Genome binding/occupancy profiling by high throughput sequencing
Summary
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
Libraries were prepared from chromatin IP and input samples using the NEBNext ultra DNA library prep kit for Illumina (NEB # 7370) and the NEBNext Multiplex Oligos for Illumina (NEB # E7335), according to the manufacturerÂ’s recommendations. No size selection was performed during sample preparation and the libraries were indexed and amplified using 12 PCR cycles, using the recommended conditions. After a final cleanup with Agencourt AmPure XP beads (Beckman # A63881), the library size distribution and concentrations were determined using a BioAnalyzer 2100 (Agilent technologies) and Qubit (Invitrogen) instrument, respectively. The final pools were prepared by mixing equimolar amounts of all individual indexed libraries and then sequenced on a HiSeq 2500 (Illumina) in Rapid mode (Paired-End 50).
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