Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression

Abstract

Plant steroid hormones, brassinosteroids (BRs), regulate essential growth and developmental processes. BRs signal through membrane-localized receptor BRI1 and several other signaling components to regulate the BES1 and BZR1 family transcription factors, which in turn control the expression of hundreds of target genes. However, knowledge about the transcriptional mechanisms by which BES1/BZR1 regulate gene expression is limited. By a forward genetic approach, we have discovered that Arabidopsis thaliana Interact-With-Spt6 (AtIWS1), an evolutionarily conserved protein implicated in RNA polymerase II (RNAPII) postrecruitment and transcriptional elongation processes, is required for BR-induced gene expression. Loss-of-function mutations in AtIWS1 lead to overall dwarfism in Arabidopsis, reduced BR response, genome-wide decrease in BR-induced gene expression, and hypersensitivity to a transcription elongation inhibitor. Moreover, AtIWS1 interacts with BES1 both in vitro and in vivo. Chromatin immunoprecipitation experiments demonstrated that the presence of AtIWS1 is enriched in transcribed as well as promoter regions of the target genes under BR-induced conditions. Our results suggest that AtIWS1 is recruited to target genes by BES1 to promote gene expression during transcription elongation process. Recent genomic studies have indicated that a large number of genes could be regulated at steps after RNAPII recruitment; however, the mechanisms for such regulation have not been well established. The study therefore not only establishes an important role for AtIWS1 in plant steroid-induced gene expression but also suggests an exciting possibility that IWS1 protein can function as a target for pathway-specific activators, thereby providing a unique mechanism for the control of gene expression.

Fig. 1.

seb1 Suppresses bes1-D phenotype and has reduced BR response. (A) Two-week-old light-grown seedlings. (B) Hypocotyl elongation assay with 2-week-old light-grown seedlings in the absence or presence of 1000 nM BRZ. (C) Two-week-old light-growing seedlings of Col-0, T-DNA insertion line atiws1 (SALK_056238) in Col-0 background, En-2, and seb1 single mutant in En-2 background. (D) Hypocotyl elongation assays with 2-week-old light-grown seedlings (0 and 100 nM BL), or 5-day-old dark-growing seedlings (0 or 1,000 nM BRZ). A total of 15–20 seedlings were used for each line for each condition and the difference between mutants and wild-type were significant, as measured by Student’s t test (P < 0.05).

Fig. 2.

BR induced gene expression is decreased in seb1 mutant. (A) Quantitative RT-PCR was performed with 2-week-old seedlings treated with or without 1,000 nM BL. The gene expression levels were significantly reduced in seb1 and increased in bes1-D as tested by two-way ANOVA F test (P < 0.01). The average and standard deviations were from three biological repeats. (B) Venn diagram shows the overlap genes among different groups. The lists of BL-induced genes and those reduced in seb1 with or without BL treatments are presented in Table S1, Table S2, and Table S3 (C) Clustering analysis of 406 BL-induced genes by GeneSpring program. Yellow rectangles indicated genes reduced in seb1 in the presence of BL.

Fig. 3.

AtIWS1 interacts with BES1 both in vitro and in vivo. In yeast two-hybrid experiments to detect direct protein–protein interactions, β-galactosidase (LacZ) activity was detected with X-gal (A) or with ONPG in a quantitative liquid-culture assay (B). (C) GST pull-down experiments using MBP-AtIWS1 and GST tagged full-length or truncated BES1. Full-length BES1 includes a DNA binding domain (DNA BD), BIN2 phosphorylation domain (Phospho), PEST motif, and the C-terminal domain. AtIWS1 was detected by a Western blot with anti-MBP antibody. (D) Cotransformation of Arabidopsis protoplasts with BES1-cYFP and AtIWS1-nYFP lead to the reconstitution of YFP activity in the nuclei, as indicated by arrows. (E) Cotransformation of cYFP and AtIWS1-nYFP did not produce any positive signals.

Fig. 4.

AtIWS1 is likely involved in transcription elongation. (A) Yeast-two-hybrid assays to detect interactions between AtIWS1 or AtIWS1 C-terminal (IWS1 domain) and Arabidopsis Spt6 (At1g65440) N-terminal domain. (B) Four-week-old plants in the media with indicated concentrations (mg/L) of 6-AU. The germination rates were determined after 3-week of culture under light. The assay was repeated three times, and representative results are shown. (C) ChIP using bes1-D seedlings with anti-BES1 antibody and control IgG. (D) ChIP using AtIWS1-TAP transgenic and WT seedlings. The 5srRNA was used as internal control for C and D. The average and standard deviations were derived from three to four biological replicates. (E) A model for AtIWS1 function in BES1 mediated gene expression.