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. 2014 Jan;26(1):210-29.
doi: 10.1105/tpc.113.115907. Epub 2014 Jan 17.

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

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

Liesbeth Vercruyssen et al. Plant Cell. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
Induction of AN3 Activity Enhances Leaf Growth and CYCB1;1 Expression. (A) to (C) Twenty-one-day-old plants, grown on control medium (Mock) or medium supplemented with DEX. (A) Leaf area of cotyledons (Cot) and leaves 1 to 8 (L1 to L8), measured from leaf series. Error bars are se (n ≥ 12). Asterisks indicate significant difference from the wild type (Col-0) (P < 0.01, Student’s t test). (B) Rosettes. (C) Cotyledons and leaves 1 and 2. Bars = 10 mm in (B) and (C). (D) to (F) GUS staining of CYCB1;1:DB-GUS and AN3-GR/CYCB1;1:DB-GUS leaves 1 and 2. Plants were transferred at 9 DAS to mock medium or medium supplemented with DEX for 24 h. (D) GUS-stained and nonstained regions, indicating the division and expansion zones, respectively, measured along the length of the leaf. Error bars are se (n ≥ 22). Asterisks indicate significant difference from DEX-treated control plants (P < 0.01, Student’s t test). (E) Leaves 1 and 2 were mounted on slides for picture taking. Bar = 1 mm. (F) GUS staining was measured with Image J in a defined area along the leaf length. Error bars are se (n ≥ 18).
Figure 2.
Figure 2.
Identification of Transcription Factors Rapidly Regulated by AN3, by Time-Course Analysis of Expression Levels. (A) Wild-type and AN3-GR plants were grown for 8 d and transferred to medium supplemented with 5 µM DEX for 8 h. The number of upregulated and downregulated genes with P value < 0.05 is shown, and differentially expressed transcription factors (TFs) are indicated in parentheses. (B) Transcription factors differentially expressed in AN3-GR leaves 1 and 2 compared with wild-type leaves 1, 2, 4, or 6 h after DEX treatment. qRT-PCR expression levels were normalized to DEX-treated wild-type expression levels, which are set at 100% for each time point. Error bars are se of three biological replicates. Asterisks indicate significant difference from DEX-treated wild-type plants (P < 0.1, Student’s t test).
Figure 3.
Figure 3.
Genome-Wide Determination of AN3 Binding Sites and Identification of Direct AN3 Target Transcription Factors by TChAP-Seq and ChIP-qPCR. (A) Genome-wide distribution of the location of the peaks called by MACS (Zhang et al., 2008), after TChAP on AN3-HBH–transformed cell cultures followed by sequencing. (B) Distance in base pairs of the peak summits relative to the translation start site (TSS) of the nearest gene. Peak summits (2040) were included which are located closest to 5′ gene ends. (C) GenomeView representation (Abeel et al., 2012) of the TChAP-seq results for CRF2, COL5, and HEC1 loci, showing read coverage in the TChAP-purified AN3-HBH versus the wild-type control samples. The reads are piled up with forward reads above the axis displayed in green and reverse reads below the axis in blue. Total coverage is indicated in yellow. Scaling was done relative to the maximum number of reads. The coding regions are indicated as pink boxes, the UTRs as black boxes, and the peaks as purple boxes above the reads. The regions amplified with ChIP-qPCR are indicated in light blue. Bar = 0.4 kb. (D) ChIP with anti-IgG and anti-GFP antibody on 14-d-old plants expressing GFP-tagged AN3. Enrichment was determined with qPCR and for each locus normalized against the input. For diagrams of the loci including the amplified regions, see Supplemental Figure 9. Error bars are sd of two biological replicates. Asterisks indicate significant difference from wild-type plants (P < 0.05, Student’s t test).
Figure 4.
Figure 4.
Tandem Affinity Purification Reveals Interaction of AN3 with SWI/SNF Complexes. (A) Images of denaturing gels of TAP experiments with C-terminal GS-tagged AN3 and N-terminal GS-tagged SWI3C and SWP73B in cell cultures. Only interactors that could be distinguished as a band are indicated. (B) Cytoscape (Shannon et al., 2003) protein interaction networks are based on the TAP experiments shown in Table 2. Pink nodes indicate proteins used as bait and purple nodes those that were pulled down with at least three out of five baits. Paralogous proteins identified by AN3 TAP are represented by blue nodes. Black edges are used when the proteins were identified by at least three baits and green edges for proteins identified by one or two bait proteins.
Figure 5.
Figure 5.
AN3 Is Essential for Optimal Binding of SWP73B to a Subset of their Target Promoters. ChIP with anti-IgG and anti-GFP antibody on Col-0 plants expressing CFP-tagged SWP73B (A) and with anti-GFP antibody on Col-0 (WT) and an3 mutants expressing CFP-tagged SWP73B (B). Enrichment was determined with qPCR and for each locus normalized against the input. In addition, for (B), the enrichment in the wild type was set arbitrarily to 1. For diagrams of the loci including the amplified regions, see Figure 3D and Supplemental Figure 9. Error bars are sd (n = 2). Asterisks indicate significant difference from wild-type plants (P < 0.05, Student’s t test).
Figure 6.
Figure 6.
BRM Is Involved in Regulation of Transcription of AN3 Target Genes and Genetically Interacts with AN3. (A) Expression levels determined by qRT-PCR in brm3 rosettes of 8-d-old plants grown in long-day (16 h light/8 h dark) conditions and in brm1 rosettes of 22-d-old plants grown in short-day (8 h light/16 h dark) conditions. Normalization of expression levels was done relative to those of the wild type (Col-0), which are set at 100% for each gene. Error bars are se (n = 3). Asterisks indicate significant difference from wild-type plants (P < 0.1, Student’s t test). (B) Leaf series of 24-d-old wild-type, brm1, an3, and an3 brm1 plants grown in long-day conditions. Bar = 10 mm. (C) Rosettes of brm3, brm1, the wild type, an3, and an3 brm1 plants at 22 DAS. Bar = 10 mm. (D) Scanning electron microscopy pictures of the abaxial epidermis of 22-d-old leaves 1 and 2 of wild-type, an3, brm1, and an3 brm1 plants. Examples of small nonlobed cells in brm1 and an3 brm1 leaves are indicated by arrowheads. Bar = 150 µm. (E) Leaf size, pavement cell sizes, and pavement cell numbers of 22-d-old leaves 1 and 2. Nonlobed and lobed cells are defined as follows: nonlobed cells < 25 µm2 < lobed cells. Normalization was done relative to the wild type (Col-0), which is set at 100% for each measurement. Error bars are se (n = 5). Single asterisks indicate significant difference from wild-type plants, and double asterisks indicate significant difference from brm1 plants (P < 0.01, Student’s t test).
Figure 7.
Figure 7.
Overexpression of SWI3C Enhances Leaf Growth. (A) Total rosette area calculated from individual leaf sizes from 21-d-old 35S:SWI3C plants. Error bars are se (n ≥ 10). Asterisks indicate significant difference from the wild type (Col-0) (P < 0.05, Student’s t test). (B) Individual cotyledon (Cot) and leaf areas (L1 to L10) measured from leaf series made at 21 DAS from plants with increased leaf growth indicated in (A). Error bars are se (n = 8). Asterisks indicate significant difference from the wild type (Col-0) (P < 0.05, Student’s t test). (C) and (D) Pavement cell area (C) and pavement cell number (D) of 21-d-old leaves 1 and 2. Error bars are se (n = 6). Asterisks indicate significant difference from the wild type (Col-0) (P < 0.01, Student’s t test). (E) Rosettes of 15-d-old Col-0 and 35S:SWI3C lines showing enhanced leaf growth. Bar = 10 mm.
Figure 8.
Figure 8.
Model for AN3 Mode of Action. AN3 associates with SWI/SNF chromatin remodeling complexes formed around a central ATPase, BRM or SYD, including SWP73A or SWP73B, SWI3C and/or SWI3D, and ARP4 and ARP7. The presence of BSH remains to be elucidated, and other putative subunits are depicted as light-gray circles. AN3 binds GRFs or possibly other, yet to be identified, transcription factors to recruit chromatin remodeling activity to induce or repress expression of downstream target genes.

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