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

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.

Figure 1. Cytosine methyltransferase DIM-2 can mediate RIP

a, The 802-bp tester construct contains one endogenous (“left”) and one ectopic (“right”) copy of an 802-bp region of the Neurospora genome (ref. 18). The “no-repeat” construct specifically lacks the ectopic repeat copy. b, RIP mutation profiles of the 802-bp construct. Top row (left to right): crosses X1{24}, X2{48} and X3{48}; bottom row (left to right): crosses X4{24}, X5{24} and X6{24}. The number of mutations is reported per site per spore. Cross X1 was published previously (ref. 18). c, RIP does not occur in the absence of the ectopic repeat copy. Cross X7{48}. The number of spores analyzed for each cross is provided in curly brackets. Strain and cross genotypes are provided in Supplementary Table 1 and 2, respectively.

Figure 2. DIM-2-mediated RIP requires the SUV39 histone methyltransferase DIM-5

a, A cartoon representation of the canonical heterochromatin pathway in N. crassa (ref. 15). b, RIP mutation profiles of the 802-bp construct. Crosses X8{60} (top) and X9{60} (bottom). c, RIP mutation profiles of the 802-bp construct. Crosses X10{60}, X11{60}, X12{60} and X13{60} (top to bottom). Only the right-most portion of the endogenous repeat copy and the entire 600-bp segment of the linker region were sequenced. d, RIP mutation profiles of the 802-bp construct. Crosses (left to right) X14{24}, X15{24} and X16{48}. e, RIP mutation profile of the 802-bp construct. Cross X17{48}. f, RIP mutation profiles of the 802-bp construct. Crosses (left to right) X18{24}, X19{24} and X20{48}. g, A fraction of “linker” mutations reports the relative activity of DIM-2-mediated RIP. Each fraction value corresponds to the number of mutations in the 600-bp segment of the linker region normalized by the total number of mutations found in this 600-bp segment and in the repeated sequences. Analyzed crosses (left to right): X1, X4, X17, X18, X19, X20. The difference between any two fraction values is evaluated for significance using the chi-squared homogeneity test (P-value is indicated above the line) and the Fisher’s exact test (P-value is indicated below the line) on the actual mutation counts. *** P ≤ 0.001, NS P > 0.05. The number of spores analyzed for each cross is provided in curly brackets. Strain and cross genotypes are provided in Supplementary Table 1 and 2, respectively.

Figure 3. DIM-2-mediated RIP responds to weak interspersed homology

a, Different homology patterns are created by only varying the base-pair sequence of the magenta region (as described in ref. 18). b, The assayed patterns of weak interspersed homology contain short units of three (“3H-8N”) and four (“4H-7N”) homologous base-pairs spaced at regular intervals of 11 base-pairs. An instance of “Random” homology corresponds to a 200-bp fragment of the GFP coding sequence. c, RIP mutation profiles. Top row (left to right): crosses X21{60}, X22{60} and X23{60}; bottom row (left to right): crosses X24{60}, X25{90} and X26{60} (as reported previously in ref. 18).

Figure 4. Recognition of short tandem repeat arrays by the DIM-5/DIM-2 pathway

a, Four copies of the dsRed coding sequence (the “4x” array) are inserted by homologous recombination as the replacement of an endogenous genomic segment (green). The ectopic csr-1 copy is inserted analogously. All DNA segments are drawn to the same scale. The restriction site 5′-CGCG-3′ can only be cut by BstUI if none of its four cytosines is methylated (ref. 29). The distal BstUI site “C” is located near the csr-1 gene. b, RIP mutation profiles of the 4x array. Crosses X27{14} (left) and X28{30} (right). The mean number of mutations in the entire sequenced region is 174.93± 30.84 and 15.20±1.69 for X26 and X27, respectively. Positions of the two proximal BstUI restriction sites (“A” and “B”, as shown in a) are indicated. c, The assay detects the failure of cleavage by BstUI at the three specified sites (“A”, “B” and “C”, as shown in a). PCR yields for Regions A and C are normalized by the corresponding PCR yield for Region B. Unnormalized PCR yields are also provided in Supplementary Fig. 6b. The analyzed repeat constructs were never subjected to RIP. Strains: FGSC#9720 (native locus), T485.4h (4x, dim-5+), T486.3h (4x, dim-5Δ) and T402.1h (ectopic csr-1).

Figure 5. DIM-2-mediated RIP can target repeats at widely separated genomic positions

a, A pair of widely-separated repeats of the csr-1 gene: the inactive ectopic copy of csr-1 is inserted near the his-3 gene, 2.7 million base-pairs away from the endogenous csr-1 locus. b, Inactivation of the endogenous csr-1 gene by RIP produces cyclosporin-resistant colonies originating from individual progeny spores. c, The percentage of cyclosporin-resistant spores produced in different genetic backgrounds. Because the ectopic repeat is present in only one of the two parental strains, the maximum expected level of cyclosporin-resistant progeny is 50 per cent. d, Patterns of RIP mutation in the endogenous csr-1 locus induced in the presence of the ectopic csr-1 copy (integrated near the his-3 gene). The per-site number of mutations is reported for sets of unique csr-1 alleles. “*” denotes the position of the existing G-to-A mutation in the ectopic csr-1 copy. The number of replica crosses, the total number of sequenced cyclosporin-resistant progeny, and the number of unique csr-1 alleles were as follows: X29 - 1 cross, 10 sequenced spores, 10 unique alleles; X30 - 9 crosses, 70 sequences spores, 34 unique alleles; X31 - 3 crosses, 28 sequenced spores, 1 unique allele. A cluster of 20 cyclosporin-resistant progeny spores was likely produced in the ridΔ/Δ, dim-2Δ/Δ background by a single gene-conversion event that have occurred early in the pre-meiotic lineage undergoing RIP. e, Dinucleotide (CpN) context of RIP mutations.

Figure 6. A general model for nucleation of heterochromatin by pairwise dsDNA/dsDNA interactions

In vegetative/somatic cells, a direct homologous interaction between two co-aligned copies of a repeated DNA sequence induces a localized deposition of H3K9me3 (green diamonds) and 5meC (red bars). Such heterochromatin nucleation sites are intrinsically unstable. However, on longer repeat arrays, multiple concomitant nucleation events may be stabilized to yield constitutive heterochromatin. During the pre-meiotic phenomenon of RIP, the same transient localized interactions result in permanent C-to-T mutations, instead of 5meC. These mutations accumulate during several cell cycles over a period of several days to give a high level of RIP in analyzed spores.