Repression domains of class II ERF transcriptional repressors share an essential motif for active repression

Abstract

We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

Figure 1.

Mapping of the Repression Domain of ERF3. (A) Scheme of the constructs used in cotransfection experiments. The GAL4-responsive reporter construct, GAL4-LUC, contained five copies of the GAL4 binding site in tandem and a minimal TATA region (starting at position −46) of the CaMV 35S promoter, the firefly gene for luciferase (LUC), and a nopaline synthase (Nos) terminator (Ohta et al., 2000). Each effector construct contained a GAL4 DNA binding domain (GAL4DB) and part of the coding region (D) of NtERF3 driven by the CaMV 35S promoter. A translational enhancer sequence from tobacco mosaic virus (Ω) was located upstream of the site of initiation of translation. (B) Relative luciferase activities in tobacco protoplasts that had been cotransfected with effector and reporter plasmids. Schemes of the deletion mutants of NtERF3 are shown at left. The indicated portions of NtERF3 were fused to GAL4DB. Closed boxes indicate the ERF DNA binding domain. All luciferase activities are expressed relative to values obtained with the reporter construct alone (None; set arbitrarily at 1). Error bars indicate ±sd.

Figure 2.

Active Repression by the Repression Domain of NtERF3. (A) Scheme of the constructs used in cobombardment experiments. The reporter gene GAL4GCC-LUC contained five copies of the GAL4-responsive element, four copies of the GCC box sequence in tandem, a minimal TATA region (starting at position −46) of the CaMV 35S promoter, the firefly gene for luciferase (LUC; shown as a closed box), and a nopaline synthase (Nos) terminator (Fujimoto et al., 2000). Effector constructs contained the coding sequence of AtERF5 (AtERF5), a GAL4 DNA binding domain (GAL4DB; shown as a closed box), the 191/225ERF3 repression domain fused with GAL4DB (ERF3RD), a VP16 activation domain fused with GAL4DB (VP16), and the VP16 activation domain fused with ERF3RD (ERF3RD•VP16). Each construct is driven by the CaMV 35S promoter, and an omega sequence, namely, a translational enhancer, from tobacco mosaic virus (Ω) was located upstream of the site of initiation of translation. (B) Relative luciferase activities after cobombardment of Arabidopsis leaves with the GAL4GCC-LUC reporter gene and the GAL4DB fusion effectors with or without the AtERF5 effector. The effectors introduced into leaves with the reporter gene are shown at left. All LUC activities are expressed relative to the value obtained with the reporter construct alone (None; set arbitrarily at 1). Error bars indicate ±sd.

Figure 3.

The ^L/_FDLN^L/_F(x)P Motif Is Conserved in the C-Terminal Regions of Class II ERF and TFIIIA-Type Zinc Finger Proteins. (A) Comparison of the amino acid sequences of the C-terminal regions of class II ERF proteins from various plants. (B) Alignment of the sequences of C-terminal regions of TFIIIA-type zinc finger proteins. Numbers in parentheses indicate the positions of the amino acid sequences. Dashes were introduced to optimize the alignment. Reverse type indicates the ^L/_FDLN^L/_F(x)P motif that was found in the class II ERF and zinc finger proteins and designated EAR. Asterisks indicate the proteins whose repression activities were analyzed in the present experiments.

Figure 4.

Repression by the Repression Domains of AtERF3, AtERF4, and OsERF3. Relative luciferase activities after cobombardment of Arabidopsis leaves with the GAL4GCC-LUC reporter gene and GAL4DB fusion effectors with or without the AtERF5 effector. The effectors were 35S-GAL4DB-AtERF3(166/225) (AtERF3RD), 35S-GAL4DB-AtERF4 (183/222) (AtERF4RD), 35S-GAL4DB-OsERF3(1/235) (OsERF3full), and 35S-GAL4DB-OsERF3(193/235) (OsERF3RD). All luciferase activities are expressed relative to the value obtained with the reporter gene alone (None; set arbitrarily at 1). Error bars indicate ±sd.

Figure 5.

The Conserved Motif in the Repression Domain Is Essential for Repression. (A) Scheme of the conserved motif and positions of amino acids replaced in 191/225 ERF3RD. The amino acids replaced by alanine residues are indicated by arrows; residue 214 (asparatic acid; m1), residue 216 (asparatic acid; m2), and both residues 214 and 216 (m3) were replaced by alanine residues. Reverse type indicates the EAR motif sequence. (B) Relative luciferase activities after cobombardment of Arabidopsis leaves with the GAL4GCC-LUC reporter gene and the GAL4DB fusion effectors with or without the AtERF5 effector. The asparatic acid residues at positions 214, 216, or both 214 and 216 were replaced by alanine residues in ERF3RDm1, ERF3RDm2, and ERF3RDm3, respectively. All luciferase activities are expressed relative to the value obtained with the reporter construct alone (None; set arbitrarily at 1). Error bars indicate ±sd.

Figure 6.

Repression by Zinc Finger Proteins That Contain the EAR Motif. (A) Scheme of the constructs used in cobombardment experiments. The effector constructs contained the coding sequence of full-length ZAT10 (ZAT10full), 174/227 ZAT10 (ZAT10RD), 1/173 ZAT10 (ZAT10ΔRD1), 1/189 ZAT10 (ZAT10ΔRD2), full-length ZAT11 (ZAT11full), 130/178 ZAT11 (ZAT11RD), and 1/129ZAT11 (ZAT11ΔRD1). Hatched boxes indicate EAR motifs. Nos, nopaline synthase terminator. (B) Relative luciferase activities after cobombardment of Arabidopsis leaves with the GAL4GCC-LUC reporter gene and the GAL4DB fusion effectors with or without the AtERF5 effector. All luciferase activities are expressed relative to the value obtained with the reporter construct alone (None; set arbitrarily at 1). Error bars indicate ±sd.