Identification and validation of maize enhancers A cartography of the maize regulatory genome

Cis-regulatory elements are crucial to establish different gene expression patterns in multicellular organisms. In Zea mays, cis-regulatory elements, including transcriptional enhancers, remain poorly characterized. This thesis constitutes an effort to bring maize enhancer into focus.
First, a summary of characteristics associated with plant enhancers, their similarities/differences with animal enhancers and techniques being used for their identification and validation is given. In addition, a list of previously identified enhancers across the plant kingdom is detailed.
Secondly, the large-scale identification of maize enhancers based on DNA and chromatin features is presented. Most importantly, the study led to the identification of 1702 candidate enhancers, which were characterized by low DNA methylation levels, high chromatin accessibility and enrichment for the H3K9ac histone mark.
The thesis also explores the role of a previously identified candidate enhancer sequence Vgt1. We show that Vgt1, and its upstream region, coined uva1, are enriched for enhancer-associated characteristics and act as cis-regulatory elements. Importantly we show that Vgt1 regulates flowering time and growth speed, potentially through regulating expression of the floral repressor gene ZmRap2.7.
Following the identification of candidate enhancers, a subset of candidates was selected for validation. Reporter assays indicated the potential of candidates to act as tissue-specific silencers or insulators. Furthermore, experiments revealed the ability of Low or UnMethylated DNA regions to also act as transcriptional enhancers, silencers or insulators thus demonstrating similarities in characteristics associated with these different types of cis-regulatory sequences.
Finally, a step-by-step protocol for Chromosome Conformation Capture (3C) in maize and Arabidopsis is presented.