Does brassinosteroid function require chromatin remodeling?

Abstract

The phytohormone brassinosteroid (BR) is crucial for plant growth and development. Although genetic and molecular approaches have improved understanding of the cellular BR signaling pathway, we still do not have sufficient knowledge about the function of BR. Therefore, proteomic analysis was used to elucidate BR signaling and gene expression in the nuclei of suspended Arabidopsis cells treated with brassinolide, a bioactive BR, or brassinazole, a BR biosynthesis inhibitor. Interestingly, chromatin remodeling-related proteins, the abundance of which was altered in response to cellular BR levels, were identified. This suggested that BR-induced gene expression is regulated not only by transcription factors directly binding to cis-elements, but also by chromatin remodeling in response to BR signaling. In this addendum, we summarize the functions of our identified nuclear proteins in chromatin remodeling and discuss the need for chromatin remodeling regulated by BR signal transduction for expression of BR-induced genes.

The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes such as cell division and expansion, seed germination, stem elongation, vascular differentiation, photomorphogenesis, and stress and disease resistance.¹ Over the past several years, genetic and molecular approaches have advanced our understanding of the BR signaling pathway.² However, the mechanisms by which BRs regulate various subsets of target genes in different tissues, organs, developmental stages, and environmental conditions are still largely unknown. As an approach to elucidating these functional mechanisms, proteomics complement recent genetic results with an exploration of protein complexes and posttranslational modification.

In our Plant Physiology and Biochemistry paper, five interesting nuclear proteins were identified and described, including a nucleosome assembly protein (NAP) 1;1 and 1;2, S-adenosylmethionine synthetase 2 (SAM syn.2), histone deacetylase 2B (HD2B), and structural maintenance of chromosome 3 (SMC3). Each of these proteins was identified by two-dimensional PAGE (2D-PAGE) and mass spectrometry, which revealed changes in the abundance of each of these proteins in response to cellular BR levels in suspended Arabidopsis T87 cells 2 d after administration of brassinolide, a bioactive BR, and brassinazole, a BR biosynthesis inhibitor (Table 1).³ In addition to these, proteins whose expression showed no correlation with cellular BR levels were also identified. Eukaryotic translation initiation factor 3E (EIF3E), vernalization independence 3 (VIP3), and TGF-β receptor interacting protein 1 (TRIP-1) were included in this class.³ Here, an overview of the individual functions of these specific nuclear proteins (HD2B, SAM syn.2, and NAP1) identified in our previous paper is presented, and the involvement of some of these proteins in chromatin remodeling in response to BR signaling is discussed.

Histone deacetylases (HDACs) remove acetyl moieties from the N-terminus of the histones, which makes the transcriptional apparatus unable to access the chromatin.⁴ Acetylation of histones is generally correlated with transcriptionally active euchromatin, whereas deacetylation is correlated with transcriptionally silent heterochromatin. Histone acetylation is a central function of transcriptional regulation, as illustrated by the association of histone acetyltransferases (HATs) with transcriptional coactivators and association of HDACs with corepressor complexes.⁵ Zhou et al.⁶ showed that HD2A, HD2B, and HD2C repress transcription by interacting with promoters in planta, where repression of the target genes by HD2s is mediated via complexes with specific transcription factors. Furthermore, it is interesting that the HDACs play important roles in plant responses to environmental stresses and to different plant hormones including abscisic acid (ABA), jasmonic acid, and ethylene.^7,8 These chromatin modifications provide an additional level of regulation of gene expression beyond that of the transcription factors that recruit RNA polymerase to target genes during signal transduction by BR and other hormones.

S-adenosylmethinine syn.2 is a cellular enzyme that catalyzes the formation of S-adenosyl-methionine (SAM) from methionine and ATP. Specific methyltransferases methylate cytosine in DNA as well as arginine and lysine residues of various proteins, including histones, using SAM as a methyl donor.⁹ Although the function of SAM syn.2 in the nucleus is not well known, Katoh et al.¹⁰ recently reported that SAM syn.2 serves as a transcriptional co-repressor of the MafK transcription factor by providing SAM locally and interacting with chromatin-related factors in mammalian cancer cells.

Nucleosome assembly protein 1, a histone chaperone, is involved in both the deposition of histones during nucleosome assembly and the eviction of histones during nucleosome disassembly, in cooperation with ATP-dependent chromatin-remodeling factors.¹¹ An ATP-dependent chromatin-remodeling factor, PICKLE (PKL), which is a switch (SWI)/sucrose non-fermenting (SNF) class chromatin remodeling factor of the chromodomain helicase-DNA-binding 3 (CHD3) type, may modify interactions between DNA and histone octamers, destabilizing nucleosomes and allowing the transcriptional complex to interact with the DNA.^12-14 Liu et al.¹⁵ showed that the Arabidopsis NAP1 proteins are positive regulators in ABA signaling, suggesting a novel link between chromatin remodeling and hormonal and stress responses. PKL plays an important role in plant development and has been implicated in plant hormone signaling via auxin, ABA, gibberellin, and cytokinin.^16-19 Together, these findings suggest that BR signaling and gene expression may involve chromatin remodeling initiated by NAP1 proteins and ATP-dependent chromatin remodeling factors.

Finally, an integrated view of chromatin remodeling in regard to BR function is discussed. As shown in Figure 1, the identified nuclear proteins are schematized as members of a chromatin remodeling and gene expression system, which may play an important role in BR-induced gene expression. It is evident that some of these identified proteins occupy primary roles in a series of chromatin remodeling processes positively or negatively controlling expression of many genes regulated by BRs.

Figure 1.

Multifunctional and fluid roles of transcription factors in regard to BR-induced gene expression. Summary of BR signaling factors potentially involved chromatin remodeling is schematized here; other transcription factors and other proteins in this scheme have not yet been discovered. Spatial and time-dependent changes in these protein complexes also have not been established. The interconnections of these various factors are not currently known. Solid lines indicate direct interactions and dotted lines indicate unknown interactions. Positive interactions are noted by an arrow. Green- stars indicate our identified nuclear proteins. BR: brassinosteroid; TF: transcription factor; NAP1: nucleosome assembly protein 1; SAM syn.2: S-adenosylmethionine synthetase 2; HD2B: histone deacetylase 2B; SMC3: structural maintenance of chromosome 3; EIF3E: eukaryotic translation initiation factor 3E; VIP3: vernalization independence 3; Ac: acetyl group; Me: methyl group; MT: methyltransferase; Met: methionine; SAM: S-adenosyl-mehionine; RNAP II: RNA polymerase II.

Katoh et al.¹⁰ have identified a number of MafK- and SAM syn.2-interacting proteins by using immunoaffinity chromatography in their proteomic study. These interactors included a variety of chromatin- and transcription-related factors and components of the SWI/SNF, nucleosome remodeling and histone deacetylase (NuRD), poly(ADP-ribose) polymerase 1, and polycomb group complexes, which mediate nucleosome remodeling, histone modification, and the formation of heterochromatin. Their results reveal a mechanism by which methyl groups are supplied and specific sites for methylation of DNA or histones on large expanses of chromatin are determined, which leads to control of gene expression. Therefore, a similar mechanism is proposed to operate in BR signaling, and the function and characterization of such nuclear protein complexes, including core transcription factors, is underway in our Arabidopsis cell system. These new findings have now focused attention on the involvement of chromatin remodeling in BR signaling in the nucleus of plant cells.

Piecing these findings together, a mechanism for BR function is proposed. During an early phase of BR function, when BRs are applied to plant cells, the BR signal transduction cascade activates, which includes BR perception by the BR-insensitive 1 (BRI1) receptor kinase at the cell surface, which leads to the accumulation of unphosphorylated bri1-EMS-suppressor 1 (BES1), brassinazole resistant 1 (BZR1), and other transcription factors in the nucleus. One group of these factors then gain access to cis-elements in the DNA on a functionally dormant part of the chromatin, probably by forming complex and compact conformations. At this point, chromatin remodeling complexes SWI/SNF, NuRD, and/or NAP1 begin to relax the higher-order architecture of chromatin via a special protein factor(s), which makes promoter regions of target genes regulated by BRs more easily accessible for transcription. Here, we propose a hypothesis highlighting the multifunctional nature of these transcription factors, as shown in Figure 1; they may have several roles in this stage of BR signaling: acting as a master by binding to cis-element as part of transcriptional control complexes; guiding methylation and/or acetylation (including the reverse reaction; de-methylation and/or de-acetylation) to recruit the relevant enzymes and proteins to specified loci on chromatin.

With the transition from one cellular physiological state to the next, BRs induce histone modification in the promoter regions of many genes. Deacetylases cause chromatin binding proteins to induce a more compact DNA conformation around specified coding regions of chromatin. In other instances, histones or DNA are methylated at particular regions of the chromatin, which silences these regions transcriptionally. In the reverse process, the activity of major genes related to the functions occurring in the subsequent cell state come into operation upon relaxation of specified loci on chromatin. Simultaneously, expression of genes for transcription factors and related proteins is also enhanced by the BR signal and these factors activate expression of secondary genes, as shown in Figure 1. These hypotheses and approaches to address them should provide important insights into the relationship between chromatin modifications and BR function.

For instance, Yu et al.²⁰ have identified two BES1-interacting proteins, early flowering 6 (ELF6) and its homolog, relative of early flowering 6 (REF6), both of which are Jumonji (Jmj) N/C domain-containing proteins. Such Jmj domain-containing histone demethylases are involved in gene expression in many developmental processes. Although both ELF6 and REF6 mainly promote BR-induced gene expression by an interaction with BES1, they appear to be able to repress gene expression in other pathways, probably by combinational control via protein complexes. We propose that this evidence may be only a small example of a greater switching system for expressing and/or repressing a group of genes.

Unfortunately, we have not yet identified any transcription factors such as BES1, BZR1, or others, which may be present in the nucleus at extremely low abundance. Identification of proteins with only higher abundance in the nucleus has been achieved in this experimental system. Therefore, improvements in preparation techniques for nuclear protein interactors are underway to allow further study of the proteins involved in BR signaling and function. It is essential to study the composition, stoichiometry, and spatial arrangement of these nuclear protein complexes that have a transcription factor at their core, as well as to investigate fluctuation in the other protein components of the BR signaling apparatus.

Shigeta T, Yasuda D, Mori T, Yoshimitsu Y, Nakamura Y, Yoshida S, et al. Characterization of brassinosteroid-regulated proteins in a nuclear-enriched fraction of Arabidopsis suspension-cultured cells. Plant Physiol Biochem. 2011 doi: 10.1016/j.plaphy.2011.04.012. In press.