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DNA sequence homology induces cytosine-to-thymine mutation by a heterochromatin-related pathway in Neurospora

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Abstract

Most eukaryotic genomes contain substantial amounts of repetitive DNA organized in the form of constitutive heterochromatin and associated with repressive epigenetic modifications, such as H3K9me3 and C5 cytosine methylation (5mC). In the fungus Neurospora crassa, H3K9me3 and 5mC are catalyzed, respectively, by a conserved SUV39 histone methyltransferase, DIM-5, and a DNMT1-like cytosine methyltransferase, DIM-2. Here we show that DIM-2 can also mediate repeat-induced point mutation (RIP) of repetitive DNA in N. crassa. We further show that DIM-2-dependent RIP requires DIM-5, HP1, and other known heterochromatin factors, implying a role for a repeat-induced heterochromatin-related process. Our previous findings suggest that the mechanism of repeat recognition for RIP involves direct interactions between homologous double-stranded DNA (dsDNA) segments. We thus now propose that, in somatic cells, homologous dsDNA–dsDNA interactions between a small number of repeat copies can nucleate a transient heterochromatic state, which, on longer repeat arrays, may lead to the formation of constitutive heterochromatin.

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Figure 1: Cytosine methyltransferase DIM-2 can mediate RIP.
Figure 2: DIM-2-mediated RIP requires the SUV39 histone methyltransferase DIM-5.
Figure 3: DIM-2-mediated RIP responds to weak, interspersed homology.
Figure 4: Recognition of short tandem-repeat arrays by the DIM-5–DIM-2 pathway.
Figure 5: DIM-2-mediated RIP can target repeats at widely separated genomic positions.
Figure 6: A general model for nucleation of heterochromatin by pairwise dsDNA–dsDNA interactions.

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Acknowledgements

We thank D. Zickler for critically reading the manuscript. This work was supported by grant GM044794 from the US National Institutes of Health to N.K. and research fellowships from the Helen Hay Whitney Foundation, the Howard Hughes Medical Institute and the Charles A. King Trust to E.G.

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Contributions

E.G. designed, performed and analyzed the data from all experiments. E.G. and N.K. wrote the manuscript.

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Correspondence to Eugene Gladyshev or Nancy Kleckner.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Analysis of RIP mutation using the 802-bp tester construct.

(a) The 802-bp tester construct is created by replacing the cyclosporin-resistant-1 (csr-1) gene with transgenic DNA containing 802 bp of perfect homology to a neighboring gene (NCU00725). (b) RIP mutations are detected by sequencing the entire tester construct (recovered by PCR) from individual ‘late-arising’ haploid spore clones. (c) When a gene of interest is analyzed for its role in RIP in the heterozygous condition, two configurations are possible: in cis, the wild-type allele of the gene is provided together with the tester construct in the same parental nucleus (maternal or paternal); in trans, the wild-type allele is provided by one parent, whereas the tester construct is provided by the other parent (along with a corresponding gene deletion allele).

Supplementary Figure 2 Numerical analysis of RIP mutation.

(a) RIP is quantified for the crosses in Figure 1b as the mean number of mutations (per spore) in the three regions of interest: the 802-bp endogenous repeat copy (solid blue), the 802-bp ectopic repeat copy (hatched blue) and the 600-bp portion of the linker region (gray). Distributions of per-spore mutation counts are compared for each region of interest by the Kolmogorov–Smirnov test (Online Methods). (b) The mean number of RIP mutations for the crosses in Figure 2d (analyzed as in a). (c) The mean number of RIP mutations for the crosses in Figure 2f (analyzed as in a). (d) RIP mutation of the 450-bp segment of the linker region is quantified for the crosses in Figure 3 as the mean number of mutations (as in a; left) and as the percentage of the mutated DNA strands (as in ref. 18; right). In the latter case, differences in the percentage of mutated strands are evaluated for significance as raw numbers using the χ2 homogeneity test (P value indicated above the line) and Fisher’s exact test (P value below the line). Interspersed homologies are compared to the corresponding instance of random homology in the same genetic background. Error bars, s.e.m. ***P ≤ 0.001, **0.001 < P ≤ 0.01, *0.01 < P ≤ 0.05; NS, P > 0.05.

Supplementary Figure 3 Pairwise correlations of mutation counts observed within the three regions of interest.

(a) All individual C-to-T (C>T; upward tickmarks) and G-to-A (G>A; downward tickmarks) mutations are identified in the sample of 24 progeny spores. Spores are ranked by the total number of mutations. Analyzed crosses X1 (left) and X5 (right). (b) Pearson product–moment correlation coefficients, calculated independently for C-to-T (C>T) and G-to-A (G>A) mutations, for the crosses in a. Only mutations in the three regions of interest (the two 802-bp repeated sequences and the 600-bp segment of the linker region) were analyzed.

Supplementary Figure 4 The H3K27 methyltransferase SET-7 has no detectable role in RIP.

(a) DIM-5-mediated H3K9me3 opposes SET-7-mediated H3K27me3 in N. crassa (refs. 25,26). (b) RIP mutation profile of the 802-bp construct. Cross X32{24}. The number of analyzed spores is provided in brackets. (c) The mean number of RIP mutations for cross X32 (analyzed as in Supplementary Fig. 2a). The wild-type cross X1 is used as the reference.

Supplementary Figure 5 DIM-2-mediated RIP mutations spread into nearby genes.

Eight progeny spores with particularly strong levels of DIM-2-mediated RIP were reanalyzed by sequencing a larger surrounding genomic region (Online Methods). The positions of the neighboring genes (corresponding to the start and stop codons of their ORFs) are indicated. The endogenous copy of the csr-1 gene (1,751 bp) is shown in blue.

Supplementary Figure 6 Detecting low levels of cytosine methylation in vegetative cells of N. crassa.

(a) A gel image with 18 individual PCR products corresponding to the three analyzed genomic regions (A, B and C) in Figure 4a. This particular experiment assays BstUI-digested DNA samples from strain T485.4h that carries the 4× array on the dim-2+; dim-5+ background. A representative graphical region of interest (ROI) that was used to quantify PCR yields in ImageJ is shown in yellow. (b) Semiquantitative PCR yields for the three genomic regions were quantified in ImageJ and are reported in raw luminosity units (mean ± s.d.). Top, DNA samples were digested with BstUI; bottom, DNA samples were mock digested. Assayed strains (left to right): FGSC#9720, T485.4h, T486.3h and T402.1h (Supplementary Table 1). The presence of intact transformed DNA was verified by PCR in mock-digested samples using primers NcHis3_F4 and NcHis3_R1 (Supplementary Table 3).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–3. (PDF 1336 kb)

Supplementary Data 1

Plasmids used in this study. (ZIP 7 kb)

Supplementary Data 2

Sequence alignments analyzed in this study. (ZIP 265 kb)

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Gladyshev, E., Kleckner, N. DNA sequence homology induces cytosine-to-thymine mutation by a heterochromatin-related pathway in Neurospora. Nat Genet 49, 887–894 (2017). https://doi.org/10.1038/ng.3857

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