The overexpression of MYC genes is frequently found in many human cancers, including adult and pediatric malignant brain tumors. Targeting MYC genes continues to be challenging due to their undruggable nature. Using our prediction algorithm, the nine-amino-acid activation domain (9aaTAD) has been identified in all four Yamanaka factors, including c-Myc. The predicted activation function was experimentally demonstrated for all these short peptides in transactivation assay. We generated a set of c-Myc constructs (1-108, 69-108 and 98-108) in the N-terminal regions and tested their ability to initiate transcription in one hybrid assay. The presence and absence of 9aaTAD (region 100-108) in the constructs strongly correlated with their activation functions (5-, 3- and 67-times respectively). Surprisingly, we observed co-activation function of the myc region 69-103, called here acetyl-TAD, previously described by Faiola et al. (Mol Cell Biol 25:10220-10234, 2005) and characterized in this study as a new domain collaborating with the 9aaTAD. We discovered strong interactions on a nanomolar scale between the Myc-9aaTAD activation domains and the KIX domain of CBP coactivator. We showed conservation of the 9aaTADs in the MYC family. In summary for the c-Myc oncogene, the acetyl-TAD and the 9aaTAD domains jointly mediated activation function. The c-Myc protein is largely intrinsically disordered and therefore difficult to target with small-molecule inhibitors. For the c-Myc driven tumors, the strong c-Myc interaction with the KIX domain represents a promising druggable target.

• Keywords
• 9aaTAD, CBP, KIX, Myc, MycN,
• MeSH
• Transcriptional Activation MeSH
• Humans MeSH
• Protein Domains MeSH
• Proto-Oncogene Proteins c-myc * metabolism   genetics   MeSH
• Amino Acid Sequence MeSH
• Protein Binding * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• MYC protein, human MeSH Browser
• Proto-Oncogene Proteins c-myc * MeSH

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.

• Keywords
• CBP, E2A, Gal4, Gcn4, KIX, KLF, MED15, MLL, Met4, TAF9, WT1, p53,
• MeSH
• Transcriptional Activation MeSH
• Gene Duplication MeSH
• Phylogeny MeSH
• Introns genetics   MeSH
• Humans MeSH
• Evolution, Molecular * MeSH
• Gene Expression Regulation * MeSH
• Amino Acid Sequence MeSH
• Sequence Homology MeSH
• Sp2 Transcription Factor antagonists & inhibitors   genetics   metabolism   MeSH
• Valine genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Sp2 Transcription Factor MeSH
• Valine MeSH

The Gal4 protein is a well-known prototypic acidic activator that has multiple activation domains. We have previously identified a new activation domain called the nine amino acid transactivation domain (9aaTAD) in Gal4 protein. The family of the 9aaTAD activators currently comprises over 40 members including p53, MLL, E2A and other members of the Gal4 family; Oaf1, Pip2, Pdr1 and Pdr3. In this study, we revised function of all reported Gal4 activation domains. Surprisingly, we found that beside of the activation domain 9aaTAD none of the previously reported activation domains had considerable transactivation potential and were not involved in the activation of transcription. Our results demonstrated that the 9aaTAD domain is the only decisive activation domain in the Gal4 protein. We found that the artificial peptides included in the original Gal4 constructs were results of an unintended consequence of cloning that were responsible for the artificial transcriptional activity. Importantly, the activation domain 9aaTAD, which is the exclusive activation domain in Gal4, is also the central part of a conserved sequence recognized by the inhibitory protein Gal80. We propose a revision of the Gal4 regulation, in which the activation domain 9aaTAD is directly linked to both activation function and Gal80 mediated inhibition.

• MeSH
• Transcriptional Activation genetics   physiology   MeSH
• DNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Protein Domains MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Transcription Factors chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• GAL4 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Transcription Factors MeSH

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.

• MeSH
• Transcriptional Activation * MeSH
• DNA-Binding Proteins chemistry   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Saccharomyces cerevisiae Proteins chemistry   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Transcription Factors chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• GAL4 protein, S cerevisiae MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Transcription Factors MeSH