Binding Site Selection for the Plant MADS Domain Protein AGL15: AN IN VITRO AND IN VIVO STUDY

Weining Tang,Sharyn E Perry

doi:10.1074/jbc.m212976200

Abstract

AGL15 (for AGAMOUS-like 15) is currently the only reported member of the plant MADS domain family of transcriptional regulators that preferentially accumulates during embryo development. Additionally, AGL15 is one of the more divergent members of the MADS domain family, including within the DNA-binding domain. Previous studies have shown that MADS domain proteins bind to DNA sequences with an overall consensus of CC(A/T)6GG (called a 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 bound 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. Next, the Arabidopsis genome data base was searched for genes containing AGL15 binding sites as candidates for direct regulation by AGL15. One gene, DTA4 (for Downstream Target of AGL15-4), was identified by this method, and then confirmed as a direct target of AGL15 in vivo.

Highlights

AGL15 is currently the only reported member of the plant MADS domain family of transcriptional regulators that preferentially accumulates during embryo development
MADS domain proteins generally bind to a consensus DNA sequence called a CArG motif1 (CC(A/T)6GG; canonical sequence); more detailed studies revealed that different MADS factors have overlapping but distinct binding site preferences
Selection of AGL15 Binding Sites in Vitro—PCR-assisted in vitro binding site selection was used to isolate DNA sequences preferentially bound by AGL15 and AG from a random population of oligonucleotides

Summary

AN IN VITRO AND IN VIVO STUDY*

MADS domain proteins generally bind to a consensus DNA sequence called a CArG motif (CC(A/T)6GG; canonical sequence); more detailed studies revealed that different MADS factors have overlapping but distinct binding site preferences (reviewed in Ref. 2). AGL15 was found to prefer a type of CArG motif with a longer central A/T-rich sequence, similar to the N-10 site of the MEF2A binding consensus [10] This preference is far unique for plant MADS domain proteins and may contribute to functional specificity of AGL15. The Arabidopsis genome data base was searched for genes containing AGL15 binding sites One such identified gene, DTA4 (for Downstream Target of AGL15-4), was further tested to verify that it represents a direct in vivo target of AGL15. Such an approach may be useful for identifying downstream target genes of other DNA-binding regulatory proteins

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION