Regulation of MYB and bHLH Transcription Factors: A Glance at the Protein Level

Accepted Manuscript Regulation of MYB and bHLH transcription factors – a glance at the protein level Marie Pireyre, Meike Burow PII:

S1674-2052(14)00048-3

DOI:

10.1016/j.molp.2014.11.022

Reference:

MOLP 47

To appear in:

MOLECULAR PLANT

Received Date: 1 September 2014 Revised Date:

10 November 2014

Accepted Date: 24 November 2014

Please cite this article as: Pireyre M., and Burow M. (2014). Regulation of MYB and bHLH transcription factors – a glance at the protein level. Mol. Plant. doi: 10.1016/j.molp.2014.11.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title:

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Regulation of MYB and bHLH transcription factors – a glance at the protein level

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Running title:

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Protein level regulation of transcription factors

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Short summary:

This review summarizes the recent progress on molecular regulatory mechanisms of plant transcription factor families MYBs and bHLHs with focus on post-transcriptional modifications such as ubiquitination, phosphorylation, acetylation, and nitrosylation. We conclude by pointing to potential applications of state of the art techniques required to better understand the molecular basis of plant responses to environmental changes.

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Marie Pireyre and Meike Burow

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DynaMo DNRF Center of Excellence , Department of Plant and Environmental Sciences,

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Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C,

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Denmark

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Meike Burow

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Department of Plant and Environmental Sciences

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Faculty of Science

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University of Copenhagen

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Thorvaldsensvej 40

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1871 Frederiksberg C, Denmark

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E-Mail: [email protected]

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Phone: +45-35 33 37 73

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Fax: +45-35 28 33 33

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Abstract In complex, constantly changing environments, plants have developed astonishing

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survival strategies. These elaborated strategies rely on rapid and precise gene regulation

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mediated by transcription factors (TFs). TFs represent a large fraction of plant genomes and

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among them, MYBs and bHLHs have unique inherent properties specific to plants. Proteins of

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these two TF families can act as homo- or heterodimers, associate with proteins from other

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protein families or form MYB/bHLH complexes to regulate distinct cellular processes. The

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ability of MYBs and bHLHs to interact with multiple protein partners has evolved to keep up

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with the increased metabolic complexity of multi-cellular organisms. Association and

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disassociation of dynamic TF complexes in response to developmental and environmental cues

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are controlled through a plethora of regulatory mechanisms specifically modulating TF activity.

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Regulation of TFs at the protein level is critical for efficient and precise control of their activity

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and thus provides the mechanistic basis for a rapid on-and-off switch of TF activity. In this

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review, examples of post-translational modifications, protein-protein interaction and subcellular

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mobilization of TFs will be discussed with regard to the relevance of these regulatory

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mechanisms for the specific activation of MYBs and bHLHs in response to a given

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environmental stimulus.

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Keywords: transcription factor, MYB, bHLH, post-translational modification, protein-protein

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interaction, transcriptional regulation

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List of abbreviations: ABA, abscisic acid; bHLH, basic Helix-Loop-Helix; COI-1, coronatine-

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insensitive protein 1; DBD, DNA-binding domain; GA, Gibberellic acid; HtH, Helix-turn-Helix;

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HR, Hypersensitive Response; JA, jasmonic acid; JAZ; jasmonate ZIM-domain protein; MBW,

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MYB/bHLH/WD40 complex; MTTF, membrane-tethered transcription factor; NLS, nuclear

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localization signal; PPI, protein-protein interaction; PTM, post-transcriptional modification; SA,

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salicylic acid; SUMO, Small Ubiquitin Modifier; TAD, transcriptional activation domain; TF,

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transcription factor; UPS, Ubiquitin-Proteasome System.

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Introduction

58 In multicellular organisms, precise gene expression in time and space is regulated by

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transcription factors (TFs). Mutants affected in spatio-temporal transcriptional regulation often

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show dramatic developmental phenotypes underlining the importance of the functions of TFs and

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the importance of efficient on-and-off switches for their activity. In Arabidopsis thaliana,

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between 6-10% of all genes encode TFs in contrast to only 3% in Drosophila melanogaster and

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5% in humans. This relatively large number of TFs might allow plants to rapidly adapt to the

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changing environments they encounter, e.g. by synthesizing, transporting, and relocating

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metabolites, RNAs, and proteins. A large proportion of the TFs in Arabidopsis are MYB (339)

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and basic Helix-Loop-Helix (bHLH; 162) TFs. The members of these two transcription factor

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families can act as homodimers, as heterodimers consisting of proteins from the same family, or

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in complexes with TFs from other protein families. Interestingly, many cases of MYB/bHLH

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complexes have been described and the parallel evolution of these two TF families has been

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associated with the developmental and metabolic plasticity found in plants (Feller et al., 2011).

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The structural, evolutionary and functional diversity of MYBs and bHLHs has been extensively

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studied with regard to the distinct pathways controlled by these TFs as well as the mechanisms

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regulating their activity (reviewed by Feller et al., 2011).

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Different MYB/bHLH complexes regulate distinct cellular processes such as cell wall

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synthesis, cell death, circadian clock, responses to abiotic and biotic stress, hormone signaling

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and the biosynthesis of specialized metabolites (Lindemose et al., 2013; Seo and Mas, 2014;

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Stracke et al., 2007; Stracke et al., 2001; Vailleau et al., 2002). In many cases, bHLH are able to

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physically interact with different MYBs to achieve their function. In addition, the activity of TFs

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appears to be regulated by cytosolic WD40 repeat proteins through formation of highly dynamic

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MYB/bHLH/WD40 (MBW) complexes (Feller et al., 2011). WD40s and bHLHs respond to a

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variety of environmental triggers and thereby offer a flexible, inducible system to activate

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transcription of multiple sets of genes, while MYB TFs confer specificity to transcriptional

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activation (Ramsay and Glover, 2005). In line with the proposition that TF complexes

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comprising MYBs and bHLHs have evolved to increase fitness in changing environments, MBW

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complexes have only been described in plants so far.

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TFs typically bind 6-12 bp long DNA sequences present in the promoters of multiple

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target genes. A sequence of six specific nucleotides has a high probability to occur in the

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genome. Moreover, the specificity of the DNA binding domain (DBD) is attributed rather to a

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family or a clade of TFs than to individual TFs. Those observations speak against precise

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transcriptional activation solely based on specificity in DNA binding and suggest instead that

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additional regulatory mechanisms occur to insure high specificity of the transcriptional process.

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DNA binding can be regulated by chromatin accessibility and nucleosome positioning (reviewed

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by Spitz and Furlong, 2012), by enhancers, cofactors and protein-protein interactions (PPIs). In

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this review, we will discuss the mechanisms underlying the spatio-temporal regulation of PPIs

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and post-transcriptional modifications (PTMs) and their importance for a fast responding

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regulatory system that includes TFs in an inactive form that can rapidly be activated upon need.

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The MYB TF family is divided into sub-clades according to the structure of the DBD that

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contains one to three repeats (R1-R3) (Figure 1-I). Each repeat consists of approximately 53

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amino acid residues that form a Helix-turn-Helix (HtH). The N-terminal R2R3 domain binds to

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DNA and is highly conserved within the whole family. In contrast, the C-terminus of MYB TFs,

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i.e. the transcriptional activation domain (TAD), is more variable (Dubos et al., 2010) and has

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been predicted to fail formation of a stable secondary structure (Lindemose et al., 2013). The

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highly conserved DBD suggests a common mechanism for the regulation of DNA binding,

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whereas the variable TAD domain might modulate TF activation and DBD accessibility and

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thereby confer specificity to DNA binding by MYB TFs. bHLH TFs are characterized by 60

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conserved amino acids at the C-terminus forming a basic DBD and a dimerization domain

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(Figure 1-II). Similar to C-terminal part of MYB proteins, the N-terminal part of bHLH proteins

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is more variable and can be grouped by differences in the TADs (Dombrecht, 2007; Cheng,

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2011; Chen, 2012). BHLH proteins bind to specific sequences called E-box (5'-CANNTG-3')

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including the well-studied G-box (5'-CACGTG) (Toledo-Ortiz, 2003). In Arabidopsis, fourteen

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R2R3 MYB and six R1 MYB proteins share a conserved bHLH binding motif

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([DE]Lx2[RK]x3Lx6Lx3R), which is critical for promoter activation because it stabilizes

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complexes of these MYBs with R/B-like bHLH proteins (Zhao et al., 2013; Zimmermann et al.,

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2004). This bHLH binding motif was used to predict MYB/bHLH interactions indicating that the

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presence of this motif in the MYB protein sequence contributes to MYB/bHLH association

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(Zimmermann et al., 2004). Nevertheless, this motif does not explain the recruitment of a

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specific bHLH protein. This review focuses on individual MYB and bHLH TFs as case studies for the regulation

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TFs and their complexes on the protein level rather than on the specificity of TFs towards their

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target promoters. As plants are well-known for their ability to control gene expression in a highly

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coordinated manner, plant MYB/bHLH complexes constitute a suitable model to study dynamic

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regulation of gene expression. In this review, the regulatory mechanisms of MYBs and bHLHs

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controlling plant metabolic pathways will serve as examples. We have chosen jasmonate

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signaling, flavonoid biosynthesis and cell death pathways to investigate functional pairs of

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MYBs and bHLH proteins. First, background on the regulation of TF activation and activity

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through MYB/bHLH complex formation will be given; second, TF regulation at the post-

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transcriptional level will be introduced; and finally, the importance of these mechanisms for

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stimuli-specific responses will be discussed.

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MYB/bHLH as regulators of plant defense pathways

As a part of jasmonate (JA)-mediated responses, the TFs MYC2-4 from the bHLH family

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activate transcription of different sets of genes involved in plant responses to environmental cues

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including hormone signaling and development (Chen et al., 2011; Cheng et al., 2011; Fernández-

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Calvo et al., 2011; Lorenzo et al., 2004). The regulation of MYC2-4 is tightly controlled at the

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protein level by a PPI/F-BOX/proteasome-based mechanism. Under uninduced conditions, MYC

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proteins are bound to the repressors NINJA, TOPLESS (TPL) and Jasmonate ZIM-domain (JAZ)

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proteins (Pauwels et al., 2010; Dombrecht et al., 2007; Lorenzo et al., 2004). Upon stimulation

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e.g. by exogenous jasmonates or herbivory, the JAZ proteins are bound by the signal receptor F-

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Box protein CORONATINE-INSENTIVE protein 1 (COI1) and thereby targeted for degradation

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by the 26S proteasome (Chini et al., 2007; Yan et al., 2013). The MYCs are thereby liberated

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from the complex to fulfill their function (Dombrecht et al., 2007; Lorenzo et al., 2004). As an

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individual protein, MYC2 has been shown to integrate different signals and to contribute to the

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regulation of multiple pathways leading to different phenotypic outputs. This makes MYC2 a

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model TF to discuss the molecular basis of multi-functionality of a single TF (Figure 2). Flavonoids are a class of secondary metabolites involved in plant-environment

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interactions (e.g. defense, pollinator attraction, UV filtration). They can be divided into three

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groups: anthocyanins, flavonols and proanthocyanidins. All steps of anthocyanin biosynthesis are

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regulated by dynamically associating and disassociating MBW complexes (Appelhagen et al.,

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2011). While the early steps of the pathway are all controlled by MYB11, MYB12 and MYB111,

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the last and anthocyanin-specific biosynthetic step is regulated by PAP1, PAP2, MYB111, or

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MYB114. These MYBs interact with different bHLHs, namely TRANSPARENT TESTA8

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(TT8), GLABROUS3 (GL3), or ENHANCER OF GL3 (EGL30) (Xu et al., 2013). The

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regulation of the anthocyanin pathway has been extensively studied because of defensive and

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anti-oxidant properties of the compounds and their role in fruit coloring. Thus, the key regulators

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as well as some PTMs modulating their interactions are known. The current knowledge on the

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pathway makes it a useful model to illustrate PPI- and PTM-mediated regulation of TF activity.

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Hypersensitive response (HR) triggered by pathogens leads to rapid cell death around the

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infected tissue and thus prevents microbes from spreading. MYB30 is a R2R3 MYB TF that has

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been shown to be involved in pathogen-induced HR and cell death (Vailleau et al., 2002).

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Depending on the process it is involved in, MYB30 interacts with different modifying enzymes

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and undergoes different PTMs leading to its activation or repression (Raffaele and Rivas, 2013).

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The regulatory network surrounding MYB30 represents a unique multi-step and multi-enzyme

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regulatory system and will be discussed to illustrate PTM-specific effects on PPIs and the

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importance of rapid TF deactivation (Figure 3).

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Post-transcriptional activation and inactivation of TFs by ubiquitination

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PTMs include all modifications on the transcript or protein level after the gene has been read

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by an RNA polymerase, e.g. alternative splicing, alternative translation start, and protein

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structure modifications. The latter are mediated by enzymes that catalyze reactions such as

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phosphorylation, acetylation, or ubiquitination. Depending on where and when a functional

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group is added, the protein will mature, be activated or inactivated, sequestered or degraded.

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Ubiquitination is a PTM process conducted by E1, E2, and E3 ligases that will lead to secondary

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structural changes or protein poly-ubiquitination and subsequent degradation of the protein by

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the 26S proteasome (Ubiquitin-Proteasome System, UPS). However, mono-ubiquitination of TFs

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can also lead to proteolysis-independent effects on TF activity and DNA binding properties

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(reviewed by Geng et al., 2012). Ubiquitination can be reversed by de-ubiquitinases, providing

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flexibility to the ubiquitin-mediated protein degradation system.

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While the first ubiquitination step catalyzed by E1 and E2 ligases shows little specificity for

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the protein targets, E3 ligases are substrate- or condition-specific. E3 ligases have been

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extensively studied in mammalian systems as they can be targeted by different drugs (Micel et

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al., 2013). In Arabidopsis, more than a thousand proteins have been predicted to possess E3

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ligase activity. Extensive review articles on the action of E3 ligases in response to environmental

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and developmental cues are available (Duplan and Rivas, 2014; Guo et al., 2013; Stone, 2014).

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The high diversity of stress-responsive E3 ligases in plants appears to be critical for balancing

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the TFs present in the cell and thus for mounting appropriate responses to environmental changes

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with sufficient specificity.

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Recently, Marino et al. (2014) showed that the Arabidopsis E3 ligase MIEL1 was able to

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leave ubiquitin marks on MYB30 leading to the degradation of MYB30 by the proteasome.

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Under normal conditions, MIEL1 is sufficiently expressed to target MYB30 to degradation and

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thus to attenuate its action to initiate cell death. Upon pathogen infection, MIEL1 expression is

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inhibited which allows for higher accumulation of the MYB30 protein and subsequently leads to

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pathogen-induced cell death (Figure 3, III) (Marino et al., 2013).

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Similarly, the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHO-GENESIS 1 (COP1)

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controls the stability of the MYB proteins PAP1 and PAP2 to repress transcription of

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anthocyanin biosynthesis genes only in dark grown plants (Maier et al., 2013). COP1 is generally

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involved in light-controlled development and gene expression by targeting TFs for proteasome-

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mediated degradation in the dark (Deng et al., 1991; Wei et al., 1994). For instance, COP1

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controls the expression of light-responsive genes by ubiquitination of unphosphorylated

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phytochrome A in response to far red light (Saijo et al., 2008). Moreover, COP1 has been

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demonstrated to affect the stability of MYC2 in a light-dependent manner (Chico et al., 2014).

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Accordingly, shading has a repressive effect on JA signaling through degradation of the MYC2

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protein by the 26S proteasome dependent on COP1 (Figure 2, I) (Chico et al., 2014). These

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observations exemplify an elegant mechanism that controls the activity of a TF in a trigger-

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specific manner, here light, while it still allows for fast activation of the downstream responses.

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Ubiquitination possesses a dual role: It can lead to decreased protein accumulation through

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proteasome-dependent degradation or it can alter TF activity. It has thus been proposed that

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ubiquitination could act as a timer for the activity of TFs. Indeed, while the first ubiquitin added

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to the TF is in some cases required for initiation of transcription by favoring DNA binding or by

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recruiting the transcriptional machinery, subsequent poly-ubiquitination of the same TF on the

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DNA can target the protein to the proteasome. Mono- followed by poly-ubiquitination thus

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represents a potential on-and-off switch of TF activity and a high turnover at the promoters

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(Kodadek et al., 2006). In this model, first described as the timer model, the TF has the time

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required to bind to a target promoter and activate gene transcription while being poly-

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ubiquitinated prior to degradation (Wu et al., 2007). Although this mechanism has not been

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described in plants, yet, it may constitute a regulatory process that facilitates rapid TF activation

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and high TF turnover to provide an efficient on-and-off switch in plant stress responses.

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Reversible modification of TF proteins by lysine acetylation

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Lysine acetylation is a reversible type of PTM mediated by lysine acetyl-transferases and

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de-acetylases. The finding that acetylation regulates the activity of the animal TF p53 marked the

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beginning of a new search for acetylated proteins. Indeed, numerous proteins involved in general

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regulation of gene expression were shown to be acetylated in yeast and humans, including TFs

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and other DNA-binding proteins (Gu and Roeder, 1997). In plants, lysine acetylation has so far

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been studied in processes such as cell wall modifications during plant infection, secondary

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metabolism as well as chromatin remodeling through histone acetylation (Xing and Poirier,

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2012). Recently, a global analysis of lysine acetylation in rice (Oryza sativa) underlined the role

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of this PTM mechanism in multiple biological processes (Nallamilli et al., 2014). The nuclear-

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localized, acetylated proteins identified in rice included primarily histones but also a few bHLHs.

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In JA signaling, the repressor TPL interacts with MYC2 to inhibit MYC2 activity (Pauwels et al.,

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2010; Dombrecht et al., 2007). TPL has been described as a Groucho (GR)/TP1 family protein,

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involved in transcriptional repression by recruiting histone de-acetylase to induce local

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chromatin compaction (Causier et al., 2012). Despite the technical limitations to study this PTM,

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numerous lines of evidence suggest an effect of acetylation on transcriptional regulation. Further

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studies are needed to understand the biological functions of acetylation of histones and TFs.

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Phosphorylation regulates TF activity and turnover

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Phosphorylation has so far been the most-studied PTM due to its implication in MAP

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kinase cascades. In mammals, the role of phosphorylation in signaling pathways was initially

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described for the regulation of the cell cycle and for oncogenesis. This PTM was intensely

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studied in the context of plant signaling during plant response to stress and immune response

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(Boudsocq and Sheen, 2013; Kaufmann et al, 2011; Ni et al., 2014; Spoel and Loake, 2011).

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Phosphorylation has moreover received a lot of attention because of the availability of a strong

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anti-body for detection of phosphorylated proteins by Western blotting and because of its value

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as a model PTM to detect modified peptides by LC-MS (Kaufman et al, 2011; Zhou et al., 2001).

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Upon JA signaling, phosphorylation of MYC2 in its TAD domain induces its proteolysis

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but also stimulates the transcriptional activity of MYC2 (Zhai et al., 2013). Based on these

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findings, two rounds of DNA binding by two MYC2 proteins have been proposed to be

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necessary to activate target gene transcription. It has been further suggested that there is a need

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for a high turnover provided by the UPS. The degradation of the MYC2 bound in the first round

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might favor binding of the next active MYC2 protein to the promoter, while on the other hand,

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the presence of the first MYC2 at the promoter might decrease the target search time for the

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second MYC2 protein. The observations made for MYC2 are not congruent with the “timer

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model” above, but rather follow the proposed model of “activation by destruction” described in

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mammals and yeast (Catic et al., 2013; Collins and Tansey, 2006; Geng et al., 2012) where TF

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degradation is not only essential to regulate TF turnover but also dependent on the TF’s

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transcriptional activity.

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REDOX-dependent regulation of TFs

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Changes in the REDOX potential of the cell can affect disulfide bridges in TF proteins and

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thereby affect their activity in response to stress conditions that lead to higher accumulation of

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hormones in the cell, i.e. JA, Salicylic Acid (SA), ethylene, Gibberellic Acid (GA), Abscisic

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Acid (ABA) and auxins. As an example, intracellular translocation of SA results in elevated

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accumulation of glutathione as well as a shift in the ratio of reduced to oxidized glutathione

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(Spoel and Loake, 2011). On the other hand, JA perception will decrease the pool of reduced

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glutathione in favor of the oxidized form (Spoel and Loake, 2011). These changes in the

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REDOX state can be perceived by reactive cysteines of regulatory proteins and thereby lead to

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changes in their structure. Heine and coworkers identified two conserved cysteine residues as a

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regulatory module in the R2R3 domain of MYB TFs (Heine et al., 2004). In the absence of

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reducing agents, MYB TFs were not able to bind DNA in vitro. While cys-53 is highly

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conserved among MYBs from different organisms, cys-49 is specific to plant R2R3 domains.

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The emergence of this second cysteine residue in plants has been suggested to change the

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accessibility of cys-53 as thus its capacity to form a disulfide bond. Cys-49 might therefore

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regulate the DNA binding capacity of the plant MYB TFs by modulating their dimerization

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capabilities. This higher complexity observed in plants talks in favor of a mechanistic adaptation

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to ensure fast and specific responses to different internal or external signals (Figure 3, II).

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Another type of REDOX-related PTMs is nitrosylation. Following REDOX changes in the

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cell, S-nitroglutathione donors change availability. In mammalian systems, nitrosylation of the

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myc oncogenic proteins has been reported to reduce their DNA binding capacity (Brendeford et

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al., 1998; Lüscher and Vervoorts, 2012). In plants, the activity of MYB30 has been shown to be

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influenced by S-nitrosylation through regulation of the secondary structure of its R2R3 domain

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(Tavares et al., 2014). Moreover and in accordance with the previous paragraph, treatment of

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nitrosylated MYB30 with reducing agent partially restored its capacity to interact with DNA,

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thus confirming that the REDOX state of the protein can change its ability to bind DNA (Tavares

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et al., 2014). In the case of MYB30, SA-induced stress decreases the pool of S-nitroglutathione

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donors leading to increased activity of MYB30 and thus to pathogen-induced cell death. On the

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other hand, a JA-induced increase of the available pool of oxidized donors might lead to an

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increase in MYB30 nitrosylation and a decrease in its activity (Figure 3, II). The REDOX state

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of the cell might thereby play a role in crosstalk between SA and JA signaling.

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Stabilization of MYB by sumoylation

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SUMO stands for Small Ubiquitin Modifier and refers to a protein able to covalently attach

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itself to another protein. SUMO has been shown to be involved in different regulatory processes

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such as nucleus-to-cytosol protein translocation, protein degradation, plant development (Conti

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et al., 2014; Elrouby, 2014), and stress responses (Miller et al., 2013). In ABA signaling, gene

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expression studies combined with phenotypic analyses have demonstrated a functional

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relationship between MYB30 and the small ubiquitin-like modifier E3 ligase SIZ1 (Zheng et al.,

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2012). Upon ABA treatment, it has been shown that SIZ1 has the capacity to stabilize MYB30

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by sumoylation of the K283 residue (Figure 3, II) (Zheng et al., 2012). This observation adds

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another mechanism to the model of stress-induced control of TFs by PTMs to mount rapid and

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specific responses in changing environment. It appears advantageous to have key regulators

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available as inactive proteins that can be activated by different PTMs occurring in response to

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different signals. Different PTMs regulate the stability/turnover, the DNA binding and the

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activation of TFs which points at the need for robust coordination of these mechanisms. This

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complexity in TF regulation by PTMs confers the capacity of plant regulatory systems to

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integrate multiple signals. It also adumbrates the importance of combinatorial effects of different

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PTMs for the activation and the deactivation of TFs.

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Membrane-tethered TFs require activation by proteolytic cleavage

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Membrane-tethered transcription factors (MTTF) are membrane-bound TFs that are activated

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through cleavage by a protease which releases the DBD and allows its translocation to the

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nucleus. Plant MTTFs have been shown to be involved in Endoplasmic Reticulum (ER) and

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mitochondrial retrograde signaling (De Clercq et al., 2013; Ng et al., 2013; Yang et al., 2014b).

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In mammals, MTTFs have been extensively studied as part of the unfolded protein responses

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associated with Alzheimer’s disease and due to their role in Notch signaling (Brown et al., 2000).

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The regulation of their proteolytic activation seems to be linked to protease activity as well as

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membrane fluidity. In plants, seven MTTFs from the bZIP and NAC TF family as well as one

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Membrane-anchored MYB (MaMYB) have been described to date (Chen et al., 2008; De Clercq

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et al., 2013; Ng et al., 2013; Seo, 2014; Yang et al., 2014b). Their rapid release upon changes in

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the cellular environment has been hypothesized to play a role in rapid stress responses (Chen et

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al., 2008).

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The identification of MaMYB was the first and is so far the only case of an ER-anchored

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MYB TF (Dunkley et al, 2006). Whereas the membrane localization of the full-length MaMYB

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and the nuclear localization of a truncated form have been demonstrated, no experimental proof

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of cleavage and re-localization has been furnished to date. This unique case of MTTF behavior in

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the MYB family could be important for induced responses. The protein might be kept at the ER

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to be quickly released upon stress. More recently, ANAC013 and ANAC017 have been shown to

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be membrane-anchored to the chloroplast and their proteolysis upon H2O2 stress liberates their

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C-terminal DBD which is then be targeted to the nucleus. (De Clercq et al., 2013; Ng et al.,

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2013). The same has been described for a membrane-bound plant homeodomain transcription

345

factor, released upon proteolysis and associated with histone modifications in response to

346

retrograde signaling (Sun et al, 2011). In both cases, stress responses appear to regulate

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transcriptional activation by cleaving membrane-bound regulatory proteins and releasing their

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DBD. Nevertheless, no direct experimental evidence for this mechanisms has been provided for

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a member of the MYB or bHLH family.

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TF localization can be modulated by interacting partners

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TFs without a membrane anchor can also reside in the cytosol, either as free, soluble proteins

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or physically associated with soluble cytoplasmic proteins until they tranlocate to the nucleus

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upon a trigger. One example is the bHLH TF MYC1, a regulator of root hair development and

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trichome patterning (Pesch et al., 2013). Despite the presence of a Nuclear Localization Signal

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(NLS) as also described for other bHLH, the soluble MYC1 protein has been shown to mostly

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reside in the cytosol, which also determines the localization of the bHLH TF GLABRA1 (GL1),

360

another regulator of trichome development. Upon interaction with MYC1, GL1 has been shown

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to be re-localized from the nucleus to the cytosol, which inhibits its function as transcriptional

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activator in the nucleus (Pesch et al., 2013). Although MYC1 induces trichome development,

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MYC1 over-expression lines showed lower trichome density. Overexpression of MYC1 might

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disturb the stoichiometry of the TF complex and thereby trichome development. GL1, likely in

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combination with other TFs, has been suggested to bind the MYC1 promoter to form a negative

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feedback loop and thereby balance MYC1expression levels (Pesch et al., 2013). MYC1 thus

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constitutes an example of TF regulation comprising multiple regulatory layers on the protein

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level. First, the GL1/MYC1 complex is formed and activated. Second, GL1 activity is repressed

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by subcellular re-localization of the protein mediated by MYC1. Finally, active TF complexes

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can feedback-regulate MYC1 transcription. In this example, a TF both activates and represses the

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same pathway dependent on its concentration and the presence of its interacting partners.

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The identification of mechanisms regulating the subcellular localization of TFs has recently

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provided new insight into retrograde signaling in response to stress. Future studies on stress-

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induced changes in membrane composition and their relevance for TF cleavage will deepen our

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understanding of the fast responses to complex triggers characteristic for plants. The regulatory

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mechanism of MTTF activation by proteolytic cleavage is likely to be extended to TFs that do

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not possess a membrane anchor themselves but are anchored through interacting partners. A

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membrane-anchored protein, potentially acting as a signal receptor, could interact with a TF and

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dissociation of the complex could release the TF and enable rapid fine-tuning of transcriptional

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regulation.

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The role of TF complexes in the nucleus

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In 2008, small TFs that repress the transcriptional activity of MYBs have been described in

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plants (Dubos et al., 2008). Those small MYB-like proteins, which only contain the R3 domain,

387

inhibit transcriptional activation by R2R3 MYBs by competing with them for formation of

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MBW complexes. Therefore, TFs possessing only a R3 domain are generally predicted to be

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repressors of transcription (Dubos et al., 2008). In the case of the flavonoid biosynthetic

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pathway, numerous small proteins negatively regulate the formation of active MBW complexes

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by interacting with the bHLHs in these complexes (Dubos et al., 2008; Matsui et al., 2008;

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Nemie-Feyissa et al., 2014). The expression of small R3 proteins is regulated by environmental

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and developmental factors. When co-expressed with the MYB TFs controlling anthocyanin

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biosynthesis, the MYB-like proteins compete for binding to the bHLHs involved, which leads to

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disruption of MBW complexes and thereby counter-balances MYB activity e.g. during nitrate

396

starvation (Nemie-Feyissa et al., 2014). Moreover, miRNA156 has been shown to influence

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MBW complexes by activating SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL)

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proteins that compete for bHLH binding and thereby coordinate anthocyanin production during

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the transition from the vegetative to the reproductive stage (Gou et al., 2011). This example

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illustrates how different conditions can affect regulation of a biosynthetic pathway by controlling

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inhibitors of TF activity. Similar to the regulation of the MYB TFs controlling anthocyanin biosynthesis, negative

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regulators modulates the transcriptional activity of MYB30. The secretory phospholipase PLA2

404

protein localizes to the Golgi where it controls auxin-dependent protein trafficking (Lee et al.,

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2010) and possibly regulates plant defense chemistry independently of its enzymatic function

406

(Froidure et al., 2010). In the nucleus, PLA2-α inhibits MYB30 through physical interaction.

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Upon pathogen attack, expression of PLA2-α is induced in periphery of the infection zone

408

suggesting a role for PLA2-α in limiting the HR response mediated by MYB30 (Figure 2, III).

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Examples of TF regulation by PPIs in the nucleus can also be found in the bHLH family.

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Recently, different labs identified a new clade of transcriptional regulators that are

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phylogenetically closely related to the MYC2-4. BHLH003, bHLH013 and bHLH017 (also

412

named JAM1, JAM2 and JAM3) bind DNA with a specificity similar to that of MYC2, MYC3,

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and MYC4 suggesting that they might compete with the MYCs for the binding of cis-regulatory

414

elements (Fonseca et al., 2014; Nakata and Ohme-Takagi, 2013; Sasaki-Sekimoto et al., 2013).

415

Importantly, physical interaction between the JAMs and MYC2 appears to play a role in the

416

nuclear targeting of JAZ1 and JAZ9 (Withers et al., 2012). Since the MYC TFs are almost

417

constitutively expressed in all tissues, their regulation at the PPI level is likely to be of primary

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importance for their biological functions. Indeed, while MYC2 is present in various tissues at

419

various stages during development, it drives transcription only under certain conditions

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dependent on its interacting partners.

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MYC2, MYC3 and MYC4, have been shown to physically associate with MYB28, MYB29,

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MYB76, MYB51, MYB34 and MYB122 to regulate the accumulation of glucosinolate defense

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compounds (Figure 1, III) (Schweizer et al., 2013; Frerigmann et al., 2014). The expression

424

patterns of the six MYB TFs show high similarity (Gigolashvili et al., 2007a; Gigolashvili et al.,

425

2007b; Hirai et al., 2007; Sønderby et al., 2007) and strongly overlap with those of the MYCs.

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Although all three MYCs can interact with all six MYBs (Frerigmann et al., 2014; Schweizer et

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al., 2013), highly specific changes in the glucosinolate output can be triggered by different

428

hormones or stresses (Beekwilder et al., 2008; Frerigmann and Gigolashvili, 2014; Sonderby et

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al., 2010). Accordingly, analysis of glucosinolate levels in myc and myb single and double

430

mutants revealed specific mutant profiles (Frerigmann and Gigolashvili, 2014; Schweizer et al.,

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2013; Sonderby et al., 2010) suggesting specific in planta roles of the individual TFs that cannot

432

be fully compensated by their closest homologues.

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Another example of TF complex-mediated transcriptional regulation is the interaction

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DELLA and JAZ proteins, which provides the molecular basis of synergism between GA and JA

435

signaling (Hou et al., 2014). Both JA and GA have been shown to induce degradation of DELLA

436

and JAZ proteins to coordinately activate the MBW complex required to induce trichome

437

initiation (Qi et al., 2014). The DELLAs compete with the MYCs to bind the JAZs, thus

438

inducing JA-independent MYC2 activity (Figure 2-III) (Davière et al., 2008; Wild et al., 2012,

439

Hou et al., 2010; Lan et al., 2014). Upon increased GA levels, DELLAs are degraded and

440

MYC2-dependent JA synthesis is inhibited. The interaction of the DELLAs with MYC2

441

mediates induction of sesquiterpene synthase genes in a JA and red light dependent manner

442

(Hong et al., 2012). JA and GA signaling thus exhibit balancing effects on each other’s pathways

443

depending on their abundance in the cell. The molecular basis of the synergy between JAZ and

444

DELLA proteins provides insight into how plants integrate different signals, how they sense

445

environmental changes and how they prioritize different processes. This allows plants to

446

orchestrate their responses precisely in space and time and thus to optimize their fitness in

447

fluctuating environments.

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Integration of regulatory mechanisms

An individual TF can be involved in multiple biological processes during the plant’s life

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cycle. Combinations of different PTMs and PPIs serve to establish different layers of TF

454

regulation on the protein level specific to environmental changes. To optimize their fitness,

455

plants have built complex regulatory systems ensuring fast and robust responses. A large number

456

of modifications occurring at the protein level modulate TF activity e.g. through altering the TF’s

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457

ability to bind interaction partners or DNA, through a change in turnover rate, or through

458

ubiquitination-dependent timing of degradation. In combination, all these mechanisms contribute

459

to the specificity of plant responses and at the same time increase their plasticity. The bHLH transcription factor MYC2 has been described as a master regulator of various

461

plant defense pathways in response to different environmental cues (Figure 2) (Kazan and

462

Manners, 2013). The idea that MYC2 has a more general effect on transcriptional activation

463

rather that high specificity to its target promoters is supported by the finding that MYC2 does not

464

act as transcriptional activator without an interaction partner (Kazan and Manners, 2013).

465

Instead, MYC2’s intrinsic property to bind different partners in different transcriptional networks

466

allows the plant to coordinate multiple signals and mount appropriate responses. The regulation

467

of DNA availability at the target loci has not been discussed in this review, but there is

468

experimental evidence suggesting that TFs also interact with histone-modifying enzymes as well

469

as mediator complexes (Badeaux and Shi, 2013; Lai et al., 2014; Yang et al., 2014a).

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As discussed above, flavonoids constitute a well-studied class of secondary plant

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metabolites. Contrary to the example of MYC2, which represents the case of a TF as a hub, the

472

anthocyanin pathway is an example of a complex TF network regulating one specific output.

473

Although multiple PPIs and specific expression patterns have been identified, the fine-tuning of

474

flavonoid accumulation keeps providing novel insight into how a multitude of regulatory

475

mechanisms insure specificity of MYB/bHLH complexes. In the future, the regulatory network

476

controlling anthocyanin biosynthesis could serve as a model pathway to build in silico tools for

477

the integration of both mechanistic models and larger gene network effects on the composition of

478

TF complexes in a pathway-specific context. As different TFs have been shown to control the

479

different parts of the pathway in a tightly controlled way, anthocyanin biosynthesis could prove

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its value as a model for other metabolic pathways, e.g. to predict the flux through a pathway for

481

engineering purposes.

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In this review, MYB30 served as example of a TF involved in different stress responses

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and modulated by different PTMs (Figure 3). The different interactors and modifying enzymes

484

allow for tight control of MYB30 activity in terms of a specific spatial and temporal regulation

485

of its targets genes. The mechanisms controlling MYB30 activity support a model in which

486

different trigger-specific signaling pathways have the same biological output, i.e. cell death. This

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type of regulation facilitates the adaptation of the regulation of a specific TF to different cellular

488

environments, e.g. maybe changing its ability to bind interaction partners depending. In different

489

cell types, the activity of MYB30 can be regulated through interaction with different partners and

490

through different PTMs. As opposed to MYC2 as an example of a TF with multiple targets and a

491

broad effect on transcriptional activation, future studies on MYB30 have the potential to

492

elucidate what confers specificity in DNA binding downstream of different internal and external

493

cues.

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Comprehension of the complexity of transcriptional regulation will strongly benefit from

499

a deeper understanding of the underlying mechanistic events. Thus, studies that investigate how

500

TFs function at the single molecule level will help to improve current models of transcriptional

501

regulation. Chen et al. (2014) have recently proposed a technique to visualize the binding of

502

single proteins to DNA which can help to resolve trial-and-error calculations of TF binding. This

503

approach allows classification of TFs in complexes based on their promoter search method and

504

binding order. Future studies using this and similar approaches to study know TF complexes will

505

provide valuable mechanistic insight into how TFs associate and disassociate to control the

506

expression of their target genes. In the case of MYC2, this level of mechanistic understanding

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may explain how MYC2 can activate different target genes dependent on its interacting partners.

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The challenges to improve our understanding of the DNA binding specificity of TFs that

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serve as regulatory hubs, like MYC2, are linked to their involvement in fast and cell-specific

510

responses. Thus, using tools for in planta visualization of bHLHs and their interactors as well as

511

investigating the chromatin landscape in the neighborhood of TF target promoters will provide

512

novel insight into the specificity of TF-DNA interaction (Sahu et al., 2013). Next generation

513

Chromatin-Immuno-Precipitation (ChIP) assays such as exo-ChIP with higher sensitivity and

514

higher spatial and temporal resolution will enable us to obtain a more comprehensive image of

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what happens in the cell (Zentner and Henikoff, 2012). This kind of in planta studies are

516

necessary because of the complexity of plants as multicellular organisms and will help to

517

integrate cell-to-cell communication with different signaling mechanisms controlling TF

518

activation (Chen and Zhu, 2004). Studies combining proteasome inhibitors and ChIP have

519

already been used in mammalian cells will also be needed in plants to detect DNA-associated

520

protein degradation, thereby e.g. linking MYC2 activity with turnover of the protein at the

521

promoter (Catic et al., 2013).

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Finally, recent advances in immuno-precipitation techniques and mass spectrometry will

523

enable the identification of novel components of regulatory protein complexes, As an example, a

524

cross-linking strategy combined with mass spectrometry was recently successful in identifying

525

protein complexes in planta (Luo et al., 2012). This methodology can in the future be extended

526

to the enrichment of cells or organelles isolated from plants grown under specific conditions at

527

different time points to study the dynamics of protein complex formation and to determine which

528

interaction partners are available in a given cell. Integrative bioinformatics will be key to

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incorporate these proteomics data with transcriptional network models.

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Acknowledgments

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The authors thank Frederik Rook and Sophie Lambertz for critical reading of the manuscript.

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Funding

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Financial support was provided by the Danish National Research Foundation DNRF grant 99.

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Figure legends

831 Figure 1: Schematic representation of MYB and BHLH TFs. A) R2R3 MYB TFs have a

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conserved DNA-Binding Domain (DBD) at the N-terminal of the protein and a more variable,

834

C-terminal Trans-Activation Domain (TAD). B) bHLH TFs of the MYC clade are characterized

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Figure 2: Signal integration by the hub TF MYC2. MYC2 has the ability to integrate multiple

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environmental and developmental signals and to control the expression levels of various

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pathways precisely in space and time. I: Constitutive expression levels and activation of MYC2

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are balanced by different PTMs. II: Upon herbivore or JA treatment, MYC2 inhibition by JAZs

843

is released leading to an accumulation of active MYC2. III: Depending on the developmental

844

stage, DELLAs interact with MYC2 to inhibit its JA-dependent transcriptional activity. GA will

845

lead to MYC2 activation to trigger GA-mediated developmental processes. IV: Active MYC2

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can bind different promoters and initiate specific responses. Squares represent MYC2 in an

847

inactive form, while circles depict active proteins. Roman numbers refer to different regulatory

848

layers described in the text. JA, jasmonic acid; GA, giberillic acid; PTM, post-transcriptional

849

modification.

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Figure 3: Combinatorial effects of PTMs and PPIs on MYB30 activity. MYB30 represents

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an example of a TF that is regulated by multiple mechanisms on the protein level. Different

853

signals are integrated on this level to ensure tight regulation of a single output, i.e. cell death. I:

854

Basal levels of transcript lead to accumulation of MYB30 protein balanced between an active

855

and inactive state by different PTMs. Upon stimulation by hormones, transcript levels incease

856

and the balance shifts towards more active protein. II: when activated, the MYB30 binds specific

857

promoters and thereby induces cell death-specific genes. III: Cell death is tightly balanced by

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modifying enzymes which will inactivate the protein to ensure locally restrict cell death. Inactive

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MYB30 is shown as square, the active form as circle. Roman numbers refer to different

860

regulatory layers that are described in the text. BRs, Brassinosteroids; SA, salicylic acid; ABA,

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abscisic acid; PTM, post-transcriptional modification.

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