An Arabidopsis Natural Epiallele Maintained by a Feed-Forward Silencing Loop between Histone and DNA

Abstract

The extent of epigenetic variation is currently well documented, but the number of natural epialleles described so far remains very limited. Determining the relevance of epigenetic changes for natural variation is an important question of research that we investigate by isolating natural epialleles segregating in Arabidopsis recombinant populations. We previously described a genetic incompatibility among Arabidopsis strains based on the silencing of a gene involved in fitness. Here, we isolated a new epiallele resulting from the silencing of a transfer-RNA editing gene in an Arabidopsis accession from the Netherlands (Nok-1). Crosses with the reference accession Col-0 show a complete incompatibility between this epiallele and another locus localized on a different chromosome. We demonstrate that conversion of an unmethylated version of this allele occurs in hybrids, associated with modifications of small RNA populations. These epialleles can also spontaneously revert within the population. Furthermore, we bring evidence that neither METHYLTRANSFERASE 1, maintaining methylation at CGs, nor components of RNA-directed DNA methylation, are key factors for the transmission of the epiallele over generations. This depends only on the self-reinforcing loop between CHROMOMETHYLASE 3 and KRYPTONITE, involving DNA methylated in the CHG context and histone H3 lysine 9 methylation. Our findings reveal a predominant role of this loop in maintaining a natural epiallele.

Fig 1. TAD3 is the candidate gene for the incompatibility between Nok-1/Est-1 and Col-0.

(A) The mapping interval localized on chromosome 5 in Col-0 contains four predicted genes. TER2 encodes a non-coding RNA associated with the telomerase [24]. AT5G24655 and AT5G24660 are homologous genes involved in sulfur response [53]. AT5G24670 encodes an enzyme involved in tRNA editing (TAD3) and GABI_141G12 was described [23]. The T-DNA positions in individual mutants isolated are represented by triangles. We recovered homozygous mutants for lines depicted in blue, and heterozygous mutants for lines depicted in red. T-DNAs in both SALK_121147 and SAIL_363_B11 are inserted in the 5’-UTR of TAD3, disrupting TER2. UTRs of TAD3 are represented by black boxes. (B) Siliques of a tad3/TAD3 heterozygous T-DNA plant (GABI_141G12) are similar to siliques of a F8 RIL plant (4RV355) carrying Col-0 alleles at chromosome 1 and heterozygous Col-0/Nok-1 for the chromosome 5 locus. Red arrows indicate aborted seeds. (C) Number of defective seeds per silique in three different RILs (F8 generation) carrying Col-0 alleles at chromosome 1 and heterozygous Col-0/Nok-1 for the chromosome 5 locus. One to five siliques per plant were phenotyped, representing a total of 400 embryos for 4RV207, 227 embryos for 4RV355 and 565 embryos for 4RV415.

Fig 2. TAD3 sequence differences between Nok-1 and Col-0.

(A) TAD3-1 polymorphisms between Col-0 and Nok-1 obtained by sequencing the whole gene (S2 Fig). The 11 exons of TAD3-1 are represented by rectangles. The two predicted transcript isoforms of Col-0 are shown, they only differ by their UTRs (white parts of the rectangles). Compared to Col-0, Nok-1 TAD3-1 contains an insertion (ins), a deletion (del) and four SNPs in the coding region (grey parts of the rectangles). Other SNPs, not presented here, were also found in introns (S2B Fig). The A>T change in the 5’-UTR is the only one shared by both Est-1 and Nok-1. The red star indicates the position of the 17 bp deletion found in other copies in Nok-1, used to design primers specific for Nok-1 TAD3 on chromosome 1 (B). The sequence corresponding to these primers is underlined. (C) Nok-1 and Est-1 are carrying extra copies of TAD3 on chromosome 1. PCR amplification on genomic DNAs extracted from plants with the indicated genotypes. K1NokK5Col corresponds to RILs fixed for the Nok-1 allele at chromosome 1 and for the Col-0 allele at chromosome 5. K1ColK5Nok corresponds to revertant plants fixed for the Col-0 allele at chromosome 1 and for the Nok-1 allele at chromosome 5 (see Fig 4). Primers are as described in (B) for the primers specific for chromosome 1 and (A) for the other primers.

Fig 3. TAD3-1 is methylated in Nok-1 and Est-1, preventing its expression.

(A) DNA methylation of TAD3-1 5’-UTR analysed by digesting the indicated genomic DNA with McrBC followed by PCR amplification. Regions amplified correspond to PCR#2 and PCR#3 (Fig 2A) and are specific for TAD3-1 (S4 Fig). (B) TAD3-1 is silenced in Nok-1 and Est-1. Plants were grown on medium containing 0 (-) or 10 (+) μg/ml of 5-aza-2’deoxycytidine (Aza) for seven days. RNAs of plants were extracted and cDNAs were amplified using primers corresponding to PCR#3 (Fig 2A), specific for chromosome 5. ATEF amplifications served as controls. (C) TAD3-1 qRT-PCR analyses using PCR#3 primers that are specific for chromosome 5 and plants described in (B).

Fig 4. The methylation of the tad3-1 epiallele is reversible.

(A) Genomic DNAs (300 ng) from the F9 plants indicated were digested with McrBC (+McrBC) and then amplified using primers specific for the regions indicated (see Fig 2A to localize the amplicons within TAD3). We verified the presence of the polymorphisms between Nok-1 and Col-0 (Fig 2A) by sequencing the PCR fragments obtained without McrBC treatment (-McrBC) for PCR#2, #3, #5 and #6, excluding a genetic recombination between Col-0 and Nok-1 in this region. All plants are from the progeny of an F8 revertant that was fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5. K1ColK5Nok are F9 plants fixed Col-0 at chromosome 1 and Nok-1 at chromosome 5. K1ColK5Col are F9 plants fixed Col-0 at both chromosomes. For the Nok-1 plants, only PCR#2 and #3 are specific for chromosome 5. (B) The methylation rates within the promoter and the gene body of TAD3 were determined in plants described in (A). Data were obtained by amplifying the regions indicated, namely ‘Prom.’ for the promoter and ‘Gene’ for the gene body, after bisulfite conversion (S7 Fig). (C) Expression of TAD3 analyzed by qRT-PCR in plants described in (A) using primers corresponding to PCR#3 (Fig 2A), specific for chromosome 5. (D) Segregation of the Nok-1 and Col-0 alleles at chromosome 5 in the progeny of the revertant. We genotyped the progeny of two F9 plants (#3 and #2), descending from the revertant, and fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5. The control is an F9 plant fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5, coming from a lineage independent of the revertant. The numbers of plants genotyped are indicated in parentheses.

Fig 5. Unmethylated TAD3-1 alleles are converted in hybrids.

(A) Methylation rates within the promoter of TAD3-1 in four hybrids obtained by crossing Col-0 and Nok-1 (Plants #1 to #4). Data (S9 Fig) were obtained by amplifying from leaf genomic DNA the promoter region corresponding to PCR#9 (S8 Fig), differentiating the Col-0 or Nok-1 alleles with the TTT insertion. (B) Expression of TAD3-1 determined by qRT-PCR in leaves of seven different hybrids (Plants #1 to #7) including the four hybrids shown in (A).

Fig 6. Sequence length distribution of the small RNAs over the TAD3-1 locus.

sRNA reads were counted in three different genomic regions: the first one (Chr5: 8,446,240–8,447,463) comprises two transposons upstream of TAD3-1, the second includes the promoter of TAD3-1 (Chr5: 8,447,463–8,447,954) and the last one corresponds to the TAD3-1 gene (Chr5: 8,447,954–8,451,218). Transposable elements are depicted in orange and TAD3-1 in grey. Counts are given in reads per million of mapped reads. The precise distribution of sRNA reads is described in S11 Fig.

Fig 7. Methylation patterns of TAD3-1 in incompatible plants carrying a cmt3 mutation.

(A) Methylation patterns of TAD3-1 in incompatible plants carrying a cmt3 mutation. Genomic DNAs (300 ng) were digested with McrBC (+McrBC) and then amplified using primers specific for the PCR fragments indicated (see Fig 2A to localise the amplicons within the TAD3-1 gene). Plants noted “CN” or “CC” are all in the cmt3-11 background. “C” indicates that the Col-0 allele is fixed, “N” indicates that the Nok-1 allele is fixed. For the Nok-1 plants, only PCR#2 and #3 are specific for chromosome 5. (B) Methylation rates within the promoter regions in incompatible plants carrying a cmt3-11 mutation. Data (S13 Fig) were obtained by amplifying from leaf genomic DNA the promoter region corresponding to PCR#7 (S7A Fig). The numbers indicate the plants analyzed, as shown in (A). In a cmt3 background, the allele inherited from Nok-1 at chromosome 5 becomes specifically hypomethylated in the CHG context compared to the Nok-1 parent (plants #6 and #46). (C) Segregation of the Nok-1 and Col-0 alleles at chromosome 5 in the progeny (n = 128) of a cmt3 mutant. The progeny of a plant fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5 and sibling of the plants presented on (A) and (B) were genotyped.