Methylation patterns of histone H3 Lys 4, Lys 9 and Lys 27 in transcriptionally active and inactive Arabidopsis genes and in atx1 mutants

Abstract

Covalent modifications of histone-tail amino acid residues communicate information via a specific 'histone code'. Here, we report histone H3-tail lysine methylation profiles of several Arabidopsis genes in correlation with their transcriptional activity and the input of the epigenetic factor ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) at ATX1-regulated loci. By chromatin immunoprecipitation (ChIP) assays, we compared modification patterns of a constitutively expressed housekeeping gene, of a tissue-specific gene, and among genes that differed in degrees of transcriptional activity. Our results suggest that the di-methylated isoform of histone H3-lysine4 (m2K4/H3) provide a general mark for gene-related sequences distinguishing them from non-transcribed regions. Lys-4 (K4/H3), lys-9 (K9/H3) and lys-27 (K27/H3) nucleosome methylation patterns of plant genes may be gene-, tissue- or development-regulated. Absence of nucleosomes from the LTP-promotor was not sufficient to provoke robust transcription in mutant atx1-leaf chromatin, suggesting that the mechanism repositioning nucleosomes at transition to flowering functioned independently of ATX1.

Figure 1

Overall histone H3-K4 methylation in wild-type and atx1 mutants. Total histones extracted from 3-week old wild-type and atx1 mutant plants were probed with antibodies specific for di- or tri-methylated K4/H3 in western-blots. Subsequent to the hybridization, membranes were stripped off and re-probed with antibodies specific for non-modified histone H3. The levels of histone H3-tail methylation of wild-type histones, defined as the ratio of mK/H3-to-H3 intensity signals, were taken as 100%.

Figure 2

Tissue-specific expression of LTP and histone H3-tail methylation patterns in wild-type and in atx1 mutants. (a) Expression of the LTP gene (At2g15050) in wild-type flowers (F), leaves (L) and stems (S) and in the respective atx1 mutant tissues (f), (l) and (s). Actin was used as a loading control for each template. (b) LTP gene structure and location of specific primers used to PCR amplify tested gene regions. Empty boxes indicate exons, black boxes indicate non-translated regions; (5′)-contain upstream sequences and, presumably, the promotor; (G)-contains sequences within the coding region. (c) Chromatins isolated from wild-type and atx1 tissues (F-flowers, L-leaf) immunoprecipitated with antibodies against specific H3-tail lysines. (I)-input DNA; 15-fold diluted samples were used as templates for the input (I) lanes. Negative controls (−), no antibody samples treated in the same way as immunoprecipitated chromatins; K9, K27, K4^d and K4^t- represent amplified bands from templates ChIP-ed with methylated histone H3- m²K9/H3, m²K27/H3, m²K4/H3 and m³K4/H3-antibodies, respectively (see Materials and Methods for dilutions, calibration and quantitation of chromatin used as template). (d) Leaf and flower chromatins, ChIP-ed with antibodies specific against unmodified histone H3 used as a general probe for nucleosome-associated DNA sequences. (e) Sensitivity to MNase digestions of the LTP and XET promotor regions in leaf (L) and flower (F) chromatins.

Figure 3

Tissue-specific expression of XET in wild-type tissues and in atx1 mutants and methylation profiles at the histone H3-tails. (a) Expression of the XET gene (At1g10550), a putative xyloglucosyl transferase of the Glycosyl hydrolases family 16. (b) LTP gene structure and location of specific primers used to PCR amplify tested gene regions. (c) Chromatins isolated from wild-type and atx1 tissues, immunoprecipitated with antibodies against specific H3-tail lysines. Annotations are as in Figure 2 (see also text for details); (d) ChIP assays for presence of 5′-XET and 5′-LTP nucleosomes from leaf, flower and stem chromatins with anti-ATX1 antibodies.

Figure 4

Tissue-specific expression and histone H3- methylation profiles of SUP and ACT genes as well as SUP-flanking intergenic sequences in wild-type and in atx1 mutant tissues. (a) Expression of the SUP gene (At3g23130) and the ACT 2/7gene (At5g09810); The panels labeled up- and down- show absence of transcripts from the intergenic regions of wild-type (F/wt) and mutant (F/atx) flower chromatins; G/wild-type and G/atx1 illustrate bands amplified with the same primers for the intergenic sequences using genomic DNA as template. (b) SUP gene structure and location of specific primers used to amplify tested regions by PCR. (c) Chromatins isolated from wild-type and atx1 tissues, immunoprecipitated with antibodies against specific H3-tail lysines. Annotations are as in Figure 2 (see also text for details); (d) Nucleosomes at the non-transcribed flanking regions in flower chromatin. (e) Structure and location of specific primers used to amplify the ACT gene. (f) histone H3-methylation profiles of the constitutively expressed ACT gene.