Unique, shared, and redundant roles for the Arabidopsis SWI/SNF chromatin remodeling ATPases BRAHMA and SPLAYED

Abstract

Chromatin remodeling is emerging as a central mechanism for patterning and differentiation in multicellular eukaryotes. SWI/SNF chromatin remodeling ATPases are conserved in the animal and plant kingdom and regulate transcriptional programs in response to endogenous and exogenous cues. In contrast with their metazoan orthologs, null mutants in two Arabidopsis thaliana SWI/SNF ATPases, BRAHMA (BRM) and SPLAYED (SYD), are viable, facilitating investigation of their role in the organism. Previous analyses revealed that syd and brm null mutants exhibit both similar and distinct developmental defects, yet the functional relationship between the two closely related ATPases is not understood. Another central question is whether these proteins act as general or specific transcriptional regulators. Using global expression studies, double mutant analysis, and protein interaction assays, we find overlapping functions for the two SWI/SNF ATPases. This partial diversification may have allowed expansion of the SWI/SNF ATPase regulatory repertoire, while preserving essential ancestral functions. Moreover, only a small fraction of all genes depends on SYD or BRM for expression, indicating that these SWI/SNF ATPases exhibit remarkable regulatory specificity. Our studies provide a conceptual framework for understanding the role of SWI/SNF chromatin remodeling in regulation of Arabidopsis development.

Figure 1.

Genomic Expression Studied in brm and syd Null Mutants. (A) Ten-day-old long-day-grown brm-101, syd-2, and wild-type (Ler) seedlings were used for genomic expression studies. Both brm-101 and syd-2 are smaller than the wild type. The size of brm-101 is reduced compared with syd-2, and the two mutants exhibit unique subtle cotyledon and leaf shape abnormalities. (B) and (C) Quantitative real-time PCR analysis of all predicted regulatory gene products (transcription factors and signaling molecules) identified using Rank Product (Breitling et al., 2004; FDR <10%; see also Table 1 and Supplemental Tables 1 to 4 online). Shown are 43 genes that were found to be misregulated by real-time PCR. The mean expression value for two biological replicates and three technical replicates per gene normalized by the value obtained for the ubiquitously expressed eukaryotic translation initiation factor EIF4A is indicated. Error bars denote the se of the mean. For ease of comparison, the value for the wild type (Ler) was set to 1. Genes downregulated in brm-101 and/or syd-2 are shown in (B), and those upregulated in brm-101 and/or syd-2 are shown in (C). (D) Genes identified as misregulated in each mutant as described above were compared in a pairwise fashion. Genes upregulated or downregulated in both mutants are shown in the overlap of the Venn diagrams. Genes misregulated in one mutant only are indicated in the nonoverlapping segments of the Venn diagrams. (E) Comparison of genes preferentially expressed during later stages of seedling development (late) and those preferentially expressed during early seedling development (early) identified from the developmental data set in AtGenExpress (Schmid et al., 2005; see Methods for details) versus those upregulated in both syd-2 and brm-101 and to those downregulated in both syd-2 and brm-101. The total number of late and early genes was 1919 and 2548, respectively. P values are based on a two-tailed Fisher's exact test.

Figure 2.

Phenotypes of syd brm Double Mutants. (A) Siliques of selfed syd brm/++, syd/+, brm/+, and the Ler wild-type plants. Parental genotypes are indicated in each panel. Several misshapen and shrunken seeds are indicated by a red arrow, and unfertilized ovules are marked with an asterisk. (B) Average percentage (mean number plus se of the mean) of misshapen and shrunken seeds in each silique after selfing. Genotypes are indicated below the graph. The total number of seeds counted (n) is shown in Table 4. (C) Representative cleared embryos from a misshapen seed (right) of selfed syd brm/++ siliques and a wild-type-looking sibling (left) from the same silique. Embryos are arrested at the heart stage (right) or earlier (data not shown).

Figure 3.

SYDN Interaction with ATSWI3 Proteins. (A) Top panel: GST pull-down assays using SYDN-GST (S) or GST alone (G) and in vitro transcribed and translated 35S Met–labeled candidate interacting proteins. Autoradiograph of a 12% polyacrylamide gel. Size marker migration is indicated at the left. Interacting proteins tested included ATSWI3A (3A), ATSWI3B (3B), ATSWI3C (3C), ATSWI3D (3D), and LAMIN (LAM). Equal amounts of these proteins were used in each reaction (data not shown). Bottom panel: Quantitation of three independent experiments of the type shown in the top panel, normalized by protein levels. Shown is the mean with se of the mean. (B) Quantitative real-time PCR analysis of ATSWI3 genes compared with SYD in different tissues. The mean and se of the mean of one representative biological replicate with three technical replicates normalized by the value obtained for the ubiquitously expressed eukaryotic translation initiation factor EIF4A are shown. Tissues tested were from plants grown in long-day conditions in soil unless otherwise indicated. Stages harvested were 10-d-old seedlings, 5-d-old roots (vertical half-strength Murashige and Skoog agar plates), second internode from 28-d-old plants, expanding leaves (8th and 9th leaves) from 21-d-old plants, inflorescences (not including fully open flowers) from 35-d-old plants, and elongating siliques from 35-d-old plants.

Figure 4.

Hypothetical SWI/SNF Core Complexes in Arabidopsis. (A) Schematic of all possible protein interactions based on individual protein–protein interactions identified in this study as well as by others (Sarnowski et al., 2002; Hurtado et al., 2006). The protein interaction network was visualized using Cytoscape 2.3.2. The edges (interactions) connecting the nodes (proteins tested) are represented by thick black lines for strong interactions and thin black lines for weak interactions. ATSWI3 proteins are referred to as SWI3A to SWI3D (red circles), BSH is the Arabidopsis SNF5 ortholog (yellow circle), and SYD and BRM are the two SWI/SNF ATPases analyzed (blue circles). (B) The protein interactions depicted in (A) theoretically allow formation of 11 potential SWI/SNF core complexes if the subunit stochiometry is the same as in metazoans (Mohrmann and Verrijzer, 2005). Each core complex consists of the central chromatin remodeling ATPase (SYD or BRM), two SWI3 subunits (ATSWI3), and one SNF5 subunit (BSH). Strong interactions are shown in orange and weaker interactions in yellow. SYD is potentially able to form a unique core complex with ATSWI3A and BSH.