The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.

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Spp1 is the H3K4me3 reader subunit of the Set1 complex (COMPASS/Set1C) that contributes to the mechanism by which meiotic DNA break sites are mechanistically selected. We previously proposed a model in which Spp1 interacts with H3K4me3 and the chromosome axis protein Mer2 that leads to DSB formation. Here we show that spatial interactions of Spp1 and Mer2 occur independently of Set1C. Spp1 exhibits dynamic chromatin binding features during meiosis, with many de novo appearing and disappearing binding sites. Spp1 chromatin binding dynamics depends on its PHD finger and Mer2-interacting domain and on modifiable histone residues (H3R2/K4). Remarkably, association of Spp1 with Mer2 axial sites reduces the effective turnover rate and diffusion coefficient of Spp1 upon chromatin binding, compared with other Set1C subunits. Our results indicate that "chromosomal turnover rate" is a major molecular determinant of Spp1 function in the framework of meiotic chromatin structure that prepares recombination initiation sites for break formation.

Lysine 27 to methionine (K27 M) mutation of the histone variant H3.3 drives the formation of an aggressive glioblastoma multiforme tumor in infants. Here we analyzed how the methionine substitution alters the stability of H3.3 nucleosomes in vitro and modifies its kinetic properties in live cells. We also determined whether the presence of mutant nucleosomes perturbed the mobility of the PRC2 subunit Ezh2 (enhancer-of-zeste homolog 2). We found that K27 M nucleosomes maintained the wild-type molecular architecture both at the level of bulk histones and single nucleosomes and followed similar diffusion kinetics to wild-type histones in live cells. Nevertheless, we observed a remarkable differential recovery of Ezh2 in response to transcriptional stress that was accompanied by a faster diffusion rate of the mobile fraction of Ezh2 and a significantly increased immobile fraction, suggesting tighter chromatin binding of Ezh2 upon transcription inhibition. The differential recovery of Ezh2 was dependent on transcription, however, it was independent from K27 M mutation status. These biophysical characteristics shed more light on the mechanism of histone H3.3 K27M in glioma genesis in relation to the kinetic properties of Ezh2.

Arabidopsis NODULIN HOMEOBOX (NDX) is a nuclear protein described as a regulator of specific euchromatic genes within transcriptionally active chromosome arms. Here we show that NDX is primarily a heterochromatin regulator that functions in pericentromeric regions to control siRNA production and non-CG methylation. Most NDX binding sites coincide with pericentromeric het-siRNA loci that mediate transposon silencing, and are antagonistic with R-loop structures that are prevalent in euchromatic chromosomal arms. Inactivation of NDX leads to differential siRNA accumulation and DNA methylation, of which CHH/CHG hypomethylation colocalizes with NDX binding sites. Hi-C analysis shows significant chromatin structural changes in the ndx mutant, with decreased intrachromosomal interactions at pericentromeres where NDX is enriched in wild-type plants, and increased interchromosomal contacts between KNOT-forming regions, similar to those observed in DNA methylation mutants. We conclude that NDX is a key regulator of heterochromatin that is functionally coupled to het-siRNA loci and non-CG DNA methylation pathways.