Phylogenetic Analyses and GAGA-Motif Binding Studies of BBR/BPC Proteins Lend to Clues in GAGA-Motif Recognition and a Regulatory Role in Brassinosteroid Signaling.

Marius L Theune,Dierk Wanke,Ulrich Bloss,Friederike Ladwig,Luise H Brand

doi:10.3389/fpls.2019.00466

Marius L Theune, Dierk Wanke + Show 3 more

Open Access

https://doi.org/10.3389/fpls.2019.00466

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Abstract

Plant GAGA-motif binding factors are encoded by the BARLEY B RECOMBINANT / BASIC PENTACYSTEINE (BBR/BPC) family, which fulfill indispensable functions in growth and development. BBR/BPC proteins control flower development, size of the stem cell niche and seed development through transcriptional regulation of homeotic transcription factor genes. They are responsible for the context dependent recruitment of Polycomb repressive complexes (PRC) or other repressive proteins to GAGA-motifs, which are contained in Polycomb repressive DNA-elements (PREs). Hallmark of the protein family is the highly conserved BPC domain, which is required for DNA binding. Here we study the evolution and diversification of the BBR/BPC family and its DNA-binding domain. Our analyses supports a further division of the family into four main groups (I–IV) and several subgroups, to resolve a strict monophyletic descent of the BPC domain. We prove a polyphyletic origin for group III proteins, which evolved from group I and II members through extensive loss of domains in the N-terminus. Conserved motif searches lend to the identification of a WAR/KHGTN consensus and a TIR/K motif at the very C-terminus of the BPC-domain. We could show by DPI-ELISA that this signature is required for DNA-binding in AtBPC1. Additional binding studies with AtBPC1, AtBPC6 and mutated oligonucleotides consolidated the binding to GAGA tetramers. To validate these findings, we used previously published ChIP-seq data from GFP-BPC6. We uncovered that many genes of the brassinosteroid signaling pathway are targeted by AtBPC6. Consistently, bpc6, bpc4 bpc6, and lhp1 bpc4 bpc4 mutants display brassinosteroid-dependent root growth phenotypes. Both, a function in brassinosteroid signaling and our phylogenetic data supports a link between BBR/BPC diversification in the land plant lineage and the complexity of flower and seed plant evolution.

Highlights

GAGA-motif binding factors (GAFs) have an indispensable function for proper growth and development in many multicellular organisms
The Basic PentaCysteine DNA-binding domain represents the sole characteristic feature shared by all BARLEY B RECOMBINANT / BASIC PENTACYSTEINE (BBR/BPC) family members
To gain an insight into its diversification in plant evolution, we first computed a cladogram on the basis of the BPC domain

Summary

INTRODUCTION

GAGA-motif binding factors (GAFs) have an indispensable function for proper growth and development in many multicellular organisms. The hallmark of the entire BBR/BPC family is the evolutionary conserved Basic PentaCysteine (BPC) DNA-binding domain at the very C-terminus of the proteins (Sangwan and O’Brian, 2002; Santi et al, 2003; Meister et al, 2004; Kooiker et al, 2005; Wanke et al, 2011) This domain contains five highly conserved Cysteine residues that were proposed to form a novel type of zinc-finger structure, which is directly involved in the DNAbinding process (Santi et al, 2003; Meister et al, 2004; Kooiker et al, 2005). A root-growth assay with different concentrations of the phytohormone Brassinolide consolidated a role for BPC6 in brassinosteroid signaling

RESULTS

DISCUSSION

MATERIALS AND METHODS