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Arabidopsis IWS1 Interacts with Transcription Factor BES1 and Is Involved in Plant Steroid Hormone Brassinosteroid Regulated Gene Expression

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Arabidopsis IWS1 Interacts with Transcription Factor BES1 and Is Involved in Plant Steroid Hormone Brassinosteroid Regulated Gene Expression Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression Lei Li, Huaxun Ye, Hongqing Guo, and Yanhai Yin1 Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011 Edited by Mark Estelle, The University of California San Diego, La Jolla, CA, and approved January 08, 2010 (received for review August 13, 2009) Plant steroid hormones, brassinosteroids (BRs), regulate essential knowledge about the transcriptional mechanisms by which BES1 growth and developmental processes. BRs signal throughmembrane- and BZR1 regulate gene expression is still limited. localized receptor BRI1 and several other signaling components to IWS1/SPN1 protein was firstly identified in yeast (13, 14). It was regulate the BES1 and BZR1 family transcription factors, which in turn reported to be involved in postrecruitment of RNAPII-mediated control the expression of hundreds of target genes. However, knowl- transcription and required for the expression of several inducible edge about the transcriptional mechanisms by which BES1/BZR1 genes (14). It was also copurified with RNAPII and several tran- regulate gene expression is limited. By a forward genetic approach, scription elongation factors, including Spt6, Spt4/Spt5, and TFIIS we have discovered that Arabidopsis thaliana Interact-With-Spt6 (13, 15, 16). Recent studies indicated that the yeast IWS1/SPN1 (AtIWS1), an evolutionarily conserved protein implicated in RNA poly- recruited Spt6 and the SWI/SNF chromatin remodeling complex merase II (RNAPII) postrecruitment and transcriptional elongation pro- during transcriptional activation (17). Human IWS1 has been cesses, is required for BR-induced gene expression. Loss-of-function shown to facilitate mRNA export and to coordinate Spt6 and the mutations in AtIWS1 lead to overall dwarfism in Arabidopsis, reduced histone methyltransferase HYBP/Setd2 to regulate histone mod- BR response, genome-wide decrease in BR-induced gene expression, ifications (18, 19). IWS1 appears to be essential, as depletion/ and hypersensitivity to a transcription elongation inhibitor. Moreover, reduction of IWS1 gene results in lethality in yeast (14) and sig- AtIWS1 interacts with BES1 both in vitro and in vivo. Chromatin immu- fi ni cant inhibition of human cell proliferation in vitro (20). How- PLANT BIOLOGY noprecipitation experiments demonstrated that the presence of ever, the biological function of IWS1/SPN1 and mechanism of its AtIWS1 is enriched in transcribed as well as promoter regions of the action remain largely elusive. target genes under BR-induced conditions. Our results suggest that In this study, we found that AtIWS1, the yeast and human AtIWS1 is recruited to target genes by BES1 to promote gene expres- counterparts of which are implicated in gene expression processes sion during transcription elongation process. Recent genomic studies after RNAPII recruitment (14, 17–19, 21), interacts with tran- have indicated that a large number of genes could be regulated at steps scription factor BES1 and is required for BR-induced gene after RNAPII recruitment; however, the mechanisms for such regula- expression. Our results therefore establish an important role for tion have not been well established. The study therefore not only AtIWS1 in plant steroid–induced gene expression and provide establishes an important role for AtIWS1 in plant steroid–induced gene insights into the functions of this conserved protein across eukar- expression but also suggests an exciting possibility that IWS1 protein yotes as well as into the mechanism of transcriptional regulation. can function as a target for pathway-specific activators, thereby pro- viding a unique mechanism for the control of gene expression. Results seb1 Suppresses bes1-D Phenotype and Has Reduced BR Response. In plant growth | transcription regulation | transcription elongation | RNA an attempt to identify BES1 downstream signaling components, polymerase II | Spt6 we screened for suppressors of bes1-D gain-of-function mutant. One of the suppressors, bes1-D seb1, was selected for further lant steroid hormones, brassinosteroids (BRs), regulate studies because of its strong suppression of the bes1-D phenotype Pdiverse plant growth and developmental processes such as cell (Fig. 1). seb1 is a single recessive mutation, as the F1 seedlings of elongation, vascular development, photomorphogenesis, repro- bes1-D seb1 and bes1-D cross displayed bes1-D phenotype and F2 duction, and stress responses (1, 2). Unlike animal steroid hor- segregated suppressors showed a 3:1 ratio. Whereas bes1-D mones that bind to nuclear receptors to directly regulate expression seedlings displayed excessive stem elongation, bes1-D seb1 of target genes, BRs function through membrane-localized recep- seedlings had reduced stem elongation (Fig. 1A). Unlike bes1-D, tor BRI1 and several other components to regulate the protein which was resistant to BRZ inhibition in hypocotyl elongation levels and activities of BES1 and BZR1 family transcription factors assays, the bes1-D seb1 double mutant restored BRZ sensitivity, (3–8), which have six members in Arabidopsis. Although knockout suggesting that this suppressor gene is required for the bes1-D of BES1 alone does not give obvious phenotype, knockdown of phenotype (Fig. 1B). BES1 and BZR1 by RNAi leads to a semidwarf phenotype, sug- We next cloned the SEB1 gene by map based cloning (Fig. gesting that this family of proteins function redundantly in BR S1A). The seb1 mutation was first narrowed down to a 60-kb signaling (9). BES1 and BZR1 have atypical basic helix–loop–helix region by a set of flanking markers. Five candidate genes in the (bHLH) DNA binding domain and bind to E-box (CANNTG) and/ interval were then sequenced. A “G” to “A” mutation in Ara- or BRRE (BR Response Element, CGTGT/CG) to regulate BR target gene expression (9, 10). In addition, BES1 interacts with other transcription factors such as BIM1 and AtMYB30 as well as Author contributions: L.L. and Y.Y. designed research; L.L. performed research; L.L. and H.Y. putative histone demethylases ELF6 and REF6 (9, 11, 12). Con- contributed new reagents/analytic tools; L.L., H.Y., and M.G. analyzed data; and L.L., M.G., sistent with the important roles of BES1 in mediating BR signaling, and Y.Y. wrote the paper. the gain-of-function mutant bes1-D, in which BES1 protein accu- The authors declare no conflict of interest. mulates to high level, displays constitutive BR response pheno- This article is a PNAS Direct Submission. types, including excessive cell elongation, resistance to the BR 1To whom correspondence should be addressed. E-mail: [email protected]. biosynthesis inhibitor brassinazole (BRZ), and increased expres- This article contains supporting information online at www.pnas.org/cgi/content/full/ sion of BR-induced genes (7). Despite the emerging findings, 0909198107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0909198107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 BR-Induced Gene Expression Is Decreased in seb1 Mutant. To understand the molecular basis for impaired BR response and growth phenotype of the seb1 mutant, we first tested the expres- sion of several known BR target genes using quantitative RT- PCR. The expression of all four genes tested were significantly reduced in the seb1 mutant and increased in the bes1-D mutant, especially under BL-induced conditions (Fig. 2A). To gain a global view of how many BR-regulated genes are affected in the mutants, we performed microarray analyses using WT, seb1, and bes1-D seedlings (all in En-2 background) with or without BL treatments. These experiments identified 3138 and 3824 differ- entially expressed genes in seb1 and bes1-D, respectively, com- pared with WT (Fig. S5). The substantial overlap (1437 genes) between AtIWS1- and BES1-regulated genes suggests that AtIWS1 plays important roles in the BR signaling pathway. To understand this further, we analyzed in greater depth the genes that were induced by BL and were reduced in seb1 mutant (Fig. 2 B and C). Of the 406 genes that were induced by BL, ≈70% were Fig. 1. seb1 Suppresses bes1-D phenotype and has reduced BR response. (A) up-regulated in bes1-D (Fig. 2C and Table S1). Comparison of the Two-week-old light-grown seedlings. (B) Hypocotyl elongation assay with 2- seb1 and WT gene expression profiles indicated that a large week-old light-grown seedlings in the absence or presence of 1000 nM BRZ. fraction of the BL-responsive genes were also regulated by (C) Two-week-old light-growing seedlings of Col-0, T-DNA insertion line AtIWS1: 31% (127/406) were reduced in seb1 in the presence of atiws1 (SALK_056238) in Col-0 background, En-2, and seb1 single mutant in BL, whereas 16% (66 / 406) were reduced in seb1 in the absence of En-2 background. (D) Hypocotyl elongation assays with 2-week-old light- B – grown seedlings (0 and 100 nM BL), or 5-day-old dark-growing seedlings (0 BL (Fig. 2 and Table S1 Table S3). Most of the BL-induced or 1,000 nM BRZ). A total of 15–20 seedlings were used for each line for each genes that were reduced in seb1 (59/66) were also included in the condition and the difference between mutants and wild-type were sig- 127 genes reduced in seb1 in the presence of BL (Fig. 2B). The nificant, as measured by Student’s t test (P < 0.05). same conclusion can be drawn from the gene clustering analysis with the 406 BR-induced genes (Fig. 2C). These genome-wide analyses revealed that AtIWS1 plays important roles in BL- bidopsis fi gene At1g32130 was identi ed, which created an early stop induced gene expression. codon in the middle of the gene (Fig. S1B). Expression of a genomic To further understand the reduced growth phenotype of seb1, clone of At1g32130 in the bes1-D seb1 mutant complemented the we examined genes implicated in cell elongation (Table 1).
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