Characterization of transcriptional enhancers in plants using Arabidopsis thaliana and Zea mays as model species

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Enhancers are non-coding regions of the genome controlling the transcription of genes located at distances ranging from kilo to mega bases. Enhancers act mainly as binding platforms for multiple transcription factors and were shown to interact directly with the promoter of their target genes. Since few enhancers are characterized in plants, we developed a pipeline to find new enhancers based on previous knowledge from plant and animal case studies. To do so, the two plant models Arabidopsis thaliana and Zea mays were used. Both functional and genome-wide approaches were used to study known enhancers in A. thaliana and Z. mays and identify putative enhancers in Z. mays. The characterization of novel enhancers in plants provides more insight about gene regulatory networks in plants. In fine, a better understanding of gene regulation could help improving important traits of cultivated plants by conventional breeding or biotechnological improvement. \nIn A. thaliana, enhancers of the FLOWERING LOCUS T (FT) were characterized by inducing DNA methylation using Inverted Repeats (IRs). Indeed, DNA methylation was often associated with the inactivation of cis-regulatory elements such as enhancers and promoters in mammals and for some cases in plants. DNA methylation deposition at the previously characterized enhancer Block C led to downregulation of FT expression and subsequent late flowering phenotype in FT-inducing growth conditions. A novel putative enhancer of FT, named Block E, was found downstream of the gene and shared several features with Block C such as accessible chromatin, conserved sequences among Brassicaceae, and putative transcription factor binding sites present at Block C. Transgenic lines containing an IR targeting a part of Block E displayed a late flowering phenotype, which was not as strong as for Block C, but significantly higher than other lines used as control and displaying a mild late flowering phenotype. We conclude that IR-targeted DNA methylation is a useful tool that can allow characterization of known enhancers and discovery of putative ones. \nIn Z. mays, differential chromatin accessibility, Histone 3 lysine 9 acetylation (H3K9ac) enrichment level, and gene expression profile were obtained for two different tissues (young seedling leaves and husk) in order to define enhancers’ location, activity, and associated genes. Enhancers were previously shown to be in most cases located in accessible chromatin, and to contain associated histone marks such as H3 lysine 27 acetylation (H3K27ac), H3 lysine 4 monomethylation (H3K4me1), or H3K9ac. We first verified whether our data could properly define known or putative enhancers and the expression of their target genes. Indeed, we could find both high chromatin accessibility and H3K9ac enrichment level for the known enhancers of teosinte branched1 (tb1) and booster1 (b1), and the corresponding expression fold change of the target genes across the two tissues. By combining chromatin accessibility and H3K9ac enrichment level data, we could determine ≈2000 candidate enhancers in intergenic regions. We could finally associate for each tissue about 20 differentially expressed genes to about 20 putative tissue-specific enhancers, which will be characterized by transient expression assays in the future.