Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin

Abstract

In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation.

Fig. 3.

Immunolocalization of H3K9^Me, H3K4^Me, and tetraacetylated histone H4 in WT and met1 nuclei. (A) Detection of H3K9^Me.(B) Detection of H3K4^Me.(C) Detection of H4 acetylation. Shown are FITC immunostaining (green) and DAPI staining (blue). Chromocenters are visible as light blue, and densely DAPI-stained structures are marked by arrowheads in the leftmost A. (D) Visualization of WT and met1 chromocenters with fluorescent in situ hybridization using 180-bp centromeric repeats probe (red) and immunostaining using 5-methylcytosine antibody (α-5MeC, green). (E) Analysis of H3 and H4 methylation and acetylation, respectively, from WT and met1 plants by Western blot probed with antibody directed against H3K9^Me (α-H3K9^Me), H3K4^Me (α-H3K4^Me), or tetraacetylated H4 (α-H4Ac). (Bottom) Coomassie blue-stained proteins.

Fig. 1.

Histone 3 methylation analyzed by ChIP and transcript levels of selected heterochromatic loci. (A) ChIP with antibodies recognizing H3K4^Me (K4) and H3K9^Me (K9) of chromatin extracts from WT and the met1 mutant (met1). Controls: chromatin extract precipitated without antibody (mock), chromatin extract (input), and PCR without template (-ve). Analyzed target loci are listed to the left of the corresponding images. (B) Quantification of ChIP analysis corresponding to images presented in A. Ordinates are fold difference. (C) RT-PCR analysis of transcripts, with (+) and without (–) reverse transcriptase (RT).

Fig. 2.

Histone 3 methylation analysis by ChIP of 180-bp centromeric repeats. (A) ChIP with antibodies recognizing H3K4^Me (K4) and H3K9^Me (K9) of chromatin extracts from WT and the met1 mutant (met1); controls as in Fig. 1. Amplification was performed using primers to a specific subset (180F4R5) or broader range of repeats, as described in the text. (B) Quantification of ChIP analysis corresponding to images presented in A. Ordinates are fold difference.

Fig. 4.

Histone 4 acetylation analyzed by ChIP of selected heterochromatic loci. (A) ChIP with antibodies recognizing histone H4 acetylated at lysines 5, 8, 12, and 16 (H4Ac) of chromatin extracts from WT and the met1 mutant; controls as in Fig. 1. Analyzed target loci are listed to the left of corresponding images. (B) Quantification of ChIP analysis corresponding to images presented in A. H4Ac was not detected at T5L23.26 and At4g03870 in either WT or met1. Ordinates are fold difference.