GSE201840
Nodulin homeobox (NDX) is required for heterochromatin homeostasis in Arabidopsis [sRNA-seq]
Nodulin homeobox (NDX) is a nuclear protein described as the regulator of specific euchromatic genes that controls multiple aspects of Arabidopsis development. At the molecular level, NDX was shown to i) interact with the Polycomb repressive complex 1 (PRC1) to promote deposition of repressive histone marks at abscisic acid-responsive gene loci, and (ii) stabilize an R-loop structure within the Flowering Locus C (FLC) to control its expression. Herein, we aimed to understand the global role of NDX in chromatin-based gene regulation and comprehensively characterized its genomic binding sites and transcriptional relationships involving sRNA profiling, DNA methylation, and 3D chromatin structure. Using quantitative microscopic and genomics approaches we show that NDX is a highly immobile heterochromatic protein that localizes primarily to centromeres and pericentromeric regions. Most NDX binding sites coincide with heterochromatic sRNA loci that are known to mediate transcriptional gene silencing via the RNA-dependent DNA methylation (RdDM) pathway. NDX localization was antagonistic with R-loop structures that were prevalent in euchromatic chromosomal arms. Functional analysis revealed that the absence of NDX led to differential hypo- and hyper-methylation in the CG / CHG / CHH sequence context, of which hypo-CHH / CHG sites were co-localized with NDX chromatin binding sites. The identified hypo-CHH/CHGs were associated with increased heterochromatic sRNA production (predominantly 24nt sRNAs) and parallel transcriptional activation of pericentric transposons. Chromosome conformation capture (Hi-C) revealed significant chromatin structural changes in the ndx mutant, with decreased intra-chromosomal interactions (chromatin decompaction) at pericentromeric regions where NDX is enriched in wild type plants, and increased inter-chromosomal contacts between KNOT-forming regions. KNOT interactions involved de novo interchromosomal contacts similar to genome structural changes observed in DNA methylation mutants. We conclude that the main role of NDX is the functional regulation of heterochromatin, which is mechanistically coupled to the effector phase of the RdDM pathway. Overall design: sRNA-seq