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

Abstract

The Sp1 transcription factor has been defined as glutamine-rich activator. The Nine amino acid TransActivation Domains (9aaTAD) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in the Sp1 and in all nine members of SP family with broad natural 9aaTAD variations. We showed by the amino acid substitutions that the glutamine residues are completely dispensable for 9aaTADs function. We described the 9aaTAD domains' origin and evolutionary history. The ancestral Sp2 gene with inactive 9aaTAD has duplicated in early chordates and created new paralogs Sp1, Sp3 and Sp4. We discovered that the accumulation of valines in the 9aaTADs correlated with the domain inactivation. The Sp2 activation domain, whose dormancy have lasted over 100 million years during chordate evolution, enabled later diversification in the Sp1-4 clade, including both repressors and activators. The new paralogs Sp1 and Sp3 activation domains have regained their original activator function by loss of valines in their 9aaTADs.Keywords: activation domain, 9aaTAD, SP, Sp1, KLF, WT1, Gal4, Met4, Gcn4 and p53. demonstrated the importance of the hydrophobic amino acids for Gcn4 (30-33) and p53 activators (8,(34)(35)(36)(37)(38)(39)(40). Similarly, in the original report for the glutamine-rich activator Sp1(41), the importance of hydrophobic amino acids was already recognized, regardless of the overrepresented glutamines.Nevertheless, the designation as acidic or glutamine-rich activation domains persists to date (42-49).In the human genome, there are about 1,691 annotated transcription factors, divided into 68 groups according to their conserved DNA binding domain (50). The C2H2-type zinc finger is the largest group (682 members), which includes the evolutionally conserved family of KLF activators (Krüppel-like factors). The SP activators (Specificity proteins Sp1-9) belong to a new evolutional branch derived from the ancestral KLF family. The SP activators have been linked to cell proliferation, embryonic development, tissue differentiation, Wnt signaling pathway, metabolism, and their dysregulation has been implicated in a number of human diseases and cancers (51)(52)(53)(54)(55)(56). This report has focused on the SP family and followed the 9aaTAD domain's evolution. MATERIALS AND METHODSConstructs. The construct pBTM116-HA was generated by an insertion of the HA cassette into the EcoRI site of the vector pBTM116. The constructs G1-G45 and H1-H45 were generated by PCR and sub-cloned into pBTM116 EcoRI and BamHI sites. All constructs have a spacer of three amino acids inserted into the EcoRI site; peptide GSG. All constructs have been sequenced by Eurofins Genomics.Further detailed information about constructs and primer sequences are available on request.Assessment of enzyme activities. The β-galactosidase activity was determined in the yeast strain L40 (57,58). The strain L40 has a chromosomally integrated lacZ reporter driven by the lexA operator. In all hybrid assays, we used 2μ vector pBTM116 for...