Targeted changes in chromatin state at thousands of genes are central to eukaryotic development. RELATIVE OF EARLY FLOWERING 6 (REF6) is a Jumonji-type histone demethylase that counteracts Polycomb repressive complex 2 (PRC2)-mediated gene silencing in plants and was reported to select its binding sites in a direct, sequence-specific manner. Here we show that REF6 and its two close paralogues determine spatial 'boundaries' of the repressive histone H3K27me3 mark in the genome and control the tissue-specific release from PRC2-mediated gene repression. Targeted mutagenesis revealed that these histone demethylases display pleiotropic, redundant functions in plant development, several of which depend on trans factor-mediated recruitment. Thus, Jumonji-type histone demethylases restrict repressive chromatin domains and contribute to tissue-specific gene activation via complementary targeting mechanisms.

Enhancers are critical for developmental stage-specific gene expression, but their dynamic regulation in plants remains poorly understood. Here we compare genome-wide localization of H3K27ac, chromatin accessibility and transcriptomic changes during flower development in Arabidopsis . H3K27ac prevalently marks promoter-proximal regions, suggesting that H3K27ac is not a hallmark for enhancers in Arabidopsis . We provide computational and experimental evidence to confirm that distal DNase І hypersensitive sites are predictive of enhancers. The predicted enhancers are highly stage-specific across flower development, significantly associated with SNPs for flowering-related phenotypes, and conserved across crucifer species. Through the integration of genome-wide transcription factor (TF) binding datasets, we find that floral master regulators and stage-specific TFs are largely enriched at developmentally dynamic enhancers. Finally, we show that enhancer clusters and intronic enhancers significantly associate with stage-specific gene regulation by floral master TFs. Our study provides insights into the functional flexibility of enhancers during plant development, as well as hints to annotate plant enhancers.