ANGUSTIFOLIA3 Binds to SWI/SNF Chromatin Remodeling Complexes to Regulate Transcription during Arabidopsis Leaf Development

Abstract

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.

SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development

“…Among plants, rice (Oryza sativa) and maize (Zea mays) have one SWP73 variant, while Arabidopsis has two, which suggests a possible functional diversification of this protein in dicots compared with monocots (Supplemental Figure 1A and Supplemental File 1). Arabidopsis SWP73s were recently confirmed to copurify with core subunits of the SWI/SNF complex (Vercruyssen et al, 2014). We used yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays to analyze the interactions of SWP73A and SWP73B variants with core components of SWI/ SNF complexes including SWI3A, SWI3B, SWI3C, SWI3D, and BUSHY (BSH) (Figure 1).…”

“…SWI/SNF, consisting of an SNF2 ATPase, two SWI3s, and an SNF5 subunit, is capable of nucleosome disruption through facilitation of reversible transition between normal (inaccessible) and altered (more accessible) conformations (Narlikar et al, 2002). In Arabidopsis thaliana, SWI/SNF complexes are involved in modulating various developmental and regulatory processes, including both vegetative and generative development, flowering time, and hormonal signaling (Sarnowski et al, 2005;Bezhani et al, 2007;Han et al, 2012;Archacki et al, 2013;Efroni et al, 2013;Sarnowska et al, 2013;Vercruyssen et al, 2014).…”

“…SWP73B was identified as an interacting partner of cyclin-dependent kinase inhibitor KRP5 (KIP-RELATED PROTEIN5), suggesting a role in controlling the cell cycle and endoreduplication (Jégu et al, 2013). Association of SWI/SNF subunits SWI3C, SWI3D, and SWP73B with AN-GUSTIFOLIA3 (AN3), recruitment of SWP73B to promoters of AN3-regulated target genes GROWTH-REGULATING FACTOR3 (GRF3), GRF5, HECATE1 (HEC1), CONSTANS-LIKE5 (COL5), CYTOKININ RESPONSE FACTOR2 (CRF2), and RESPONSE REGULATOR4, and altered expression of these genes in the brahma (brm) SNF2-ATPase mutant suggest an important role of SWI/SNF complexes in the regulation of leaf development (Vercruyssen et al, 2014). Another major role of SWI/SNF complexes in flowering time control is indicated by the recent finding that SWP73B (BAF60) is involved in chromatin loop formation of the flowering time repressor FLOWERING LOCUS C (FLC) gene (Jégu et al, 2014).…”

Combined Large-Scale Phenotyping and Transcriptomics in Maize Reveals a Robust Growth Regulatory Network

“…In addition to the covalent histone modifications, chromatin remodeling also depends on ATP-dependent chromatin remodeling complexes that move, eject, or restructure nucleosomes. For instance, the transcription of GRFs is regulated by recruitment of the SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes to their promoters by the transcriptional coactivator AN3 (Vercruyssen et al, 2014;Nelissen et al, 2015). The phenotypes upon differential expression of AN3, BRAHMA, or SWI/SNF-ASSOCIATED PROTEIN73B, subunits of the SWI/SNF complex, reflect the importance of this chromatin-remodeling complex in the regulation of leaf growth (Farrona et al, 2004;Horiguchi et al, 2005;Vercruyssen et al, 2014), and constitutive overexpression of GRF1 and GRF2 also increased leaf and cotyledon size (Kim et al, 2003).…”

Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues

“…In contrast to Y2H and PCA, AP-MS has been traditionally unable to detect transient interactions, which are often lost during the washing steps necessary to remove nonspecific binding. The technique already proved its merits though, in various plant species (Arabidopsis, rice, petunia, tomato, tobacco) for different cellular processes, including the cell cycle (Van Leene et al 2010), flowering (Smaczniak et al 2012), leaf development (Vercruyssen et al 2014) and endocytosis (Gadeyne et al 2014). …”