Background

Gene transcription is controlled in part by cis-regulatory elements (CREs). CREs are short, non-coding DNA motifs that comprise specific binding sites for regulatory proteins like transcription factors. CREs and transcription factor binding sites are often evolutionarily conserved. We identified evolutionary conserved non-coding sequences (CNS) in the promoters and introns of LEC1 genes from the different dicot species CRISPR-Cas9 mutagenesis of these CNS in the model plant Arabidopsis thaliana (arabidopsis) resulted in ectopic expression phenotypes, like the development of embryogenic tissue on seedlings and enhanced in vitro regeneration.

Project description

We would like to understand how the mutations in the LEC1 CNS alter LEC1 expression and to identify the associated DNA binding proteins and CREs. Fluorescent protein fusions driven by the mutated CNS alleles will be used to understand the effect of the mutation on LEC1 expression. For example, do these mutations result in ectopic LEC1 expression or do they change LEC1 expression in
response to certain plant hormone or physiological signals? Phylogenetic footprinting identified many potential DNA binding motifs - DNA binding protein pairs in the LEC1 CNS. CRISPR-Cas9 mutagenesis will be used to narrow down the regulatory regions in the CNS that are responsible for the observed phenotypes/change in LEC1 expression pattern. Yeast one-hybrid assays and electromobility shift assays (EMSAs) will be used to identify/confirm the DNA binding motif - DNA binding protein pairs. Functional analysis of these LEC1 DNA binding proteins will be used to understand their role in regulating LEC1 expression.