MORC family ATPases required for heterochromatin condensation and gene silencing

Abstract

Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause derepression of DNA-methylated genes and TEs but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, which are predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes.

Fig. 1

Mutations of two MORC homologs induce SDC::GFP and TE overexpression. (A) Wild type (wt), drm2 mutant and cmt3 mutant plants carrying SDC::GFP showing no GFP fluorescence under UV light (insets show each plant under white light) and drm2 cmt3 double mutant and EMS-mutagenized lines wt #67, cmt3 #49, and cmt3 #7 plants showing strong GFP fluorescence. (B) Western blot using anti-GFP antibody confirms SDC::GFP overexpression in the EMS mutants. Coomassie staining of the large Rubisco subunit (rbcL) is used as loading control. (C) Number of TEs overexpressed in atmorc1 and atmorc6 and classified by superfamily. For each mutant, only TEs with a ≥ 4 fold increase in both the EMS and T-DNA alleles over wild type and with a p-value ≤ 0.05 are represented. (D) Relative fold increase of four TE transcripts in atmorc1-4 and atmorc6-3 over wild type assayed by Real-Time quantitative PCR (RT-qPCR) and normalized to ACTIN7. Errors bars indicate standard deviation based on three independent biological replicates.

Fig. 2

DNA methylation is not impaired in atmorc1 and atmorc6 mutants. (A and B) Metaplot analyses showing DNA methylation level in atmorc1-4, atmorc6-3 and wild type for the set of TEs upregulated in atmorc1-4 (A) and atmorc6-3 (B). The gray vertical lines mark the boundaries between 1 kilobase upstream and downstream regions of TEs. (C) Southern blot analyses assaying CG methylation level at CEN180 repeats using HpaII-treated genomic DNAs. m, methylated; u, unmethylated. met1-3 genomic DNA is used as positive control for loss of CG methylation (23). (D) Percent DNA methylation at SDC and 4 TEs overexpressed in atmorc1-4 and atmorc6-3 assayed by bisulfite sequencing. 24 clones were analyzed for each individual analysis.

Fig. 3

AtMORC1 and AtMORC6 are required for maintenance of chromatin architecture, form nuclear bodies near chromocenters, and morc-1 is involved in gene silencing in C. elegans. (A) Interaction matrix of the wild type Arabidopsis genome from Hi-C analysis. Positions along the 5 chromosomes are shown from left to right and top to bottom, and each pixel represents interactions from uniquely mapping paired end reads in 200 kilobase bins. Black bars and circles mark the positions of the pericentromeric and telomeric regions, respectively. Light grey regions represent areas masked out due to problematic mapping. Black bars show separation between chromosomes. (B) Difference plot showing enrichment of Hi-C interactions in atmorc6-1 in red and interactions depleted in atmorc6-1 in blue. (C) Anti-Myc immunostaining showing localization of pAtMORC6::AtMORC6-Myc and pAtMORC1::AtMORC1-Myc in nuclear bodies adjacent to chromocenters. AtMORC1 and AtMORC6 showed an average of 2.0 +/− 1.0 (average plus or minus standard deviation) and 2.5 +/− 1.2 bodies per chromocenter, respectively. DAPI staining shows chromocenter location. Bottom panels are merges. (D) A silenced seam cell-specific GFP transgene in the eri-1 (mg366) sensitized background is overexpressed in worms fed with bacteria expressing double-stranded RNA targeting morc-1 or rde-4 but not in worms fed with bacteria expressing a control empty vector. Results are representative of five independent replicates.