NODULIN HOMEOBOX is required for heterochromatin homeostasis in Arabidopsis

Abstract

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.

Coding and noncoding transcriptomes of NODULIN HOMEOBOX (NDX)-deficient Arabidopsis inflorescence

“…3b–d . (2) The transcriptomes of inflorescence and seedlings 6 , 16 were compared in Col-0 (control) samples (Fig. 4a ), identifying 5555 (17.37%) up- and 5618 (17.56%) downregulated genes in flowers relative to seedlings.…”

“…Recent genomic data challenged the idea that NDX acts as a general R-loop regulator that controls the formation of chromosomal R-loops throughout the Arabidopsis genome. Instead, NDX appears to be linked to heterochromatin function by regulating the activity of heterochromatic siRNAs (het-siRNAs) and non-CG DNA methylation pathways at pericentromeric regions 6 . This observation is consistent with earlier data showing that NDX coincides with a heterochromatic patch of H3K9 dimethylation and Pol IV-dependent siRNA transcripts within the FLC terminator/ COOLAIR promoter region 5 , 7 , linking NDX to heterochromatin function.…”

“…Our analysis identified specific sets of genes differentially expressed in ndx1-4 , as well as noncoding heterochromatic siRNA (het-siRNA) loci whose activity was significantly changed in the absence of NDX. Transcriptomic data obtained from inflorescence were also compared with previously published seedling data 6 , highlighting development-specific changes in gene expression profiles.

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“…The seedling data were published in ref. 6 , which were used for comparison with the flower transcriptomic data generated in this study. …”

R‐loop: The new genome regulatory element in plants

“…In rice experiments based on H3K4me3 chromatin‐associated RNA–DNA interactions followed by paired‐end‐tag sequencing (ChRD‐PET) and ssDRIP‐seq analysis, ~20% of H3K4me3‐associated DNA–RNA interactions were shown to be derived from R‐loops, suggesting that R‐loops are involved in chromatin loop conformations (Figure 3C) (Xiao et al, 2022). Recently, work by Karanyi et al (2022) demonstrated that AtNDX, an R‐loop regulator at the FLC locus, regulated heterochromatin organization (Karanyi et al, 2022). However, it is still unclear whether AtNDX‐mediated (hetero)chromosomal contacts in Arabidopsis are R‐loop dependent, as R‐loop structures were shown to be critical for the integrity of topologically associated domains (TADs) in acute myeloid leukemia (AML) cells (Luo et al, 2022).…”