DDM1-facilitated R-loop resolution and H2A.Z exclusion primes heterochromatin formation in Arabidopsis [ssDRIP-seq, ChIP-seq, pNET-seq, ATAC-seq]

Programmed constitutive heterochromatin silencing is essential for eukaryotic genome regulation, yet the initial step of this process is ambiguous. A large proportion of R-loops (RNA:DNA hybrids) had been unexpectedly identified within Arabidopsis pericentromeric heterochromatin with unknown functions. Through a genome-wide R-loop profiling screen, we find DDM1 (Decrease in DNA Methylation 1) is the primary restrictor of pericentromeric R-loops via its RNA:DNA helicase activity. Low levels of pericentromeric R-loops resolved by DDM1 co-transcriptionally can prime constitutive heterochromatin silencing. Furthermore, we demonstrate that DDM1 physically excludes histone H2A variant H2A.Z, and promotes H2A.W deposition for faithful heterochromatin initiation soon after R-loop clearance. The dual functions of DDM1 in R-loop resolution and H2A.Z eviction are essential for sperm nuclei structure maintenance in mature pollen. Our work unravels the co-transcriptional R-loop resolution coupled with accurate H2A variants deposition is the primary step of constitutive heterochromatin silencing in Arabidopsis, which might be conserved across eukaryotes. To compare the genome-wide R-loop levels among different genotypes , ssDRIP-seq was conducted using 12-day-old seedlings under long day conditions. Native ChIP (see details in extract protocol) -seqs were conducted to examine the enrichment levels of H2A.W-Flag, H2A.Z, H3K27me1 and H3K9me2 in wide-type and ddm1 mutant (12-day-old seedlings under long day conditions). To test the nascent RNA abundance in wide-type and ddm1 mutant, pNET-seq was conducted using 12-day-old seedlings under long day conditions. And ATAC-seq was used to compare the chromatin condensation state between wide-type and ddm1 mutant.