Induction of H3K9me3 and DNA methylation by tethered heterochromatin factors in Neurospora crassa

Abstract

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1-mediated histone deacetylation in heterochromatin spreading and gene silencing.

The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

“…Abundant research data show that some special proteins, which can recognize methylated DNA and methylated histones, are essential for recruiting methyltransferases to correct genomic sites in fungi [100]. For example, the H3K9me3 marker is established by the DCDC histone methyltransferase complex, including DIM-5, DIM-7, DIM-9, CUL4, and DDB1 [102,103], which is targeted to AT-rich DNA and typical of RIP [102]. After H3K9me3 is generated by DIM-5, heterochromatin protein 1 can accurately identify the H3K9me3 marker and contributes to recruiting DIM-2 to catalytic methylation at the same locus [100].…”

DNA methylation in Verticillium dahliae requires only one of three putative DNA methyltransferases, yet is dispensable for growth, development and virulence

“…Similar effects on gene and transposon expression occurs in the V. dahliae Hp1 mutant, indicating that the differential expression observed in the Hp1 and Dim5 mutants are related to disrupted Hp1 functioning. In N. crassa, and also fission yeast Schizosaccharomyces pombe that lacks DNA methylation, Hp1 was found to be involved in the formation of H3K9me3-associated heterochromatin (Freitag et al, 2004;Hayashi et al, 2012;Gessaman & Selker, 2017). As such, it is possible that the transcriptional changes in the V. dahliae Hp1 and Dim5 mutants are due to pleiotropic effects of chromatin decondensation.…”

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

“…To bypass DIM-5 localization and address the role of the DIM-7 C-terminus on DIM-5 activity, we tethered DIM-5 to the his-3 locus with the LexA operator system (Gessaman and Selker 2017). Establishment of DNA methylation via ectopic localization of DIM-5 requires all the heterochromatin machinery, and its activity can be indirectly assessed by Southern blotting for the his-3 locus using the DNA methylation-specific AvaI restriction enzyme; higher molecular weight products indicate heterochromatin formation (Gessaman and Selker 2017). The presence of the DIM-7 stop666L+16aa allele in the genetic background did not support tethered DIM-5 activity (compare with activity in dim-7 + strain; Figure 5C).…”