simultaneous loss of ATX1 and CLF restored AG repression and normalized leaf phenotypes. At the molecular level, disrupted ATX1 and CLF functions did not lead to erasure of the CLF- and ATX1-generated epigenetic marks, as expected: instead, in the double mutants, H3K27me3 and H3K4me3 tags were partially restored. We demonstrate that ATX1 and CLF physically interact linking mechanistically the observed effects.

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Figures

Figure 1.
Figure 1.
Phenotypes and expression of the homeotic gene AG in wild type, in single, and in double mutant plants. (a) Fourth rosette leaf from single (clf or atx1), from double (atx1/clf) mutant, and from wild type (wt) plants. (b) Same age plants are at different stages of development: atx1 and clf mutants are flowering, while atx1/clf and wt plants are still at vegetative stages and (c) expression of the CLF, ATX1 and AG genes in the different genetic backgrounds; ACTIN7, amplified from the respective templates under the same number of cycles is shown as a loading control. All samples were amplified from the same template.
Figure 2.
Figure 2.
H3K27me2 and H3K27me3 profiles of three Arabidopsis genes. (a) ChIP assays with antibodies specific against di- and tri-methylated H3K27; superscripts ‘t’ and ‘d’ stand for the tri-, or di-methylated isoforms, respectively. (I)-input sample is 15% of the immunoprecipitated DNA; (-) is negative control without antibody. Quantified bands from three independently performed ChIP assays are shown as graphs. Changes in H3K27me3 levels of wild type and clf SUP and AP1 nucleosomes are insignificant (p > 0.5).
Figure 3.
Figure 3.
Methylation profiles of histone H3-tail lysines of AG-nucleosomes. (a) ChIP assays with antibodies specific against tri-methylated H3K4 di- and tri-methylated H3K27, and di-methylated H3K9. Amplified regions are from the 5′-transcription start site and from downstream gene (G)-regions (see b). The characteristic leaf phenotypes are shown in parallel with the methylation patterns at 5′-end and downstream nucleosomes, and corresponding expression of AG is indicated; (b) schematic representation of the AG gene: empty vertical boxes represent exons, horizontal boxes represent introns. Bars below the line show regions amplified in the PCR-assay; 5′-represents the region upstream of the transcription start site; G (gene) stands for downstream coding sequences; (c) quantified bands showing the relative change in methylation modifications at the 5′-end and the downstream (G)-nucleosomes; results are from three independently performed ChIP assays; (d) the AP1-methylation profiles are shown as controls illustrating the quality of the templates; the same amounts of template DNA were used in the respective amplification experiments and (e) ACTIN7, a constitutively expressed gene carries only ‘activating’ m3K4/H3 tags.
Figure 4.
Figure 4.
ChIP assays with antiATX1 antibodies. Bands corresponding to the 5′-end AG-region amplified after a ChIP with antiATX1 antibodies; downstream region nucleosomes were only weakly represented in the precipitated fraction, while AP1 nucleosomes were not found in the ATX1-bound fraction.
Figure 5.
Figure 5.
Interaction of ATX1- and CLF-in yeast and in plant cells. (a) ATX1 and CLF cloned as bait and prey in the pGBKT9 and the pGADT7 vectors; yeast cells transformed with the constructs, as shown, and grown on selective SD media lacking leucine, trypotphan, histidine and adenine (high stringency); (b) BiFC assay of ATX1-YN/CLF-YC. The yellow signal indicates binding of ATX1 and CLF fusion proteins inside the nucleus; (c) BiFC assay of ATX1-YC and CLF-YN (ATX1 and CLF expressed in the opposite combinations of YFP-peptides). Fluorescence is shown in green, using a filter, to distinguish interactions of a different combination of complementing YFP-halves. (d) Cells transformed with the control vectors ATX1-YN/(-)YC and CLF-YN/(-)YC did not generate fluorescence. The images on the right represent merges of the left images with the DIC images.
Figure 6.
Figure 6.
A model for the interaction of the ATX1-containing TrxG and CLF-containing PcG complexes at the AG locus. In wild type leaf chromatin, CLF and ATX1 counterbalance each other. At the molecular level, this is associated with non-transcribed AG and with presence of m3K4/H3 and m3K27/H3. Both ATX1 and CLF activities are required for the ‘normal’ repression of AG generating, a bivalent chromatin state. ATX1 and CLF participate in antagonistic complexes functioning as a specific pair (nucleosome on the left). Loss of either CLF or ATX1 cannot be substituted by a homologous activity within the pair. ATX1 at the AG- 5′-nucleosomes may recruit CLF but not vice versa. This may account for the m3K4 and m3K27 patterns in the single mutants (see text). Elimination of both ATX1 and CLF allows a different pair of antagonists to label AG (nucleosome on the right). However, they modify only downstream nucleosomes because the specific factor taking ATX1/CLF to the start site fails to recruit their homologs. Assembly of substitute complexes restoring the methylation tags might not be occurring 100% of the time accounting for the variability in rescued phenotypes.
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