Precise localization and dissection of gene promoters are key to understanding transcriptional gene regulation and to successful bioengineering applications. The core RNA polymerase II initiation machinery is highly conserved among eukaryotes, leading to a general expectation of equivalent underlying mechanisms. Still, less is known about promoters in the plant kingdom. In this study, we employed cap analysis of gene expression (CAGE) at three embryonic developmental stages in barley to accurately map, annotate, and quantify transcription initiation events. Unsupervised discovery of de novo sequence clusters grouped promoters based on characteristic initiator and position-specific core-promoter motifs. This grouping was complemented by the annotation of transcription factor binding site (TFBS) motifs. Integration with genome-wide epigenomic data sets and gene ontology (GO) enrichment analysis further delineated the chromatin environments and functional roles of genes associated with distinct promoter categories. The TATA-box presence governs all features explored, supporting the general model of two separate genomic regulatory environments. We describe the extent and implications of alternative transcription initiation events, including those that are specific to developmental stages, which can affect the protein sequence or the presence of regions that regulate translation. The generated promoterome dataset provides a valuable genomic resource for enhancing the functional annotation of the barley genome. It also offers insights into the transcriptional regulation of individual genes and presents opportunities for the informed manipulation of promoter architecture, with the aim of enhancing traits of agronomic importance.

Computational Regulatory Genomics MRC London Institute of Medical Sciences London UK

Department of Cell Biology and Genetics Faculty of Science Palacky University Slechtitelu 27 78371 Olomouc Czech Republic

DNAFORM Precision Gene Technologies 230 0046 Yokohama Kanagawa Japan

Institute of Clinical Sciences Faculty of Medicine Imperial College London Hammersmith Hospital Campus London UK

Institute of Experimental Botany of the Czech Academy of Sciences Slechtitelu 31 77900 Olomouc Czech Republic

Merck Sharp and Dohme Limited 120 Moorgate London EC2M 6UR UK

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