Strong activation domain

Abstract

A new and strong transcriptional activation domain was identified from the Arabidopsis protein Ethylene Response Factor 98 (AtERF98). This domain has been designated as the “EDLL domain” and has a number of highly conserved amino acid residues that are found throughout the members of the AtERF98 family from plants, including in monocot and eudicot orthologs. The EDLL domain was shown to be highly active when it was fused to transcription factors from plant and yeast, and was also shown to have activation potential comparable to the widely-used VP16 activation domain derived from Herpes simplex. The EDLL domain was also active when it was targeted to a gene promoter by a sequence-specific DNA binding protein or by protein-protein interactions. Unlike other known activation domains such as VP16 and GAL4, the EDLL domain is relatively small in size, and being of plant origin, it is favored as a strong transcriptional activation tool for application in transgenic food crops.

1 - 24 . (canceled) 
     
     
         25 . A synthetic chimeric polypeptide comprising a transcription activation domain of:
 (i) SEQ ID NO: 55;   (ii) SEQ ID NO: 56;   (iii) SEQ ID NO: 94;   (iv) SEQ ID NO: 95;   (v) an amino acid sequence with a minimum percentage identity to any of SEQ ID NO: 37-54, wherein the minimum percentage identity is selected from the group consisting of 100%, 95%, 93.8%, 90%, 87.5%, 85%, 81.2%, 80%, 75.0%, 70%, 68.7%, 65%, 62.5%, 60%, 56.2%, 50%, and 43.7%;   (vi) an amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 57-63 and 76-93; or   (vii) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO: 57-63 or 76-93, wherein the stringent conditions comprise at least 6×SSC and 1% SDS at 65° C. with a first wash step for 10 minutes at about 42° C. with about 20% (w/v) formamide in 0.1×SSC and with a subsequent wash step for 10 minutes with 0.2×SSC and 0.1% SDS at 65° C.;   wherein the transcription activation domain is covalently fused to a transcription regulatory polypeptide; and   wherein the transcription activation domain and the transcription regulatory polypeptide are mutually heterologous and do not occur in nature in the same protein.   
     
     
         26 . The chimeric polypeptide of  claim 25 , wherein the transcription regulatory polypeptide is a transcription factor polypeptide. 
     
     
         27 . The chimeric polypeptide of  claim 25 , wherein the chimeric polypeptide comprises any of SEQ ID NOs: 37-54 or SEQ ID NO: 56. 
     
     
         28 . The chimeric polypeptide of  claim 25 , wherein expression of the chimeric polypeptide is regulated by an inducible, developmental or tissue-specific promoter. 
     
     
         29 . A recombinant polynucleotide encoding the chimeric polypeptide according to  claim 25 . 
     
     
         30 . A host plant cell comprising a nucleic acid construct encoding a chimeric polypeptide comprising a transcription activation domain covalently fused to a transcription regulatory polypeptide, wherein the transcription activation domain and the transcription regulatory polypeptide are mutually heterologous and do not occur in nature in the same protein, or do not occur in the same copy number or configuration in nature; and
 wherein the transcription activation domain comprises:   (i) SEQ ID NO: 55;   (ii) SEQ ID NO: 56;   (iii) SEQ ID NO: 94;   (iv) SEQ ID NO: 95;   (v) an amino acid sequence with a minimum percentage identity to any of SEQ ID NO: 37-54, wherein the minimum percentage identity is selected from the group consisting of 100%, 95%, 93.8%, 90%, 87.5%, 85%, 81.2%, 80%, 75.0%, 70%, 68.7%, 65%, 62.5%, 60%, 56.2%, 50%, and 43.7%;   (vi) an amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 57-63 and 76-93; or   (vii) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO: 57-63 or 76-93, wherein the stringent conditions comprise at least 6×SSC and 1% SDS at 65° C. with a first wash step for 10 minutes at about 42° C. with about 20% (w/v) formamide in 0.1×SSC and with a subsequent wash step for 10 minutes with 0.2×SSC and 0.1% SDS at 65° C.   
     
     
         31 . The host cell of  claim 30 , wherein the transcription regulatory polypeptide is a transcription factor polypeptide. 
     
     
         32 . The host cell of  claim 30 , wherein the chimeric polypeptide comprises any of SEQ ID NOs: 37-54 or SEQ ID NO: 56. 
     
     
         33 . The host cell of  claim 30 , wherein expression of the chimeric polypeptide is regulated by an inducible, developmental or tissue-specific promoter. 
     
     
         34 . A transgenic plant comprising a host plant cell according to  claim 30 . 
     
     
         35 . A method for increasing the expression of a target polynucleotide sequence, the methods steps comprising:
 (a) generating a nucleic acid construct encoding a chimeric polypeptide comprising a transcription activation domain covalently fused to a transcription regulatory polypeptide, wherein the transcription activation domain and the transcription regulatory polypeptide are mutually heterologous and do not occur in nature in the same protein, or do not occur in the same copy number or configuration in nature;   wherein the chimeric polypeptide activates transcription of the target polynucleotide sequence to which the chimeric polypeptide binds, and   wherein the transcription activation domain comprises:   (i) SEQ ID NO: 55;   (ii) SEQ ID NO: 56;   (iii) SEQ ID NO: 94;   (iv) SEQ ID NO: 95;   (v) an amino acid sequence with a minimum percentage identity to any of SEQ ID NO: 37-54, wherein the minimum percentage identity is selected from the group consisting of 100%, 95%, 93.8%, 90%, 87.5%, 85%, 81.2%, 80%, 75.0%, 70%, 68.7%, 65%, 62.5%, 60%, 56.2%, 50%, and 43.7%;   (vi) an amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 57-63 and 76-93; or   (vii) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO: 57-63 or 76-93, wherein the stringent conditions comprise at least 6×SSC and 1% SDS at 65° C. with a first wash step for 10 minutes at about 42° C. with about 20% (w/v) formamide in 0.1×SSC and with a subsequent wash step for 10 minutes with 0.2×SSC and 0.1% SDS at 65° C.; and   (b) introducing the nucleic acid construct into a host cell.   
     
     
         36 . The method of  claim 35 , wherein the transcription regulatory polypeptide is a transcription factor polypeptide. 
     
     
         37 . The method of  claim 35 , wherein the chimeric polypeptide comprises any of SEQ ID NOs: 37-54 or SEQ ID NO: 56. 
     
     
         38 . The method of  claim 35 , wherein expression of the chimeric polypeptide is regulated by an inducible, developmental or tissue-specific promoter.