Abstract
BackgroundSET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction.Methodology/Principal FindingsA slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to ∼90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2.Conclusions/SignificanceThe severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences.
Highlights
Active transcription of eukaryotic genes is dependent on a permissive chromatin structure that allows transcription factors access to promoter and enhancer regions
Plants homozygous for ashh2-1, ashh2-2 and ashh2-5 show a characteristic dwarf and bushy phenotype (Figures 1C and 1H, and Figure S1). This phenotype was not found in ashh2-6 plants, the only allele that can be expected to produce a normal transcript and for which the expression level in inflorescences was only reduced to 55% (Figure 1B and Figure S1)
Stamens were shorter with thinner filaments that often were bent just beneath the anther, while the carpels were shorter and thicker compared to wt (Figure 1F). ashh2 siliques developed poorly (Figure 1G), and flowering and growth continued for at least 100 days (Figure 1H)
Summary
Active transcription of eukaryotic genes is dependent on a permissive chromatin structure that allows transcription factors access to promoter and enhancer regions. Enzymes that can add methyl groups to lysine residues on histone tails can be grouped in evolutionarily conserved classes named after the Drosopohila proteins SU(VAR), E(Z), TRITHORAX (TRX) and ASH1, that all have a similar 130 amino acid (aa) long SET domain [3,4]. Proteins belonging to the ASH1 class have properties similar to TRX in maintenance of transcription during development. These four classes have been identified in Arabidopsis thaliana [5]. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein ( called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set, a H3K36 HMTase. We focus on the role of ASHH2 in plant reproduction
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