STH2 has 2 B there

Abstract

Recently we reported the identification of a novel B-box transcription factor SALT TOLERANCE HOMOLOG 2 (STH2) that interacts genetically with two key regulators of the light-signaling pathway, HY5 and COP1. We also provided phenotypic and genetic characterization of the sth2 mutant suggesting that STH2 plays a positive role in regulating photomorphogenesis both independently or together with HY5. Functional assays in protoplasts revealed that STH2 could act as a transcriptional activator. To our knowledge this is the first report of a B-box domain containing protein playing a direct role in activating transcription in plants. Here we discuss the possible position of STH2 in the transcriptional network and comment on the role of the B-box domain in plants.

Biology of Interactions between STH2, HY5 and COP1

Light induces massive reprogramming of the plant transcriptome, and many of the early light-responsive genes are transcription factors. LONG HYPOCOTYL 5 (HY5) is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis and acts downstream to the photoreceptors.2 It is constitutively nuclear, binds to the promoters of light inducible genes and regulates their expression during photomorphogenesis. The activity of HY5 is in turn controlled by a key negative regulator, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) that degrades HY5 in the dark.3 We recently identified STH2, a B-box containing protein that interacts genetically with both HY5 and COP11. Phenotypes like hyposensitivity to blue, red and far-red light, enhanced number of lateral roots and reduced anthocyanin accumulation indicate that STH2 plays a positive role in photomorphogenesis. We found that STH2 could activate transcription and showed that the B-boxes in STH2 and a functional G-box element in the promoter are required for the activity. The fact that HY5 has been found to constitutively bind to the promoters of a set of genes related to photosynthesis and circadian regulation, such as RbcS1A, CHS, CCA1 and TOC1 irrespective of the light-dark transition or the daily rhythm suggests that HY5 binding is not sufficient for transcriptional activation and might require some additional cofactors for regulation.2 Our result suggests that STH2 and possibly other B-box containing proteins could be the additional factors HY5 needs for transcriptional regulation. Furthermore STH2 might also act as a cofactor for other G-box binding proteins such as HYH regulating HY5 independent processes.4,5 Our data showing COP1-dependent localization of STH2 to speckles and genetic interactions between sth2 and cop1 both in the dark and in light suggests that the B-box protein STH2, the bZIP transcription factor HY5 and the RING finger and coiled-coil domain containing COP1 protein work in close proximity to regulate photomorphogenesis in Arabidopsis.

RBCC/TRIM Family Proteins

The B-box is a zinc-ligating domain consisting of conserved Cys and His residues.6 In animals B-boxes are mostly found in conjugation with a RING finger domain (originally termed an A-box) and a coiled-coil domain forming RBCC or tripartite motif proteins. The RBCC family includes a large number of proteins involved in various cellular processes like apoptosis, cell cycle regulation and viral response.7 Recently a number of TRIM/RBCC proteins (Fig. 1A) have been found to play a role in ubiquitination and the B-boxes proposed to participate in substrate recognition. Other functions of this domain involve localization into nuclear bodies as in the tumor suppressor protein PML (Promyelocytic Leukemia), transcriptional regulation8 and protein-protein interaction. The B-box domain has been classified into two subgroups, B-box1 and B-box2, both of which have seven or eight conserved Cys and His residues to coordinate two zinc atoms in a ring-like fold. B-boxes are usually found as single domains or as tandem repeats. While B-box2 is present in all RBCC proteins, B-box1 is absent in proteins containing only one B-box and is present N-terminal to B-box2 in proteins containing two tandemly repeated B-boxes.

B-box Proteins in Plants

Although RBCC proteins are absent in Arabidopsis, there are 32 proteins with N-terminal B-boxes (Fig. 1B). In contrast to animals all Arabidopsis B-box containing proteins have at least one B-box with an Asp as the fourth zinc-coordinating residue. The consensus sequence of this B-box is shown in Figure 1C. A large subgroup (the 17 COL proteins) of this family contains an additional CCT domain in the C-terminal part of the protein.9 STO, STH1 and STH2 together with five other proteins form a small subgroup, with two tandem repeated B-boxes with high homology. A third subgroup consists of five proteins, which contains only one B-box. Some other variants of B-boxes in Arabidopsis contain a Glu or His residue instead of the Asp as the fourth zinc-coordinating residue (Fig. 1B).

Role of B-Boxes

Although the B-box has been proposed to be a protein interaction domain, its molecular function in plants is not well understood. Recently it was reported that the CCT domain of the B-box containing protein CONSTANS (CO) was involved in the formation of a heterotrimeric DNA binding complex suggesting that in plants B-box proteins have evolved to attain different functional roles.10 Our yeast data showed that a structurally intact B-box domain was important for interaction with HY5, providing evidence for the role of B-box domain in protein-protein interaction. Furthermore, transient transfection assay in protoplasts indicated that a functional B-box domain is required to activate transcription, thus providing a novel molecular function for this domain. We propose a model wherein STH2 acts as a transcriptional cofactor that interacts with HY5 through its B-boxes and is able to activate transcription through the G-box promoter element.

Conclusions and Outlook

The fact that COP1 could recruit STH2 to nuclear speckles and the genetic interaction between sth2 and cop1 is interesting. Previously we had reported interaction between COP1 and the B-box containing COL3, STH1 and STO proteins.11,12 Recent results suggest that coiled-coil domain containing SPA proteins are important for the stability of the B-box containing protein CO.13 All these interactions between B-box containing proteins and the RING, coiled-coil domain containing COP1-SPA proteins suggest a mechanism of creating a functional equivalent of RBCC protein in an organism that lacks such proteins. Whether these interacting B-box proteins together with COP1 play a role in ubiquitination and proteolysis or act as a substrate for COP1 mediated degradation is a question yet unanswered. However studies of these biochemical complexes might help unravel the functional intricacies of manifold cellular processes regulated by B-box containing proteins.

Figures and Tables

Figure 1 (A) Schematic representation of domains present in PML and LIN-41, two RBCC proteins found in animals and the Ring finger, coiled-coil domain containing COP1 protein. NHL and WD40 are protein interaction domains. (B) Classification and schematic representation of the 32 B-box containing proteins in Arabidopsis. Black boxes represent B-boxes present in all Arabidopsis proteins, whereas graded boxes indicate a variant of B-box containing Glu or His as the fourth zinc-coordinating residue. CCT represents the CO, CO-like, TOC1 domain. Numbers indicate members in each class. (C) Consensus sequence of the B-box found in all Arabidopsis proteins containing an Asp as the fourth zinc-coordinating residue.

Addendum to: