biostudies-literatureUnknown13(1)Karanyi ZNational Research, Development and Innovation Fund of HungaryArabidopsis 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 pericNature communications5058https://www.ebi.ac.uk/biostudies/studies/S-EPMC9420119biostudies-literatureNODULIN HOMEOBOX is required for heterochromatin homeostasis in Arabidopsis.PMC9420119Karanyi ZHetey SSzaker HMHorvath ASzekvolgyi LMosolygo-L ANagy EFero OMiskei MHolb IBoros-Olah BCsorba TfalseNODULIN HOMEOBOX is required for heterochromatin homeostasis in Arabidopsis.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 peric2022-01-01T00:00:00Z2022 Aug2025-04-18T19:06:22.149Z2025-04-07T06:47:40.385ZS-EPMC94201193603024010.1038/s41467-022-32709-y