Genome Analysis
Genome-Wide Analysis of Ethylene-Responsive Element Binding Factor-Associated Amphiphilic Repression
Motif-Containing Transcriptional Regulators
in Arabidopsis1[W][OA]
Sateesh Kagale,Matthew G.Links,and Kevin Rozwadowski*
Saskatoon Rearch Centre,Agriculture and Agri-Food Canada,Saskatoon,Saskatchewan,Canada S7N0X2
The ethylene-responsive element binding factor-associated amphiphilic repression(EAR)motif is a transcriptional regulatory motif identified in members of the ethylene-responsive element binding factor,C2H2,and auxin/indole-3-acetic acid families of transcriptional regulators.Sequence comparison of the core EAR motif sites from the proteins revealed two distinct conrvation patterns:LxLxL and DLNxxP.Proteins containing the motifs play key roles in diver biological functions by negatively regulating genes involved in developmental,hormonal,and stress signaling pathways.Through a genom
e-wide bioinformatics analysis,we have identified the complete repertoire of the EAR repressome in Arabidopsis(Arabidopsis thaliana) comprising219proteins belonging to21different transcriptional regulator families.Approximately72%of the proteins contain a LxLxL type of EAR motif,22%contain a DLNxxP type of EAR motif,and the remaining6%have a motif where LxLxL and DLNxxP are overlapping.Published in vitro and in planta investigations support approximately40%of the proteins functioning as negative regulators of gene expression.Comparative quence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential posttranslational modification sites that may influence the functionality of the EAR motif.Homology arches against protein databas of poplar(Populus trichocarpa), grapevine(Vitis vinifera),rice(Oryza sativa),and sorghum(Sorghum bicolor)revealed that the EAR motif is conrved across the diver plant species.This genome-wide analysis reprents the most extensive survey of EAR motif-containing proteins in Arabidopsis to date and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation.
Plants respond to various developmental and envi-ronmental cues by regulating gene expression at the transcriptional and posttranscriptional levels.Gene regulation at the transcriptional level is orches
trated by a complex and coordinated network of activators, repressors,coactivators,and corepressors.In general, there has historically been greater rearch focus on transcriptional activation mechanisms and positive control of gene regulation.In comparison,relatively little is known about transcriptional repression and negative regulation of gene expression.Transcrip-tional repression mechanisms are currently under inten investigation,and results obtained in recent years have convincingly shown that transcriptional repression is a major regulatory mechanism and plays a key role in many biological process(for review,e Thiel et al.,2004).Transcriptional repressors are basi-cally classified as passive or active repressors(Hanna-Ro and Hann,1996).Passive repressors do not posss an intrinsic repression domain and inhibit activation of transcription by competing with tran-scriptional activators for cognate DNA-binding sites or by directly interacting with them to form inactive heterodimers.Converly,active repressors generally contain a distinct,small,and portable repression do-main(s)that inhibits the activation of transcription either by interacting with components of basal tran-scription machinery or positive transcriptional regu-lators and/or by recruiting histone deacetylas (HDACs),which modify chromatin structure and pre-vent other transcriptional activators from binding to their target cis-elements(Hanna-Ro and Hann, 1996;Pazin and Kadonaga,1997).A repression do-main was initially identified almost two decades ago in the Drosophila active repressors Engrailed(Han and Ma
nley,1993)and Kruppel(Licht et al.,1994).Since then,numerous active repression domain-containing proteins have been reported in yeast,Drosophila,and animals(Hanna-Ro and Hann,1996),but in plants only a few active repression domains esntial for transcriptional repression have been identified(Ohta et al.,2001;Matsui et al.,2008;Ikeda and Ohme-Takagi,2009).
1This work was supported by Agriculture and Agri-Food Canada (grant to K.R.).
*Corresponding author;a.
The author responsible for distribution of materials integral to the findings prented in this article in accordance with the policy described in the Instructions for Authors(www.plantphysiol)is: Kevin a).
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www.plantphysiol/cgi/doi/10.1104/pp.109.151704
A subt of class II ethylene-responsive element binding factors(ERFs)and TFIIIA-type zincfinger pro
teins(ZFPs)in various plant species,including Arabidopsis(Arabidopsis thaliana),tobacco(Nicotiana tabacum),wheat(Triticum aestivum),petunia(Petunia hybrida),and soybean(Glycine max),have been identi-fied as active repressors(Ohta et al.,2001;Zhang et al.,2010).The repression domain located in their C-terminal region is designated as the ERF-associated amphiphilic repression(EAR)motif and contains a conrved connsus quence of L/F DLN L/F(x)P (Ohta et al.,2001).The complete deletion of the C-terminal EAR motif region or targeted mutation of Asp residue within the EAR motif of NtERF3,an ERF protein from tobacco,completely abolishes its capacity for repression of transcription(Ohta et al.,2001). Similarly,the deletion of C-terminal EAR motif regions of Zinc Finger of Arabidopsis10(ZAT10)and ZAT11 results in complete loss of their repression activity (Ohta et al.,2001),confirming an esntial role for the EAR motif in the repressor activity of the proteins. Several other ERFs and ZFPs containing an EAR motif function as transcriptional repressors and are known to play roles in hormone signaling,dia resistance, and abiotic stress resistance(Fujimoto et al.,2000; Sakamoto et al.,2004;McGrath et al.,2005;Song et al., 2005;Yang et al.,2005;Kazan,2006;Jiang et al.,2008).
A quence similar to the EAR motif in ERFs and ZFPs is also found in the C-terminal region of SUPER-MAN(SUP),a negative regulator of transcription involved inflower development(Sakai et al.,1
995). SUP contains a quence(QDLDLELRLGFA)that when fud to the GAL4DNA-binding domain con-verts it into a dominant repressor(Hiratsu et al.,2002, 2003).Transient expression assays demonstrated that the repression domain of SUP,when tethered to a GAL4DNA-binding domain,had approximatelyfive times stronger repressive activity than the EAR motif of ERFs(Hiratsu et al.,2002).The minimal functional unit responsible for the repressive activity of SUP was identified as an amphiphilic motif compod of six amino acids,DLELRL(Hiratsu et al.,2004),which is similar to the EAR motif found in ERFs and ZFPs. Mutational analysis within the DLELRL hexapeptide motif revealed that the Leu residues within this motif are esntial and sufficient for strong repressive activ-ity(Hiratsu et al.,2004).
The AUXIN/INDOLE-3-ACETIC ACID(AUX/ IAA)proteins,another group of active repressors comprising29proteins in Arabidopsis,also contain a LxLxL type of motif that bears remblance to the EAR motif from SUP,some ERFs,and ZFPs(Tiwari et al., 2001,2004).The repression motifs within AUX/IAAs have been shown to confer repression of primary auxin-responsive genes,and the Leu residues within the hexapeptide motif were found to be crucial for their repressive activity(Tiwari et al.,2001,2004).
A recent study(Szemenyei et al.,2008)has further confirmed the role of the EAR motif in suppressing
cgtn直播在线观看auxin-regulated gene expression during embryonic apical-basal cell fate determination.Perhaps the most interesting discovery of this study was demonstrating the direct interaction of the EAR motif of INDOLE-3-ACETIC ACID INDUCIBLE12(IAA12),an AUX/ IAA protein,with the CTLH(C-Terminal to LISH) domain of TOPLESS(TPL),a member of the Groucho/ Tup1family of corepressors.Genetic evidence sug-gests that TPL works in conjunction with HISTONE DEACETYLASE19(HDA19)during the transition stage of embryogenesis(Long et al.,2006).Consider-ing the general role of HDACs in suppressing gene expression by deacetylation of Lys residues on his-tones,it appears likely that TPL may facilitate EAR motif-mediated gene regulation through chromatin modification.
Apart from ERFs,ZFPs,and AUX/IAAs,veral other repressor proteins,including Arabidopsis MYB DOMAIN PROTEIN4(AtMYB4)and AtMYB32be-longing to the MYB family(Jin et al.,2000;Preston et al.,2004),HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2(HSI2)and related proteins of the ABSCISIC ACID-INSENSITIVE3/VIVIPAROUS1(ABI3/ VP1)family(Tsukagoshi et al.,2005),AGAMOUS-LIKE15(AGL15)of the MADS(for MCM1,AGA-MOUS,DEFICIENS,and SRF[rum respon factor]) family(Hill et al.,2008),and NIM1-INTERACTING1 (NIMIN1;Weigel et al.,2005),have been reported to contain EAR-like motifs.Among the,AtMYB4and AtMYB32are required for normal pollen development (Preston et al.,2004).In addition,AtMYB4also plays a role in t
he development of UV light-protecting sun-screens(Jin et al.,2000).The B3domain and EAR motif-containing proteins such as HSI2and related proteins are involved in the repression of ed matu-ration genes during the transition from ed matura-tion to edling growth(Tsukagoshi et al.,2007).The MADS factor protein AGL15along with AGL18is also known to act as a repressor offloral transition in Arabidopsis(Adamczyk et al.,2007).NIMIN1inter-acts with NONEXPRESSOR OF PR GENES1(NPR1),a key regulator of systemic acquired resistance in Arabi-dopsis,and modulates the expression of pathogenesis-related(PR)genes(Weigel et al.,2005). Considering the diver t of biological traits that the currently known EAR motif-containing proteins influence and their distribution across multiple fami-lies,it is reasonable to hypothesize that the EAR motif is a distinct regulatory motif that is utilized by many additional transcriptional repressor proteins involved in different signaling pathways.From this perspective, a genome-wide survey in Arabidopsis would aid in the identification of the complete repertoire of EAR motif-containing proteins and thereby shed light on the transcriptional regulatory mechanisms influenced by them.In this study,we report the identification, classification,and structural analysis of219Arabidop-sis EAR motif-containing proteins through a system-atic genome-wide bioinformatics analysis.Alignment of the large data t of EAR motifs prented here enabled the identification of additional conrved
Kagale et al.
amino acids proximal to the EAR motif,of which some residues may provide a means for posttranslational modification and regulation of the function of EAR motifs.Putative orthologs for each of the proteins are identified from other plant species,such as poplar (Populus trichocarpa),grapevine(Vitis vinifera),rice (Oryza sativa),and sorghum(Sorghum bicolor),and evidence supporting the broad evolutionary conr-vation of EAR motifs is provided.Finally,the mech-anism of action of EAR motif-containing proteins and their u in generating dominant-negative chimeric repressors are discusd.
RESULTS
Identification of EAR Motif-Containing Proteins
in Arabidopsis
A survey of published literature identified49EAR motif-containing proteins(Supplemental Table S1)be-longing to various transcription factor(TF)and other transcriptional regulator(OTR)families such as ERFs (eight proteins;Ohta et al.,2001;McGrath et al.,2005; Yang et al.,2005),ZFPs(four proteins;Ohta et al.,2001; Sakamoto et al.,2004;Mittler et al.,2006),ABI3/VP1 family(three proteins;Tsukagoshi et al.,2005,2007), MY
B family(five proteins;Jin et al.,2000;Preston et al., 2004),AUX/IAA family(28proteins;Tiwari et al.,2004), Homeobox family(one protein;Kieffer et al.,2006), MADS family(one protein;Hill et al.,2008),and NPR family(one protein;Weigel et al.,2005).The protein quence comparison of the proteins in the con-rved EAR motif region(Fig.1)revealed two distinct degenerate connsus quence patterns:DLNxxP (where X can be one of the20common amino acids), consisting of a conrved DLN box and a Pro residue at thefifth or sixth position,wherein the Asp and Leu residues are implicated in repression activity(Ohta et al.,2001;Tsukagoshi et al.,2005);and LxLxL,con-sisting of three conrved Leu residues in alternate positions,which have been shown to be critical for both repressor activity and interaction with corepres-sors(Hiratsu et al.,2004;Tiwari et al.,2004;Szemenyei et al.,2008).To gain further insight into the potential utilization of the EAR motif in plant gene regulation, we conducted a genome-wide analysis of Arabidopsis to identify novel EAR motif-containing proteins.We generated an initial collection by performing a non-weighted degenerate pattern matching(DPM)analy-sis of the Arabidopsis proteome using quence patterns DLNxxP or LxLxL as queries(Yan et al., 2005).This arch identified187and5,471nonredun-dant proteins containing at least one occurrence of the DLNxxP or LxLxL type of motif,respectively,within their protein quences(Supplemental Table S2).Only 73out of the187group of proteins and352out of the 5,471group of proteins were found to be potential transcriptional regulators bad on The Arabidopsi
十六大报告全文s Information Resource(TAIR)Gene Ontology(GO)annotations,providing an initial t of398(consider-ing an overlap of27proteins between the187and352 protein groups)candidate transcriptional regulators potentially employing an EAR motif to influence gene expression.Since the evidence from the current liter-ature suggests a unique role for the EAR motif in transcriptional repression,identification of this motif in a large number of nontranscriptional regulator pro-teins(approximately5,200proteins)by DPM analysis reflects the possibility that the connsus quence of EAR motifs identified in many of the proteins in the initial data ts may not function as transcriptional repressors.The relatively short and partially degener-ate quences of the LxLxL type of EAR motif might have contributed to the identification of fal positives using the initial analysis parameters.To asss the incidence of fal positives,we performed additional DPM arches using control quence patterns de-rived from LxLxL by replacing Leu residues with one of the remaining19amino VxVxV).Except for using SxSxS as a query employing the structurally dissimilar Ser,all other pattern arches returned substantially fewer hits compared with LxLxL(Sup-plemental Table S2);notably,the query patterns with similar hydrophobic residues Ile and Val returned a much lower number of hits(approximately9%and 22%,respectively)than LxLxL.However,the recovery of hits with the alternate amino acid patterns suggested that the LxLxL pattern may occur to some degree unrelated to its function in regulating gene expression. Therefore,we concluded that th
e nonrandom and potentially functional EAR repressome in Arabidopsis is possibly smaller than the approximately400tran-scriptional regulators originally identified by DPM analysis.
In an attempt to identify bonafide EAR motif-containing proteins in Arabidopsis,we employed a strategy containing hidden Markov model(HMM; Eddy,1998)analysis combined with pattern hit-initi-ated BLAST(PHI-BLAST;Zhang et al.,1998;Fig.2)in arching the Arabidopsis proteome.Becau of sound statistical foundation,as well as modularity andflex-ibility to incorporate prior knowledge into the model architecture,HMM-bad strategies have been rou-tinely and reliably employed in genome-wide arches for target proteins,motifs,and domains.The protein quences of the t of49known EAR motif-contain-ing proteins derived from a literature survey(Fig.1) were grouped into six subgroups bad on motif type (DLNxxP or LxLxL)and location(N-terminal[N], middle[M],or C-terminal[C]region).To build HMM profiles,protein quences were truncated to an ap-proximately30-amino acid region of each protein comprising the core EAR motif site and adjoining quence of612amino acids.The subgrouping of proteins by location of the EAR motif was carried out to minimize quence variations within the EAR motif region that may be due to the relative positions of EAR motif sites in their protein quences.The rationale for including adjoining quences in HMM analysis was护理学专业排名
The Arabidopsis EAR Repressome
bad on the assumption that the quences flanking the EAR motif may posss additional signals required for its proper function,which may be newly identified by our whole proteome analysis.A role for flanking quences in transcriptional repression by the EAR motif has been demonstrated previously;for example,an Ala substitution for Thr or Glu residues at 22or 23positions of the EAR motif in IAA17resulted in partial loss of its transcriptional repression activity (Tiwari et al.,2004).The individual HMM profiles generated from the six subgroups of quences were ud to scan the Arabidopsis proteome for putative EAR motif-containing proteins via HMMER (HMM software for biological quence analysis).The HMM hits with a score of 5or greater were manually inspected and included in further steps if they appeared to be bona fide EAR motif-containing proteins bad on two criteria:(1)the prence of an amphiphilic pentapep-tide (LxLxL)and/or hexapeptide (DLNxxP)EAR mo-tif(s);and (2)the prence,bad on GO annotation or literature evidence,of a well-defined DNA-binding domain or prior knowledge of interaction with pro-teins containing a DNA-binding domain,suggesting a role in transcriptional regulation.The first iteration of HMM analysis recovered 33novel EAR motif-contain-ing proteins in addition to the proteins ud for training HMM profiles.In the next step,quences of the novel proteins and the t of 49EAR motif-containing pr
定语从句教案
oteins derived from our literature survey (Fig.1)were ud to perform PHI-BLAST analysis (Zhang et al.,1998).This step was carried out to help ensure that no distantly related homologs of EAR motif-containing proteins were misd.The PHI-BLAST hits with an E-value significantly below thresh-old were manually assd for the prence of an EAR motif.The novel positive hits recovered by PHI-BLAST and the first iteration of HMM analysis were reincorporated into the six subgroups of EAR motif-containing proteins classified by type and location of EAR motif,and the HMM profiles were refined.Sub-quent iterations of HMM/PHI-BLAST analysis were carried out until no more novel EAR motif-containing proteins were identified.Combined,the iterative HMM/PHI-BLAST strategy identified an additional 209EAR motif-containing proteins,out of which 170proteins have been previously annotated as transcrip-tional regulators and the remaining 39proteins were either ambiguously annotated or lacked prior
knowl-
机械工程及自动化就业前景
沪江论坛Figure 1.Arabidopsis EAR motif-containing proteins described in the literature.The 49proteins are divided into two groups bad on the quence conrvation pattern within the core EAR motif sites (high-lighted in color).The alignment includes 12amino acid residues upstream and downstream of the EAR motif,or up to where the nominal 12-amino acid quence is abridged by encountering the first or last amino acid of the protein.A,The DLNxxP motif is conrved in some members of class II ERFs,TFIIIA-type ZFPs,and ABI3/VP1family proteins.B,The LxLxL motif is conrved in AUX/IAAs and some members of the MYB and HD-Zip family proteins.Sequence logos (Crooks et al.,2004)illustrating the frequency of amino acids within the EAR motifs are prented below the respective alignments.
Kagale et al.
edge of their involvement in transcriptional regula-tion.Since the focus of our study was to identify transcriptional regulators containing bona fide EAR motifs,only 170proteins were added to the list of 49EAR motif-containing proteins that were previously validated in the literature.The remaining 39proteins lacking transcription-related annotation may not func-tion as transcriptional repressors an
interest的用法d were hence considered as fal positives.Unlike DPM analysis (fal discovery rate [FDR]=0.92),only a small pro-portion of the predictions made by HMM/PHI-BLAST analysis were found to be fal positives (FDR =0.15).Overall,this analysis identified 219distinct,nonredun-dant Arabidopsis EAR motif-containing transcription-related proteins (Table I;Supplemental Table S3).
A comparison between this t of 219proteins and the protein t identified by DPM analysis revealed that the DPM arch data t has 179additional transcriptional regulator proteins.A majority of the proteins contain a LxLxL type of EAR motif (Supple-mental Table S4).The high-stringency parameters in-cluded in our HMM/PHI-BLAST analysis may have excluded the proteins from being retrieved.To rule out this possibility,we did an additional arch,using the multiple expectation maximization for motif elic-itation (MEME)method (Bailey and Elkan,1995),for significantly overreprented motifs within the pro-tein quences of the t of 179additional proteins discovered by DPM analysis.Interestingly,the most
highly overreprented quence identified by MEME output was similar to the connsus quence of the EAR motif;however,the combined E-value of the output was too high to convincingly define the con-nsus quence as not being a potential artifact (data not shown).Owing to this lack of statistical evidence,the 179proteins were not included in the final list of EAR motif-containing trans
cription-related proteins (Table I)but are reported in Supplemental Table S4to provide a complete list of possible candidates.It is worth noting that the bHLH class of transcription factors with 23proteins is one of the most prevalent TF families in this protein t.
Classification of EAR Motif-Containing Proteins
A list of Arabidopsis Genome Initiative (AGI)codes and the quences of core EAR motif sites of all 219proteins are prented in Table I.Additional informa-tion,such as protein name and description,protein identifiers,and location of EAR motif sites in each quence,is listed in Supplemental Table S3.Upon careful inspection of the protein quences of all 219proteins,about 279“high-confidence”(bad on HMM/PHI-BLAST analysis)EAR motif sites were identified (Fig.3A).The number of EAR motif sites found in each protein varies from one to four (Fig.3B).Approximately 75%(165out of 219)of the proteins contain only one EAR motif site,while the remaining 25%contain two (49proteins),three (four proteins),or four (one protein)EAR motif sites.The EAR motif-containing proteins were further classified bad on type of the motif:LxLxL (containing three or more Leu residues in alternate positions),DLNxxP,and an ad-ditional class containing overlapping (not mutually exclusive)LxLxL and DLNxxP motif sites.The LxLxL,DLNxxP ,and overlapping LxLxL and DLNxxP type of EAR motif sites were found in 165,51,and 14proteins,respect
ively,of which 11proteins contained at least two different types of EAR motifs (Fig.3C).The EAR motif sites were mainly found in the C-terminal region (131out of 219proteins)and at slightly lower fre-quency in the N-terminal (71proteins)and middle (48proteins)regions (Fig.3,D and E).
To obtain insight into the biological functions of the EAR motif-containing proteins,they were further classified according to families.A total of 180out of 219proteins contain distinct DNA-binding domains and were classified into 18different TF families (Fig.3F;Table I).The remaining 39proteins do not posss a defined DNA-binding domain but are known in the literature to regulate transcription by interacting with TFs;hence,they were designated as OTRs and cate-gorized into three distinct OTR families:AUX/IAA,JAZ (for Jasmonate;ZIM domain),and NPR.The fam-ilies with the greatest number of EAR motif-containing proteins include C2H2(56proteins),HOMEOBOX (29proteins),AUX/IAA (28proteins),MADS (23pro-teins),and ERF (17proteins);the remaining families contain one to six proteins each (Table I).
Proteins
青春演讲稿Figure 2.Strategy for the identification of EAR motif-containing proteins in Arabidopsis.An iterative approach including literature survey,HMM analysis,and PHI-BLAST was ud to arch through the Arabidopsis protein databa.Details of each step are given in the text.N,M,and C refer to locations of the EAR motifs as being in the N-terminal,middle,and C-terminal regions of the protein,respectively.
The Arabidopsis EAR Repressome