Selection and Characterization of Mutants Defective in DNA Methylation in Neurospora crassa

Abstract

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 defective in methylation (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.

Histone deacetylation and cytosine methylation compartmentalize heterochromatic regions in the genome organization ofNeurospora crassa

“…Given that HDA-1 is targeted to heterochromatic nucleosomes by CHAP binding AT-rich DNA and HP1/CDP-2 binding H3K9me3 (39, 40), these factors would render the HCHC complex as a heterochromatin-specific HDAC. This explains the copious HCHC mutants recovered in a selection for strains with compromised heterochromatin silencing (94); it is possible the HCHC complex is the only heterochromatin specific HDAC in Neurospora. Our data also suggest that unknown HAT(s) specifically acetylate heterochromatic H4K16 – which is only seen with compromised HCHC HDAC activity – given that euchromatic acetylation is minimally altered in HCHC mutants.…”

“…We speculate the additional methyl groups on AT-rich DNA in a Δ cdp-2 strain allow heterochromatic regions to remain associated with the nuclear periphery, either by active recruitment by a methyl-DNA binding protein (MRBP-1) (103, 104) or passively by Liquid-Liquid Phase Separation (105). The identity of MRBP-1 in Neurospora is unknown, as Neurospora does not encode a protein with a strong MBD domain homologous to human MeCP-2, but perhaps DIM-10, which compromises heterochromatin silencing but not 5 m C deposition or H3K9me3 enrichment (94), could bind methylated DNA in Neurospora. Regardless, our results suggest a new role for 5 m C in maintaining chromatin compartmentalization when heterochromatin is compromised, thereby safeguarding genome organization and function.…”

“…Given that HDA-1 is targeted to heterochromatic nucleosomes by CHAP binding AT-rich DNA and HP1/CDP-2 binding H3K9me3 (39,40), these factors would render the HCHC complex as a heterochromatin-specific HDAC. This explains the copious HCHC mutants recovered in a selection for strains with compromised heterochromatin silencing (94); it is possible the HCHC complex is the only heterochromatin specific HDAC in Neurospora.…”

Advances in Approaches to Study Chromatin-Mediated Epigenetic Memory

“…The disruption of either Chp1 or RITS damages heterochromatin-mediated transcriptional silencing at the centromeric region. , In N. crassa , the primary components of the H3K9 methylation pathway are similar to those in S. pombe . The NcDCDC (DIM-5, 7, 9, CUL4, DDB1) complex acts as H3K9 methylation “writer,” but its recruitment does not rely on the RNAi pathway in N. crassa . , The HP1 protein “reads” these H3K9 methylations to recruit NcHCHC complex (HP1, CDP2, HDA1, CHAP) and DNA methyltransferase DIM-2, respectively. NcHCHC defects lead to hyperacetylation of centromeric histones and hypermethylation of centromeric DNA, impairing centromeric silencing and centromere function .…”

“…The NcDCDC (DIM-5, 7, 9, CUL4, DDB1) complex acts as H3K9 methylation "writer," but its recruitment does not rely on the RNAi pathway in N. crassa. 48,49 The HP1 protein "reads" these H3K9 methylations to recruit NcHCHC complex (HP1, CDP2, HDA1, CHAP) and DNA methyltransferase DIM-2, 50 respectively. NcHCHC defects lead to hyperacetylation of centromeric histones and hypermethylation of centromeric DNA, 51 impairing centromeric silencing and centromere function.…”