Two parallel pathways recruit the H3K9me3 HMT in somatic cells, requiring the Argonaut NRDE-3, or the MBT-domain protein, LIN-61 (RNA-seq)

The establishment and maintenance of chromatin domains shape the epigenetic memory of a cell, with histone H3 lysine 9 methylation defining repressed heterochromatin. We show that in C. elegans the SET-25 (SUV39/G9a) HMT that catalyzes H3K9me1-3, is able to establish repressed domains de novo. We identify here two distinct pathways that recruit SET-25 to its targets. One requires LIN-61 (L3MBTL2), a conserved protein with 4 MBT domains that recognizes H3K9me2 deposited by the HMT MET-2 (SETDB1). The second pathway is MET-2-independent and requires a somatic Argonaut NRDE-3 and 22nt small nuclear RNAs. This NRDE-3 pathway targets ~10% of all SET-25-modified loci genome-wide including intact RNA and DNA transposons. Removal of both pathways in the met-2;nrde-3 double mutant synergistically derepresses transposons in early embryos and elevates embryonic lethality. The redundancy of these pathways illustrates the key role played by chromatin-mediated silencing in protecting the genome against inherent threats. Overall design: Total RNAseq of RNA isolated from early C. elegans embryos (~1-300 cell stage) in N2 (Bristol), met-2(n4256) III, set-25(n5021) III, met-2(n4256) III;lin-61(tm2649) I, lin-61(tm2649) I, set-25(n5021) III;lin-61(tm2649) I, met-2(n4256) III;nrde-3(tm1116) X, set-25(n5021) III;nrde-3(tm1116), nrde-3(tm1116) mutants. Extraction of RNA was performed according to the WormBook protocol (Stiernagle, 2006). Total RNA was purified using RNeasy kit (QIAGEN 74104) including DNase treatment. Depletion of ribosomal RNA was done for 5 lg of total RNA with Ribo-Zero? Margnetic Gold Kit (Epicentre MRGZG12324) and further concentrated with RNA Clean & Concentrator? kit (Zymo Research R1015) according to corresponding manufacturer?s instructions.