DNA methylation, a prototypical epigenetic modification implicated in gene silencing, occurs in many eukaryotes and plays a significant role in the etiology of diseases such as cancer. The filamentous fungus Neurosporacrassa places DNA methylation at regions of constitutive heterochromatin such as in centromeres and in other A:T-rich regions of the genome, but this modification is dispensable for normal growth and development. This and other features render N. crassa an excellent model to genetically dissect elements of the DNA methylation pathway. We implemented a forward genetic selection on a massive scale, utilizing two engineered antibiotic-resistance genes silenced by DNA methylation, to isolate mutants d efective i n m ethylation (dim). Hundreds of potential mutants were characterized, yielding a rich collection of informative alleles of eleven genes important for DNA methylation, most of which were already reported. In parallel, we characterized the pairwise interactions in nuclei of the DCDC, the only histone H3 lysine 9 methyltransferase complex in Neurospora, including those between the DIM-5 catalytic subunit and other complex members. We also dissected the N- and C-termini of the key protein DIM-7, required for DIM-5 histone methyltransferase localization and activation. Lastly, we identified two alleles of a novel gene, dim-10 - a homolog of Clr5 in Schizosaccharomyces pombe - that is not essential for DNA methylation, but is necessary for repression of the antibiotic-resistance genes used in the selection, which suggests that both DIM-10 and DNA methylation promote silencing of constitutive heterochromatin.

中文翻译：

---

粗糙脉孢菌 DNA 甲基化缺陷突变体的选择和表征。


DNA甲基化是一种与基因沉默有关的典型表观遗传修饰，发生在许多真核生物中，并且在癌症等疾病的病因学中发挥着重要作用。丝状真菌粗糙脉孢菌将 DNA 甲基化置于组成型异染色质区域，例如着丝粒和基因组中其他富含 A:T 的区域，但这种修饰对于正常生长和发育来说是可有可无的。这一特征和其他特征使粗糙脉孢菌成为从基因角度剖析 DNA 甲基化途径元件的绝佳模型。我们利用两个通过 DNA 甲基化沉默的工程抗生素抗性基因进行了大规模的正向遗传选择，以分离出甲基化缺陷的突变体 ( dim )。对数百个潜在突变体进行了表征，产生了对 DNA 甲基化很重要的 11 个基因的丰富信息等位基因集合，其中大部分已被报道。与此同时，我们表征了 DCDC（脉孢菌中唯一的组蛋白 H3 赖氨酸 9 甲基转移酶复合体）细胞核中的成对相互作用，包括 DIM-5 催化亚基和其他复合体成员之间的相互作用。我们还解剖了 DIM-5 组蛋白甲基转移酶定位和激活所需的关键蛋白 DIM-7 的 N 端和 C 端。 最后，我们鉴定了新基因dim-10 （粟酒裂殖酵母Clr5 的同源物）的两个等位基因，这对于 DNA 甲基化不是必需的，但对于抑制选择中使用的抗生素抗性基因是必需的，这表明DIM-10 和 DNA 甲基化都会促进组成型异染色质的沉默。