| AGAMOUS-Like 15 (AGL15) is a member of the plant MADS domain family of transcriptional regulators. AGL15 accumulates preferentially during embryo development. Previous studies have supported a role for AGL15 during embryogenesis in promotion and maintenance of embryonic potential. To better understand how AGL15 functions, it is imperative to identify what genes are regulated by AGL15, how they are regulated, and what roles their products play in development.; MADS domain proteins regulate gene expression by binding to DNA sequences with an overall consensus of CC(A/T)6GG (CArG motif). Nonetheless, different MADS domain proteins exhibit similar yet distinct binding site preferences that may be critical for differential gene regulation. To determine the consensus sequence preferentially recognized by AGL15 in vitro, PCR-assisted binding site selection assays were performed. AGL15 was observed to prefer a CArG motif with a longer A/T-rich core and is to date the only plant MADS domain protein having such a preference. AGL15 was also found to bend DNA upon binding. DNA-bending may be important for regulating gene expression.; Two approaches were taken to identify direct target genes of AGL15. The Arabidopsis genome was searched for genes containing AGL15 binding sites as candidates for direct regulation by AGL15. DTA4 (for D&barbelow;ownstream T&barbelow;arget of A&barbelow;GL15-4&barbelow;) was identified this way. A chromatin immunoprecipitation (ChIP) approach that allows purification of in vivo protein-DNA complexes was developed to isolate genes regulated by AGL15. Additionally, the foundation for a ChIP-chip (ChIP in combination with microarray chip) approach to analyze global AGL15 binding sites was established. DTA5 was identified by mapping a co-precipitated DNA fragment to its regulatory region. The identity of DTA4 and DTA5 as direct in vivo targets of AGL15 was confirmed by a rigorous set of tests.; In addition, an AGL15 ortholog from soybean was isolated. By sequence comparison, conserved protein motifs were identified, which may be important for AGL15 function. The developmental roles of soybean AGL15 and its potential as a biotechnology tool for soybean transformation are being assessed. |
