The 9aaTAD Is Exclusive Activation Domain in Gal4

Abstract

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.

The evolution of the 9aaTAD domain in Sp2 proteins: inactivation with valines and intron reservoirs

“…Previously, we have identified the 9aaTAD activation domains in the Gal4 (17) and in p53 acidic activators (14). In this study, we aimed to determine the 9aaTAD presence in other members of the SP family, where the 9aaTAD position would correspond with the Sp1 region 467-472 that has been shown to be essential for transcriptional activation (41).…”

“…The predicted Sp1 activation domain includes glutamines, which are not conserved in Sp5, 6, 7, 8, 9 ( Figure 2) and are not generally conserved in other 9aaTAD domains. Similarly, the acidic activation domain of the Gal4 activator includes acidic aspartic acids, which are not generally conserved in other 9aaTAD domains (12,14,17). In order to prove that the function of the 9aaTADs was not solely dependent on overrepresented aspartates nor on glutamines (which are present in the Gal4 and Sp1 activation domains), we have substituted all Gal4 aspartic residues for glutamines (construct Gal4-Q, non-acidic Gal4 9aaTAD) and all Sp1 glutamines and asparagine for aspartic acid residues (construct Sp1-Ac, acidic Sp1 9aaTAD), and showed their ability to activate transcription (Figure 1).…”

“…We and others have shown that the activation domains have the competence to activate transcription as small peptides (above 9 to 14 amino acids long) in over 40 activators including Gal4, E2A, MLL and p53 (12)(13)(14)(15)(16)(17). Although the activation domains have enormous variability, they are universally recognized by transcriptional machinery throughout eukaryotes (18).…”

The 9aaTAD activation domains in the four Yamanaka Oct4, Sox2, Myc, and Klf4 transcription factors essential during the stem cell development

“…The 9aaTAD family is represented by Gal4, p53, MLL, TCF3/E2A and SREBP1. The 9aaTADs were found in the SP/KLF family (Sp1, Sp2, Sp3, Sp4, Klf1, Klf2, Klf3, Klf4, Klf5, Klf6, Klf7, Klf8, Klf12, Klf15, WT1), the SOX family (Sox18 and SoxE), in hormone receptors (RARa, HNF4, PPAR, VDR, NHR49), in yeast transcription factors (Oaf1, Pip2, Pdr1, Pdr3, Rtg3, Gln3, Gcn4, Pho4, Msn2, Msn4, Met4), and in artificial activators of transcription (P201, B42, p53-ECapLL, KBP 2.20, pRJR200, G80BP-A, G80BP-B (12)(13)(14)(15)(16)(17)(18)(19).…”

How to overcome the ATRA resistance with the 9aaTAD activation domains in retinoic acid receptors

“…The 9aaTAD activation domains are universal activators of transcription in numerous transcription factors, which are recognized throughout the eukaryotes [1][2][3] . The 9aaTAD domains are recognized by multiple mediators of transcription including TAF9, MED15, CBP and p300 [4][5][6][7][8][9] .…”