Structure-Based Design of a Potent Artificial Transactivation Domain Based on p53

Langlois, Chantal; Gatto, Annarita Del; Arseneault, Geneviève; Lafrance‐Vanasse, Julien; Simone, Mariarosaria De; Morse, Thomas M.; Paola, Ivan de; Lussier-Price, Mathieu; Legault, Pascale; Pedone, Carlo; Zaccaro, Laura; Omichinski, James G.

doi:10.1021/ja208999e

Cited by 13 publications

(11 citation statements)

References 84 publications

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“…Similar result has been reported for p53 mimetic ECapLL [ 28 ]. We recognized analogical modification of the second p53 9aaTAD-II domain towards the first 9aaTAD motif (in the p53 9aaTAD-I domain) [ 16 ].…”

Section: Discussionsupporting

confidence: 91%

The 9aaTAD Transactivation Domains: From Gal4 to p53

et al. 2016

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The family of the Nine amino acid Transactivation Domain, 9aaTAD family, comprises currently over 40 members. The 9aaTAD domains are universally recognized by the transcriptional machinery from yeast to man. We had identified the 9aaTAD domains in the p53, Msn2, Pdr1 and B42 activators by our prediction algorithm. In this study, their competence to activate transcription as small peptides was proven. Not surprisingly, we elicited immense 9aaTAD divergence in hundreds of identified orthologs and numerous examples of the 9aaTAD species' convergence. We found unforeseen similarity of the mammalian p53 with yeast Gal4 9aaTAD domains. Furthermore, we identified artificial 9aaTAD domains generated accidentally by others. From an evolutionary perspective, the observed easiness to generate 9aaTAD transactivation domains indicates the natural advantage for spontaneous generation of transcription factors from DNA binding precursors.

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Section: Discussionsupporting

confidence: 91%

The 9aaTAD Transactivation Domains: From Gal4 to p53

et al. 2016

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“…Importantly, these same studies have shown that the p53 activation domain appears to use a similar structural motif (IExWF). Indeed, the design of an efficient artificial transcription activation domain based on the structural details of the VP16/p53 interaction with TFIIH was recently reported (Langlois et al ., ).…”

Section: Resultsmentioning

confidence: 97%

Activation domains for controlling plant gene expression using designed transcription factors

Li¹,

Blue²,

Zeitler

et al. 2013

Plant Biotechnology Journal

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SummaryTargeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.

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“…Tfb1PH and CBP KIX were expressed and purified as previously described [39], [55]. EBNA2 431–487 , EBNA2 448–471 and mutants were expressed as GST fusion proteins in E. coli host strain TOPP2, purified over glutathione-sepharose resin (GE Healthcare) and cleaved with thrombin (Calbiochem) as previously described [39].…”

Section: Methodsmentioning

confidence: 99%

Structural and Functional Characterization of a Complex between the Acidic Transactivation Domain of EBNA2 and the Tfb1/p62 Subunit of TFIIH

et al. 2014

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Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431–487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448–471 (EBNA2448–471) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2448–471 complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2448–471 complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of intrinsically disordered acidic TADs in recognizing common target host proteins.

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Structure-Based Design of a Potent Artificial Transactivation Domain Based on p53

Cited by 13 publications

References 84 publications

The 9aaTAD Transactivation Domains: From Gal4 to p53

The 9aaTAD Transactivation Domains: From Gal4 to p53

Activation domains for controlling plant gene expression using designed transcription factors

Structural and Functional Characterization of a Complex between the Acidic Transactivation Domain of EBNA2 and the Tfb1/p62 Subunit of TFIIH

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