Molecular Plant Advance Access published October 31, 2013
The many faces of plant SWI/SNF complex Jose C. Reyes
Molecular Biology Department. Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER). Consejo Superior de Investigaciones Científicas (CSIC). Av. Americo Vespucio 41092 Seville. Spain.
Downloaded from http://mplant.oxfordjournals.org/ at Grand Valley State Univ on November 1, 2013
*Corresponding author: José C. Reyes:
Tel.: +34 954467842; fax: + 34 954461664 E-mail address: [email protected]
Running title: Plant SWI/SNF complexes
1
The SWI/SNF complexes In plants, as well as in metazoans, the execution of developmental programs and the response to environmental stimuli depend on changes in gene transcription programs that require extensive chromatin modifications of basically two types: posttranslational modification of histones and ATPdependent reorganization of histones-DNA interactions. Many different nuclear machineries are necessary to carry out chromatin modifications at the appropriate time and place. Yeast SWI/SNF (switch/sucrose nonfermentable) was the first ATP-dependent chromatin-remodelling complex to be described in the 80´s. Since then, SWI/SNF complexes have been found in many eukaryotes, with functions ranging from control of development to genome stability (recently reviewed in (Euskirchen et al., 2012)). About a decade ago, the first Arabidopsis genes encoding orthologs to SWI/SNF complex subunits were reported (Brzeski et al., 1999; Farrona et al., 2004; Wagner and Meyerowitz, 2002). A large body of genetic and biochemical evidence strongly suggests that SWI/SNF complexes are also present in plants (Bezhani et al., 2007; Farrona et al., 2004; Hurtado et al., 2006; Sarnowski et al., 2005). However, since an endogenous complex has not yet been purified from plants, plant SWI/SNF subunits have only been identified based in sequence conservation. As in metazoans, Arabidopsis SWI/SNF subunits are often encoded by more than one gene, thus allowing combinatorial assembly and a diversity of related complexes with different functions, which are now beginning to be elucidated. These complexes are formed by one DNA-dependent ATPase, responsible for the nucleosome remodelling activity, and by several other subunits involved in complex targeting and activity regulation. Two canonical ATPases has been described in Arabidopsis, namely SPLAYED (SYD) and BRAHMA (BRM) (Farrona et al., 2004; Hurtado et al., 2006; Wagner and Meyerowitz, 2002). In addition, two closely-related ATPases, CHR12 (also called MINU1) and CHR23 (also called MINU2), are also present in Arabidopsis (Cong-Cong et al., 2012; Mlynarova et al., 2007; Sang et al., 2012); however, no interactions between these enzymes and the SWI/SNF subunits have been observed to date. Therefore, whether CHR12 and CHR23 are bona fide SWI/SNF subunits is still unclear. It is thought that Arabidopsis SWI/SNF 2
complexes also contain one ortholog of SNF5/INI1, called BUSHY (BSH) (Brzeski
et
al.,
1999),
two
of
the
four
possible
orthologs
of
SWI3/BAF170/BAF155 (SWI3A, SWI3B, SWI3C, and SWI3D) (Sarnowski et al., 2005) and one of the two possible orthologs of SWP73/BAF60 (SWP73A and SWP73B)
(Figure
1A).
Interactions
demonstrated
between
SWI/SNF
components and other factors during the last years have allowed to outline a number of possible complexes (Figure 1A) (Bezhani et al., 2007). In this Research Highlight, I will mostly describe recent advances in the knowledge of the multiple roles that SWI/SNF complexes play in development, phytohormone response and RNA-mediated silencing.
SWI/SNF in development Leaf development is intensely altered in SWI/SNF-deficient plants. Thus, brm and swi3c rosette leaves are strongly curled downwards under long-day conditions and uneven and serrated in short days (Archacki et al., 2009; Farrona et al., 2004; Hurtado et al., 2006; Sarnowski et al., 2005). The groups of Eshed and Wagner recently uncovered a role for BRM and SWI3C as coactivators of TCP4 (TEOSINTE BRANCHED1, CYCLOIDEA, PCF4) (Efroni et al., 2013). While cytokinin (CK) promote cell division and the formation of marginal leaf serrations, TCP4 promotes leaf growth arrest and maturation. BRM interacts with TCP4 (Figure 1A), and together they activate the expression of the ARR16 gene, an A-class ARR factor that inhibits CK signalling. This provides a signal to reduce CK responses in the margins of the lamina and to promote the progressive loss of morphogenetic potential. Efroni et al. did not address the origin of the curled leaves of brm and swi3c plants; however, it is easy to speculate that an uncoupled maturation of adaxial with respect to the abaxial surfaces of the lamina are responsible for the phenotype. Interestingly BRM and SYD positively control expression of CUP-SHAPED COTYLEDON (CUC) genes, at least in the cotyledon boundary region (Kwon et al., 2006). CUC genes are required for leaf serration (Hasson et al., 2011) and CK has been shown to activate CUC genes (Li et al., 2010). Therefore, it is unclear how BRM promotes at the same time reduction of CK response and activation of CUC gene expression. The transition from vegetative to reproductive development also seems to 3
be dependent on the SWI/SNF complexes. In fact, BRM is involved in the regulation of three out of the four flowering pathways in Arabidopsis, namely, the photoperiod, the gibberellin and the autonomous pathways (Archacki et al., 2013; Farrona et al., 2011) (Figure 1B). Consistently, brm mutants display a complex flowering phenotype characterized by early flowering in long days and late flowering in short days. Besides its role in flowering time control, SWI/SNF complexes also affect flower development. Mutations in BRM and SYD provoked subtle floral homeotic transformations and an small reduction in the level of the MADS factors AP3 and AG (Farrona et al., 2004; Hurtado et al., 2006; Wagner and Meyerowitz, 2002; Wu et al., 2012). Since brm syd double mutants are embryonic lethal, the group of Wagner generated a conditional double mutant in flower primordial germ cells (Wu et al., 2012). Interestingly, these plants displayed severe floral homeotic transformations, demonstrating that BRM- and SYD-containing complexes have redundant roles in flower patterning. LFY and SEP3 seems to recruit both ATPases to the promoter regions of AP3 and AG, which is required to counteract the repressive effects mediated by the Polycomb complex. Thus, clf mutations partially suppress the severe defects caused by the combined loss of SYD and BRM. It is worth remembering that brahma mutant in Drosophila was originally identified as a suppressor of homeotic transformations present in Polycomb mutations. Therefore, these data indicate that the Polycomb-Trithorax antagonistic regulation of homeotic genes is conserved between plants and metazoans (Wu et al., 2012), although the structures of these genes are not conserved. This suggests that during evolution, animals and plants have converged to use similar chromatin machineries to control similar functions. Several SWI/SWF subunits have also been shown to co-purify with the five major floral homeotic MADS proteins (AP1, AP3, PI, AG, and SEP3) (Figure 1A), suggesting that the SWI/SNF complexes are coactivators of MADS factor target genes (Smaczniak et al., 2012). Consistently, expression of NAP, an AP3 and PI target gene, was strongly impaired in brm plants (Hurtado et al., 2006). Taken together, these results indicate that SWI/SNF complexes play a dual role in flower development: they first activate transcription of several ABC transcription factors, and then they cooperate with them to regulate downstream targets (Figure 1B). 4
In addition to counteracting Polycomb repression, SWI/SNF complexes also appear to cooperate together with Polycomb complexes to repress transcription. For example, brm mutants display high levels of FLC expression that is accompanied by a reduction in the level of the characteristic Polycombdependent mark H3K27me3 at the FLC promoter (Farrona et al., 2011). On the other hand, BRM, SWI3C and BSH also seem to cooperate with Polycomb to repress seed maturation genes in leaves (Tang et al., 2008).
SWI/SNF in the phytohormone response Several SWI/SNF subunits have been recently involved in abscisic acid (ABA), gibberellic acid (GA), jasmonate (JA), and ethylene (ET) signalling pathways (Archacki et al., 2013; Han et al., 2012; Walley et al., 2008). Jasmonate, and ethylene pathways signal biotic and abiotic stresses, such as wounding and pathogen infections. Walley et al. found that syd mutants present an increased susceptibility to fungal pathogens. Consistently, SYD is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways such as PDF1.2a, VPS2 and MYC2 (Walley et al., 2008). Brm mutants were shown to have a constitutively active abscisic acid (ABA) signalling pathway, and consequently, they displayed hypersensitivity to ABA and enhanced drought tolerance (Han et al., 2012). Expression of the transcription factor ABA INSENSITIVE5 (ABI5) is de-repressed in the absence of ABA and accumulates to high levels upon ABA addition. BRM is required to maintain high occupancy of the +1 nucleosome at the 5´ part of the coding region of the ABI5 locus, but BRM was not involved in the ABA-dependent reorganization of the -1 nucleosome at the promoter around the ABA response elements. This was in agreement with the fact that induction by ABA was not impaired in brm mutants. Interestingly, BRM binds to the ABI5 promoter both in the absence and presence of exogenous ABA, suggesting the possibility that BRM is inactivated in the presence of ABA (Han et al., 2012). It was reported that SWI3B interacted with the negative regulators of ABA signalling, HAB1, ABI1, ABI2 and PP2CA (Figure 1A), but, surprisingly, swi3b hypomorphic mutants displayed a reduced ABA response (Saez et al., 2008). It is worth noting that SWI3C and SWI3B interact with BRM (Hurtado et al., 2006); however, while swi3c and brm mutants display very similar phenotypes 5
(Archacki et al., 2009; Hurtado et al., 2006; Tang et al., 2008), swi3b deficiency provokes different effects, suggesting that BRM and SWI3C are in the same SWI/SNF complexes but that SWI3B is mostly in different complexes. Alternatively, the opposite functions in ABA signalling of BRM and SWI3B may suggest that SWI3B negatively controls the activity of BRM-containing complexes (at least in this pathway), highlighting the importance of the combinatorial subunit composition of the complexes in regulating its activities. Gibberellic acid (GA) counteracts many of the effects of ABA signalling in seed germination and root elongation. Interestingly, brm and swi3c mutants show GA-related growth and developmental defects (Archacki et al., 2013; Sarnowska et al., 2013), at least partially suppressed by GA addition. Consistently, these mutants display a reduced level of GA 4. Jerzmanowski and collaborators have also found that swi3c mutants display low expression of the GA-receptor GID1a and of four DELLA proteins (Sarnowska et al., 2013). Furthermore, SWI3C interacts with several DELLA proteins and with SPY, although the molecular consequences of these interactions are still unknown (Sarnowska et al., 2013). Therefore, it seems that BRM containing SWI/SNF complexes positively influences germination and root growth by acting at two levels: inhibiting ABA signalling and promoting GA response (Figure 1C). However, since GA and ABA pathways reciprocally down-regulate each other, whether GA- and ABA-related brm phenotypes are two faces of the same coin or whether BRM directly regulates both pathways in a coordinated fashion require further investigation.
SWI/SNF in lncRNA-mediated transcriptional silencing RNA-mediated transcriptional silencing pathways in plants involves two classes of noncoding RNAs: small interfering RNAs (siRNAs) and long noncoding RNAs (lncRNA). The mechanism by which lncRNAs silence transcription is still unclear. The group of Wierzbicki has recently reported that the SWI/SNF complex is involved in this silencing mechanism (Zhu et al., 2013). Thus, SWI3B interacts with the lncRNA binding protein IDN2 (Figure 1A). More importantly, transcription of the solo-LTR locus was significantly de-repressed in the swi3b/+, swi3d and syd mutants. In contrast, another silencing target, At2TE78930, was de-repressed in swi3b/+, swi3c, and brm mutants, indicating 6
that different SWI/SNF complexes are involved in silencing specific targets. Derepression is often accompanied by loss of DNA methylation and nucleosome destabilization at the affected locus. Since it has been shown that DNA methylation correlates with nucleosome positioning, Zhu et al. suggest that lncRNAs affect gene silencing by promoting nucleosome positioning with the help of chromatin remodelling complexes; then, maintenance of silencing would be reinforced by a feedback loop between DNA methylation and nucleosome positioning.
Future directions The SWI/SNF complexes affect many different aspects of plant biology, and can play distinct roles in different pathways or even opposite roles in the same pathway. We are still far from understanding how the activity and composition of the complexes are regulated at the molecular level, or what determines whether a complex is an activator or repressor of transcription. To address this, it is essential that we obtain a better understanding of the network of interactions of SWI/SNF subunits with multiple transcription factors, as well as proteomic analyses of the endogenous family of complexes. In this sense a great tool has been recently published by Efroni et al. In this study a total of 400 pairwise interactions by yeast-two-hybrid were found between six SWI/SNF subunits and 210 transcription factors (Efroni et al., 2013). In vivo confirmation of these interactions and its functional consequences will be of paramount importance. Promoter specific chromatin characteristics will also strongly determine the activity of SWI/SNF complexes as activators or repressors. Genome-wide nucleosome mapping studies comparing wild-type with SWI/SNF mutant lines will be essential to understand SWI/SNF chromatin activity. Additional layers of complexity, such as how post-transcriptional modifications alter the activity of the complexes, or the mechanisms by which lncRNAs recruit specific complexes to specific loci, are still unknown. A further challenge will be to understand the interplay between different chromatin machineries. For instance, how can SWI/SNF cooperates with Polycomb complexes to repress seedspecific genes (Tang et al., 2008) yet counteract Polycomb activity in expressing flower homeotic genes (Wu et al., 2012)? Why do brm mutations overcome the requirement of H2A.Z and the SWR1 complex for gene activation 7
(Farrona et al., 2011)? The answers to these intriguing questions that are currently under investigation will provide us with a clearer understanding of the critical roles of SWI/SNF complexes in plant gene regulation.
FUNDING This work was supported by Ministerio de Educación y Ciencia [BFU201123442 and CSD2006-00049]; Junta de Andalucía [P06-CVI-4844] and Fundación Ramón Areces.
ACKNOWLEDGEMENTS I thank S. Farrona for critically reading of the manuscript and discussion.
Figure legends
Figure 1. The many faces of the SWI/SNF complex. A) Protein-protein interaction network of SWI/SNF subunits. Interactions between SWI/SNF subunits are shown as red lines. Interactions of SWI/SNF subunits with other proteins, or between non-SWI/SNF subunits, are shown as blue lines. Proteins of the same family have the same colour. The protein interaction network was visualized using Cytoscape 2.6.3. This interactome combines yeast two-hybrid, pull down, far-western and mass spectrometry and BiFC data from different origins (Bezhani et al., 2007; Efroni et al., 2013; Farrona et al., 2004; Hurtado et al., 2006; Saez et al., 2008; Sarnowska et al., 2013; Sarnowski et al., 2005; Smaczniak et al., 2012; Wu et al., 2012; Zhu et al., 2013), BioGRID 3.2 and our unpublished data). B) BRM and SYD control flower development at several levels. C) Model of control of seed germination and root growth by BRM.
REFERENCES Archacki, R., Buszewicz, D., Sarnowski, T.J., Sarnowska, E., Rolicka, A.T., Tohge, T., Fernie, A.R., Jikumaru, Y., Kotlinski, M., Iwanicka-Nowicka,
8
R., et al. (2013). BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of GibberellinMediated Responses in Arabidopsis. PLoS ONE 8:e58588. Archacki, R., Sarnowski, T.J., Halibart-Puzio, J., Brzeska, K., Buszewicz, D., Prymakowska-Bosak, M., Koncz, C., and Jerzmanowski, A. (2009). Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes. Planta 229:1281-1292. Bezhani, S., Winter, C., Hershman, S., Wagner, J.D., Kennedy, J.F., Kwon, C.S., Pfluger, J., Su, Y., and Wagner, D. (2007). Unique, Shared, and Redundant Roles for the Arabidopsis SWI/SNF Chromatin Remodeling ATPases BRAHMA and SPLAYED. Plant Cell 19:403-416. Brzeski, J., Podstolski, W., Olczak, K., and Jerzmanowski, A. (1999). Identification and analysis of the Arabidopsis thaliana BSH gene, a member of the SNF5 gene family. Nucleic Acids Res. 27:2393-2399. Cong-Cong, L., Jun-Feng, Z., Ying-Jie, G., and Su-Juan, C. (2012). Two Putative Chromatin-Remodeling ATPases Play Redundant Roles in Seed and Embryo Development of Arabidopsis. Plant Physiol J 11:1084-1090. Efroni, I., Han, S.K., Kim, H.J., Wu, M.F., Steiner, E., Birnbaum, K.D., Hong, J.C., Eshed, Y., and Wagner, D. (2013). Regulation of leaf maturation by chromatin-mediated modulation of cytokinin responses. Dev Cell 24:438445. Euskirchen, G., Auerbach, R.K., and Snyder, M. (2012). SWI/SNF chromatinremodeling factors: multiscale analyses and diverse functions. J Biol Chem 287:30897-30905. Farrona, S., Hurtado, L., Bowman, J.L., and Reyes, J.C. (2004). The Arabidopsis thaliana SNF2 homolog AtBRM controls shoot development and flowering. Development 131:4965-4975. Farrona, S., Hurtado, L., March-Diaz, R., Schmitz, R.J., Florencio, F.J., Turck, F., Amasino, R.M., and Reyes, J.C. (2011). Brahma is required for proper expression of the floral repressor FLC in Arabidopsis. PLoS ONE 6:e17997. Han, S.K., Sang, Y., Rodrigues, A., Wu, M.F., Rodriguez, P.L., and Wagner, D. (2012). The SWI2/SNF2 chromatin remodeling ATPase BRAHMA represses abscisic acid responses in the absence of the stress stimulus in Arabidopsis. Plant Cell 24:4892-4906. Hasson, A., Plessis, A., Blein, T., Adroher, B., Grigg, S., Tsiantis, M., Boudaoud, A., Damerval, C., and Laufs, P. (2011). Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development. Plant Cell 23:54-68. Hurtado, L., Farrona, S., and Reyes, J.C. (2006). The putative SWI/SNF complex subunit BRAHMA activates flower homeotic genes in Arabidopsis thaliana. Plant Mol Biol 62:291-304. Kwon, C.S., Hibara, K., Pfluger, J., Bezhani, S., Metha, H., Aida, M., Tasaka, M., and Wagner, D. (2006). A role for chromatin remodeling in regulation of CUC gene expression in the Arabidopsis cotyledon boundary. Development 133:3223-3230. Li, X.G., Su, Y.H., Zhao, X.Y., Li, W., Gao, X.Q., and Zhang, X.S. (2010). Cytokinin overproduction-caused alteration of flower development is
9
partially mediated by CUC2 and CUC3 in Arabidopsis. Gene 450:109120. Mlynarova, L., Nap, J.P., and Bisseling, T. (2007). The SWI/SNF chromatinremodeling gene AtCHR12 mediates temporary growth arrest in Arabidopsis thaliana upon perceiving environmental stress. Plant J 51:874-885. Saez, A., Rodrigues, A., Santiago, J., Rubio, S., and Rodriguez, P.L. (2008). HAB1-SWI3B interaction reveals a link between abscisic acid signaling and putative SWI/SNF chromatin-remodeling complexes in Arabidopsis. Plant Cell 20:2972-2988. Sang, Y., Silva-Ortega, C.O., Wu, S., Yamaguchi, N., Wu, M.F., Pfluger, J., Gillmor, C.S., Gallagher, K.L., and Wagner, D. (2012). Mutations in two non-canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects. Plant J 72:1000-1014. Sarnowska, E.A., Rolicka, A.T., Bucior, E., Cwiek, P., Tohge, T., Fernie, A.R., Jikumaru, Y., Kamiya, Y., Franzen, R., Schmelzer, E., et al. (2013). DELLA-interacting SWI3C core subunit of SWI/SNF chromatin remodeling complex modulates gibberellin responses and hormonal crosstalk in Arabidopsis. Plant Physiol. Sarnowski, T.J., Rios, G., Jasik, J., Swiezewski, S., Kaczanowski, S., Li, Y., Kwiatkowska, A., Pawlikowska, K., Kozbial, M., Kozbial, P., et al. (2005). SWI3 Subunits of Putative SWI/SNF Chromatin-Remodeling Complexes Play Distinct Roles during Arabidopsis Development. Plant Cell 17:24542472. Smaczniak, C., Immink, R.G., Muino, J.M., Blanvillain, R., Busscher, M., Busscher-Lange, J., Dinh, Q.D., Liu, S., Westphal, A.H., Boeren, S., et al. (2012). Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc Natl Acad Sci U S A 109:1560-1565. Tang, X., Hou, A., Babu, M., Nguyen, V., Hurtado, L., Lu, Q., Reyes, J.C., Wang, A., Keller, W.A., Harada, J.J., et al. (2008). The Arabidopsis BRAHMA chromatin-remodeling ATPase is involved in repression of seed maturation genes in leaves. Plant Physiol 147:1143-1157. Wagner, D., and Meyerowitz, E.M. (2002). SPLAYED, a Novel SWI/SNF ATPase Homolog, Controls Reproductive Development in Arabidopsis. Curr. Biol. 12:85-94. Walley, J.W., Rowe, H.C., Xiao, Y., Chehab, E.W., Kliebenstein, D.J., Wagner, D., and Dehesh, K. (2008). The chromatin remodeler SPLAYED regulates specific stress signaling pathways. PLoS Pathog 4:e1000237. Wu, M.F., Sang, Y., Bezhani, S., Yamaguchi, N., Han, S.K., Li, Z., Su, Y., Slewinski, T.L., and Wagner, D. (2012). SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors. Proc Natl Acad Sci U S A 109:3576-3581. Zhu, Y., Rowley, M.J., Bohmdorfer, G., and Wierzbicki, A.T. (2013). A SWI/SNF chromatin-remodeling complex acts in noncoding RNA-mediated transcriptional silencing. Mol Cell 49:298-309.
10
TCP transcription factors
A
GA signaling
Histone octamer
ABA signaling
Floral homeotic MADS factors
SWI/SNF complexes
B
lncRNA-mediated silencing
C Photoperiod pathway
Gibberellin pathway
Autonomous pathway
Vernalization pathway
ABA
Floral pathways integrator
BRM
BRM Floral meristem identity genes
SYD Floral organ identity genes
ABC factors target genes
GA
The many faces of plant SWI/SNF complex Jose C. Reyes
Molecular Biology Department. Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER). Consejo Superior de Investigaciones Científicas (CSIC). Av. Americo Vespucio 41092 Seville. Spain.
Downloaded from http://mplant.oxfordjournals.org/ at Grand Valley State Univ on November 1, 2013
*Corresponding author: José C. Reyes:
Tel.: +34 954467842; fax: + 34 954461664 E-mail address: [email protected]
Running title: Plant SWI/SNF complexes
1
The SWI/SNF complexes In plants, as well as in metazoans, the execution of developmental programs and the response to environmental stimuli depend on changes in gene transcription programs that require extensive chromatin modifications of basically two types: posttranslational modification of histones and ATPdependent reorganization of histones-DNA interactions. Many different nuclear machineries are necessary to carry out chromatin modifications at the appropriate time and place. Yeast SWI/SNF (switch/sucrose nonfermentable) was the first ATP-dependent chromatin-remodelling complex to be described in the 80´s. Since then, SWI/SNF complexes have been found in many eukaryotes, with functions ranging from control of development to genome stability (recently reviewed in (Euskirchen et al., 2012)). About a decade ago, the first Arabidopsis genes encoding orthologs to SWI/SNF complex subunits were reported (Brzeski et al., 1999; Farrona et al., 2004; Wagner and Meyerowitz, 2002). A large body of genetic and biochemical evidence strongly suggests that SWI/SNF complexes are also present in plants (Bezhani et al., 2007; Farrona et al., 2004; Hurtado et al., 2006; Sarnowski et al., 2005). However, since an endogenous complex has not yet been purified from plants, plant SWI/SNF subunits have only been identified based in sequence conservation. As in metazoans, Arabidopsis SWI/SNF subunits are often encoded by more than one gene, thus allowing combinatorial assembly and a diversity of related complexes with different functions, which are now beginning to be elucidated. These complexes are formed by one DNA-dependent ATPase, responsible for the nucleosome remodelling activity, and by several other subunits involved in complex targeting and activity regulation. Two canonical ATPases has been described in Arabidopsis, namely SPLAYED (SYD) and BRAHMA (BRM) (Farrona et al., 2004; Hurtado et al., 2006; Wagner and Meyerowitz, 2002). In addition, two closely-related ATPases, CHR12 (also called MINU1) and CHR23 (also called MINU2), are also present in Arabidopsis (Cong-Cong et al., 2012; Mlynarova et al., 2007; Sang et al., 2012); however, no interactions between these enzymes and the SWI/SNF subunits have been observed to date. Therefore, whether CHR12 and CHR23 are bona fide SWI/SNF subunits is still unclear. It is thought that Arabidopsis SWI/SNF 2
complexes also contain one ortholog of SNF5/INI1, called BUSHY (BSH) (Brzeski
et
al.,
1999),
two
of
the
four
possible
orthologs
of
SWI3/BAF170/BAF155 (SWI3A, SWI3B, SWI3C, and SWI3D) (Sarnowski et al., 2005) and one of the two possible orthologs of SWP73/BAF60 (SWP73A and SWP73B)
(Figure
1A).
Interactions
demonstrated
between
SWI/SNF
components and other factors during the last years have allowed to outline a number of possible complexes (Figure 1A) (Bezhani et al., 2007). In this Research Highlight, I will mostly describe recent advances in the knowledge of the multiple roles that SWI/SNF complexes play in development, phytohormone response and RNA-mediated silencing.
SWI/SNF in development Leaf development is intensely altered in SWI/SNF-deficient plants. Thus, brm and swi3c rosette leaves are strongly curled downwards under long-day conditions and uneven and serrated in short days (Archacki et al., 2009; Farrona et al., 2004; Hurtado et al., 2006; Sarnowski et al., 2005). The groups of Eshed and Wagner recently uncovered a role for BRM and SWI3C as coactivators of TCP4 (TEOSINTE BRANCHED1, CYCLOIDEA, PCF4) (Efroni et al., 2013). While cytokinin (CK) promote cell division and the formation of marginal leaf serrations, TCP4 promotes leaf growth arrest and maturation. BRM interacts with TCP4 (Figure 1A), and together they activate the expression of the ARR16 gene, an A-class ARR factor that inhibits CK signalling. This provides a signal to reduce CK responses in the margins of the lamina and to promote the progressive loss of morphogenetic potential. Efroni et al. did not address the origin of the curled leaves of brm and swi3c plants; however, it is easy to speculate that an uncoupled maturation of adaxial with respect to the abaxial surfaces of the lamina are responsible for the phenotype. Interestingly BRM and SYD positively control expression of CUP-SHAPED COTYLEDON (CUC) genes, at least in the cotyledon boundary region (Kwon et al., 2006). CUC genes are required for leaf serration (Hasson et al., 2011) and CK has been shown to activate CUC genes (Li et al., 2010). Therefore, it is unclear how BRM promotes at the same time reduction of CK response and activation of CUC gene expression. The transition from vegetative to reproductive development also seems to 3
be dependent on the SWI/SNF complexes. In fact, BRM is involved in the regulation of three out of the four flowering pathways in Arabidopsis, namely, the photoperiod, the gibberellin and the autonomous pathways (Archacki et al., 2013; Farrona et al., 2011) (Figure 1B). Consistently, brm mutants display a complex flowering phenotype characterized by early flowering in long days and late flowering in short days. Besides its role in flowering time control, SWI/SNF complexes also affect flower development. Mutations in BRM and SYD provoked subtle floral homeotic transformations and an small reduction in the level of the MADS factors AP3 and AG (Farrona et al., 2004; Hurtado et al., 2006; Wagner and Meyerowitz, 2002; Wu et al., 2012). Since brm syd double mutants are embryonic lethal, the group of Wagner generated a conditional double mutant in flower primordial germ cells (Wu et al., 2012). Interestingly, these plants displayed severe floral homeotic transformations, demonstrating that BRM- and SYD-containing complexes have redundant roles in flower patterning. LFY and SEP3 seems to recruit both ATPases to the promoter regions of AP3 and AG, which is required to counteract the repressive effects mediated by the Polycomb complex. Thus, clf mutations partially suppress the severe defects caused by the combined loss of SYD and BRM. It is worth remembering that brahma mutant in Drosophila was originally identified as a suppressor of homeotic transformations present in Polycomb mutations. Therefore, these data indicate that the Polycomb-Trithorax antagonistic regulation of homeotic genes is conserved between plants and metazoans (Wu et al., 2012), although the structures of these genes are not conserved. This suggests that during evolution, animals and plants have converged to use similar chromatin machineries to control similar functions. Several SWI/SWF subunits have also been shown to co-purify with the five major floral homeotic MADS proteins (AP1, AP3, PI, AG, and SEP3) (Figure 1A), suggesting that the SWI/SNF complexes are coactivators of MADS factor target genes (Smaczniak et al., 2012). Consistently, expression of NAP, an AP3 and PI target gene, was strongly impaired in brm plants (Hurtado et al., 2006). Taken together, these results indicate that SWI/SNF complexes play a dual role in flower development: they first activate transcription of several ABC transcription factors, and then they cooperate with them to regulate downstream targets (Figure 1B). 4
In addition to counteracting Polycomb repression, SWI/SNF complexes also appear to cooperate together with Polycomb complexes to repress transcription. For example, brm mutants display high levels of FLC expression that is accompanied by a reduction in the level of the characteristic Polycombdependent mark H3K27me3 at the FLC promoter (Farrona et al., 2011). On the other hand, BRM, SWI3C and BSH also seem to cooperate with Polycomb to repress seed maturation genes in leaves (Tang et al., 2008).
SWI/SNF in the phytohormone response Several SWI/SNF subunits have been recently involved in abscisic acid (ABA), gibberellic acid (GA), jasmonate (JA), and ethylene (ET) signalling pathways (Archacki et al., 2013; Han et al., 2012; Walley et al., 2008). Jasmonate, and ethylene pathways signal biotic and abiotic stresses, such as wounding and pathogen infections. Walley et al. found that syd mutants present an increased susceptibility to fungal pathogens. Consistently, SYD is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways such as PDF1.2a, VPS2 and MYC2 (Walley et al., 2008). Brm mutants were shown to have a constitutively active abscisic acid (ABA) signalling pathway, and consequently, they displayed hypersensitivity to ABA and enhanced drought tolerance (Han et al., 2012). Expression of the transcription factor ABA INSENSITIVE5 (ABI5) is de-repressed in the absence of ABA and accumulates to high levels upon ABA addition. BRM is required to maintain high occupancy of the +1 nucleosome at the 5´ part of the coding region of the ABI5 locus, but BRM was not involved in the ABA-dependent reorganization of the -1 nucleosome at the promoter around the ABA response elements. This was in agreement with the fact that induction by ABA was not impaired in brm mutants. Interestingly, BRM binds to the ABI5 promoter both in the absence and presence of exogenous ABA, suggesting the possibility that BRM is inactivated in the presence of ABA (Han et al., 2012). It was reported that SWI3B interacted with the negative regulators of ABA signalling, HAB1, ABI1, ABI2 and PP2CA (Figure 1A), but, surprisingly, swi3b hypomorphic mutants displayed a reduced ABA response (Saez et al., 2008). It is worth noting that SWI3C and SWI3B interact with BRM (Hurtado et al., 2006); however, while swi3c and brm mutants display very similar phenotypes 5
(Archacki et al., 2009; Hurtado et al., 2006; Tang et al., 2008), swi3b deficiency provokes different effects, suggesting that BRM and SWI3C are in the same SWI/SNF complexes but that SWI3B is mostly in different complexes. Alternatively, the opposite functions in ABA signalling of BRM and SWI3B may suggest that SWI3B negatively controls the activity of BRM-containing complexes (at least in this pathway), highlighting the importance of the combinatorial subunit composition of the complexes in regulating its activities. Gibberellic acid (GA) counteracts many of the effects of ABA signalling in seed germination and root elongation. Interestingly, brm and swi3c mutants show GA-related growth and developmental defects (Archacki et al., 2013; Sarnowska et al., 2013), at least partially suppressed by GA addition. Consistently, these mutants display a reduced level of GA 4. Jerzmanowski and collaborators have also found that swi3c mutants display low expression of the GA-receptor GID1a and of four DELLA proteins (Sarnowska et al., 2013). Furthermore, SWI3C interacts with several DELLA proteins and with SPY, although the molecular consequences of these interactions are still unknown (Sarnowska et al., 2013). Therefore, it seems that BRM containing SWI/SNF complexes positively influences germination and root growth by acting at two levels: inhibiting ABA signalling and promoting GA response (Figure 1C). However, since GA and ABA pathways reciprocally down-regulate each other, whether GA- and ABA-related brm phenotypes are two faces of the same coin or whether BRM directly regulates both pathways in a coordinated fashion require further investigation.
SWI/SNF in lncRNA-mediated transcriptional silencing RNA-mediated transcriptional silencing pathways in plants involves two classes of noncoding RNAs: small interfering RNAs (siRNAs) and long noncoding RNAs (lncRNA). The mechanism by which lncRNAs silence transcription is still unclear. The group of Wierzbicki has recently reported that the SWI/SNF complex is involved in this silencing mechanism (Zhu et al., 2013). Thus, SWI3B interacts with the lncRNA binding protein IDN2 (Figure 1A). More importantly, transcription of the solo-LTR locus was significantly de-repressed in the swi3b/+, swi3d and syd mutants. In contrast, another silencing target, At2TE78930, was de-repressed in swi3b/+, swi3c, and brm mutants, indicating 6
that different SWI/SNF complexes are involved in silencing specific targets. Derepression is often accompanied by loss of DNA methylation and nucleosome destabilization at the affected locus. Since it has been shown that DNA methylation correlates with nucleosome positioning, Zhu et al. suggest that lncRNAs affect gene silencing by promoting nucleosome positioning with the help of chromatin remodelling complexes; then, maintenance of silencing would be reinforced by a feedback loop between DNA methylation and nucleosome positioning.
Future directions The SWI/SNF complexes affect many different aspects of plant biology, and can play distinct roles in different pathways or even opposite roles in the same pathway. We are still far from understanding how the activity and composition of the complexes are regulated at the molecular level, or what determines whether a complex is an activator or repressor of transcription. To address this, it is essential that we obtain a better understanding of the network of interactions of SWI/SNF subunits with multiple transcription factors, as well as proteomic analyses of the endogenous family of complexes. In this sense a great tool has been recently published by Efroni et al. In this study a total of 400 pairwise interactions by yeast-two-hybrid were found between six SWI/SNF subunits and 210 transcription factors (Efroni et al., 2013). In vivo confirmation of these interactions and its functional consequences will be of paramount importance. Promoter specific chromatin characteristics will also strongly determine the activity of SWI/SNF complexes as activators or repressors. Genome-wide nucleosome mapping studies comparing wild-type with SWI/SNF mutant lines will be essential to understand SWI/SNF chromatin activity. Additional layers of complexity, such as how post-transcriptional modifications alter the activity of the complexes, or the mechanisms by which lncRNAs recruit specific complexes to specific loci, are still unknown. A further challenge will be to understand the interplay between different chromatin machineries. For instance, how can SWI/SNF cooperates with Polycomb complexes to repress seedspecific genes (Tang et al., 2008) yet counteract Polycomb activity in expressing flower homeotic genes (Wu et al., 2012)? Why do brm mutations overcome the requirement of H2A.Z and the SWR1 complex for gene activation 7
(Farrona et al., 2011)? The answers to these intriguing questions that are currently under investigation will provide us with a clearer understanding of the critical roles of SWI/SNF complexes in plant gene regulation.
FUNDING This work was supported by Ministerio de Educación y Ciencia [BFU201123442 and CSD2006-00049]; Junta de Andalucía [P06-CVI-4844] and Fundación Ramón Areces.
ACKNOWLEDGEMENTS I thank S. Farrona for critically reading of the manuscript and discussion.
Figure legends
Figure 1. The many faces of the SWI/SNF complex. A) Protein-protein interaction network of SWI/SNF subunits. Interactions between SWI/SNF subunits are shown as red lines. Interactions of SWI/SNF subunits with other proteins, or between non-SWI/SNF subunits, are shown as blue lines. Proteins of the same family have the same colour. The protein interaction network was visualized using Cytoscape 2.6.3. This interactome combines yeast two-hybrid, pull down, far-western and mass spectrometry and BiFC data from different origins (Bezhani et al., 2007; Efroni et al., 2013; Farrona et al., 2004; Hurtado et al., 2006; Saez et al., 2008; Sarnowska et al., 2013; Sarnowski et al., 2005; Smaczniak et al., 2012; Wu et al., 2012; Zhu et al., 2013), BioGRID 3.2 and our unpublished data). B) BRM and SYD control flower development at several levels. C) Model of control of seed germination and root growth by BRM.
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TCP transcription factors
A
GA signaling
Histone octamer
ABA signaling
Floral homeotic MADS factors
SWI/SNF complexes
B
lncRNA-mediated silencing
C Photoperiod pathway
Gibberellin pathway
Autonomous pathway
Vernalization pathway
ABA
Floral pathways integrator
BRM
BRM Floral meristem identity genes
SYD Floral organ identity genes
ABC factors target genes
GA
