Genome-wide binding of SEPALLATA3 and AGAMOUS complexes determined by sequential DNA-affinity purification sequencing.

Xuelei Lai,François Parcy,Jérémy Lucas,Véronique Hugouvieux,Chloe Zubieta,Arnaud Stigliani

doi:10.1093/nar/gkaa729

Abstract

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3–AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3–AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3–AG tetramer. SEP3 and SEP3–AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3–AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3–AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.

Highlights

Transcription factors (TF) often act cooperatively by forming heteromeric complexes, with proper oligomerization patterns thought to be a key component of gene regulation [1]
In order to exploit the versatility of DNA-affinity purification sequencing (DAP-seq) and apply this technique to the MADS TF family, we developed a modified DAP-seq protocol incorporating protein tagging and sequential protein complex purification called seq-DAP-seq (Figure 1A)
Different epitope tags were fused to the C-terminus of the MADS TFs to reduce potential interference with protein–DNA interactions mediated by the N-terminal MADS DNA-binding domain (DBD) and to allow for sequential purification of the in vitro transcription-translation produced proteins

Summary

Introduction

Transcription factors (TF) often act cooperatively by forming heteromeric complexes, with proper oligomerization patterns thought to be a key component of gene regulation [1]. The MADS TFs, named for founding members MCM1 (Saccharomyces cerevisiae), AGAMOUS (Arabidopsis thaliana), DEFICIENS (Antirrhinum majus) and Serum response factor (Homo sapiens), provide a key example of a TF family that is present in almost all eukaryotes and binds a highly conserved DNA sequence called a CArG-box (CC-’Adenine-rich’-GG) as an obligate dimer [2,3,4]. It is hypothesized that the K domain, was critical in the functional diversification of the family in plants by allowing the binding or accessing of novel genomic sites [8]. This hypothesis has been challenging to test

Methods

Results

Conclusion