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 four evolutionary conserved non-coding sequences (CNS) in the promoters of BBM genes from the different dicot species and the associated transcription factor binding sites. CRISPR-Cas9 mutagenesis of these four CNS in the model plant Arabidopsis thaliana (arabidopsis) resulted in novel phenotypes, like the development of embryogenic tissue on seedlings and enhanced in vitro regeneration.

Project description

We would like to know how mutations in the BBM CNS alter BBM expression and would like to identify the associated DNA binding proteins and CREs that regulate BBM expression. Fluorescent protein fusions driven by the mutated CNS alleles will be used to understand the effect of the mutation on BBM expression. For example, do these mutations result in ectopic BBM expression or do they change BBM expression in response to certain plant hormones? Phylogenetic footprinting identified many potential DNA binding motifs - DNA binding protein pairs in the BBM 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 BBM 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 BBM DNA binding proteins will be used to understand their role in regulating BBM expression.