Dynamic Control of Enhancer Activity Drives Stage-Specific Gene
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ARTICLE https://doi.org/10.1038/s41467-019-09513-2 OPEN Dynamic control of enhancer activity drives stage-specific gene expression during flower morphogenesis Wenhao Yan 1,7, Dijun Chen 1,2,7, Julia Schumacher 1,2, Diego Durantini2,5, Julia Engelhorn3,6, Ming Chen4, Cristel C. Carles 3 & Kerstin Kaufmann 1 fi 1234567890():,; Enhancers are critical for developmental stage-speci c 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 devel- opment, as well as hints to annotate plant enhancers. 1 Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, 10115 Berlin, Germany. 2 Institute for Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany. 3 Université Grenoble Alpes (UGA), CNRS, CEA, INRA, IRIG-LPCV, 38000 Grenoble, France, 38000 Grenoble, France. 4 Department of Bioinformatics, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. 5Present address: Ernst Benary Samenzucht GmbH, Friedrich-Benary-Weg, 134346 Hann, Muenden, Germany. 6Present address: Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany/Institute for Molecular Physiology, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany. 7These authors contributed equally: Wenhao Yan, Dijun Chen. Correspondence and requests for materials should be addressed to D.C. (email: [email protected]) or to K.K. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:1705 | https://doi.org/10.1038/s41467-019-09513-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09513-2 ulticellular development is controlled by precise spa- H3K27me3 was reported in the genomic regions of predicted tiotemporal regulation of gene expression, which is tissue-specific enhancers, with a positive correlation for H3K27ac M 20 largely accomplished through the activation and and a negative correlation with H3K27me3 . More recently, by repression via cis-regulatory elements within the promoter- combining the information of genome-wide DNA methylation, proximal or distal regulatory regions, such as enhancers. Pro- chromatin accessibility, and H3K9ac profiling, 1500 intergenic moters and enhancers are noncoding DNA sequences that can be enhancers were predicted in maize21. Although these studies bound by multiple regulatory proteins, especially transcription spark the renewed interest in the investigation of plant enhancer factors (TFs) to activate the expression of target genes1. Active elements, genome-wide mechanisms of dynamic enhancer activ- promoters and enhancers are devoid of nucleosomes, thereby ities to trigger spatiotemporal gene expression in plant develop- rendering the DNA accessible for TF binding. While promoters ment have not yet been elucidated. locate near the transcription start site (TSS), enhancers can be up Here, we used flower development as an ideal system to study to several Mbs away from their target genes1. A single gene can be enhancer dynamics and to dissect their roles in the control of regulated by multiple enhancers with different spatiotemporal stage-specific gene expression patterns by integrative analysis of activities, resulting in a large combinatorial complexity of genome-wide H3K27ac locations (ChIP-seq), chromatin accessi- expression repertoires for a given set of genes2. Large-scale ana- bility (DNase-seq)18, and transcriptome dynamics (RNA-seq) in lyses in animal genomes showed that promoters and enhancers Arabidopsis thaliana at four representative stages. We found that are distinguishable based on their stereotypical DNA features and H3K27ac prevalently marks promoters but rarely localizes in chromatin signatures3. For example, enhancers are often asso- distal regulatory regions, suggesting that H3K27ac is not a hall- ciated with mono-methylation of H3 at lysine 4 (H3K4me1) and mark for active enhancers in Arabidopsis thaliana as it is in H3 acetylation marks at lysine 9 and 27 (H3K9ac and H3K27ac)4, metazoans22, and as data from rice and maize indicate23,24. while gene promoters differ from enhancers by their enrichment Examination of DHS-predicted distal intergenic enhancers reveals in H3K4me33. On the other hand, there is an increasing number their high-stage specificity across flower development, their of reports5,6 showing that promoters and enhancers share similar functional importance in the regulation of flowering genes, as well structural and functional features1. as their high evolutionary conservation across crucifer and dicot Enhancers have recently been subjected to intensive investi- species. gation as there is increasing evidence showing their importance not only in developmental control but also in evolution and diseases, including cancer2. The advent of next-generation Results sequencing technologies and the identification of enhancer Distribution of H3K27ac and DHSs during flower develop- chromatin properties enable genome-wide prediction of enhan- ment. H3K27ac is a primary epigenetic mark of active enhancers cers in a high-throughput manner3, leading to the discovery of in animals1,3,22, and it was recently shown to highly correlate with thousands of enhancer candidates in human6,7 and other animal active gene expression in plants23–25. Here, we further investi- genomes8. Enhancers appear to function as integrated platforms gated the role of H3K27ac dynamics in stage-specific gene reg- for binding of multiple TFs, often including lineage- and signal- ulation. We generated a genomic atlas of H3K27ac modification determining (intrinsic or extrinsic cue-dependent signal) TFs2,9. across flower development in the model plant species Arabidopsis Remarkably, cell identity gene loci are frequently associated with thaliana. We took advantage of a previously described floral dense clusters of enhancers or super-enhancers with both over- induction system, which allows collection of synchronized flower lapping and distinct spatiotemporal activities10,11. Genome-wide tissue of specific developmental stages26 and that proved to be analyses of enhancer functions have revealed that enhancers are highly suitable for depicting quantitative changes in chromatin functionally complex and developmentally dynamic during cell marks, accessibility, and TF binding18,27 (Fig. 1a). The inflores- differentiation12. Lineage-regulating factors, including pioneer cence meristem (before induction, stage 0) as well as floral tissues factors and their associated TF networks, contribute to shaping at stages of meristem specification (2 days after induction (DAI), enhancer repertoires during development12–14. stage 2), floral whorl specification (4 DAI, stages 4–5), and organ Contrary to the fast growing knowledge of the regulatory differentiation (8 DAI, stages 7–8) were harvested for ChIP-seq to function of enhancers in metazoans, knowledge of genome-wide map H3K27ac modification patterns (Fig. 1b; Supplementary landscapes of plant enhancers and their dynamic activities Fig. 1). A set of controls confirmed the high quality of the ChIP- remains limited in the context of developmental stage-specific seq data (Supplementary Fig. 2). Time-series transcriptomics gene regulation15. Enhancers are predominantly found in regions (RNA-seq) and genome-wide open chromatin profiles18 gener- of accessible chromatin3, which is more sensitive to DNase I ated on the same developmental series were integrated into the digestion than condensed chromatin. Genome-wide mapping of analysis (Fig. 1b). Comparative analyses show that both H3K27ac open chromatin regions has been conducted in the selected model and DNase І accessibility highly positively correlate with the and crop plant species16–21. In 2012, Zhang et al. reported the expression of nearby genes at all four developmental stages mapping of open chromatin in rice seedlings and callus using (Supplementary Fig. 3), and highly expressed genes contained DNase-seq. They found that DNase I hypersensitivity sites both H3K27ac sites and DHSs around their TSSs (Supplementary (DHSs) are associated with highly expressed genes in both tissues, Fig. 4). For example, dynamic changes of H3K27ac and DHS and that 42.3% and 44.5% of DHSs in seedlings and in calli, profiles at the key floral regulatory genes APETALA1 (AP1,a respectively, reside in intergenic regions16. The same group later floral meristem identity gene) and APETALA3 (AP3,afloral identified a high overlap between TF occupancy and DHSs in organ identity gene) correlate with their expression dynamics.