INTRODUCTION TEA Domain proteins: Transcription Factors Unknown 3D structure

1. GBD GBD + GAD GBD-NAD GBD-NAD + GAD GBD-Tead1 GBD-Tead1 + GAD 0 25 50 75 100 125 0 mM 3AT Colonies 10 mM 3AT (% of control grown with histidine) 25 20 (Units) 6 7 5 3 4 8 1 2 9 15 13 11 12 10 14 15 16 galactosidase 17 18 10 23 19 24 25 2 0 21 22 26 Beta- 5 0 3 4 1 9 20 16 21 26 11 13 25 14 19 6 5 8 12 18 7 23 24 2 15 22 10 17 A B C D Mutated Clones Selected Tead4 N term Intrinsic activation and repression domains of TEAD proteins are functional and modulated by their neighbor domains in yeast cellLuciana B. Rodrigues, Ygor M. P. Pessoa, Mônica B. Rodriguez* & J. Miguel OrtegaDep. Bioquímica & Imunologia, Dep. Biologia Geral*, UFMG, Antonio Carlos 6627, Belo Horizonte, MG 31270-010, Brazil • INTRODUCTION • TEA Domain proteins: • Transcription Factors • Unknown 3D structure • Present in diverse organisms • yeast (TEC-1 protein) • fruit fly (SD protein) • mouse and man (Tead1, 2, 3, 4a and 4b) • Known interacting proteins • MAX, TBP, PARP, TONDU and VG (fruit fly) METHODOLOGY We investigated the intrinsic activity of Tead1 domains in yeast cells with the one-hybrid system Different domains were assayed as a fusion to Gal4p DNA binding domain (GBD) NAD TEA - - - +++ PR STY ZF GAL UAS Promoter Lac Z reporter gene 52 134 236 342 426 GBD NAD = N terminal Acidic Domain TEA = DNA binding domain PR = Proline Rich domain STY = Ser/Thr/Tyr rich domain ZF = putative Zinc Finger domain TGTGGAATG product X-gal beta-galactosidase X-gal (GT-IIC) TEAD1 (NAD) Activation by NAD is squelched by GAD RESULTS We observed that residues 1-52 constitute a typical acidic domain that activates transcription in yeast Addition of TEA suffices for cis-modulation, and full length protein does not activate transcription in yeast Mild activation is observed with NAD-TEA-PR, due to 3D bumps or is there a second activation domain? Surprisingly, under squelching, Tead1(1-52) turns into a repressor of basal transcription Thus, addition of the neighbor domain TEA abolishes activation as well as repression of basal transcription under squelching Residues 135-236 (Proline-Rich Domain) caused repression of basal transcription of two different promoters, but this activity was absent when residues 135-426 were used PR inhibits HIS3 basal transcription (HF7c) Promoter Promoter GAL UAS HIS3 GAL UAS HIS3 GBD GBD enzime PR His3 PR STY cis-modulation ZF histidine NAD TEA - - - +++ PR STY ZF NAD TEA - - - +++ PR STY ZF + histidine - histidine NAD TEA 52 134 236 342 426 PR STY ZF - - - +++ TGTGGAATG 52 134 236 342 426 (GT-IIC) TGTGGAATG 52 134 236 342 426 (GT-IIC) PROMOTER PR STY ZF 134 236 342 426 PROMOTER PR Transcription factor with acidic domain 134 236 (Gal4) Using yeast one-hybrid selection we isolated several mutants of Tead1, Tead4a and Tead4b that lack either total or partially the modulation by neighbor domain • CONCLUSIONS • Our data show that several intrinsic activities of Transcription Factors might be conserved between mouse and yeast cells but modulation by neighbor domains can avoid detection of these activities • Moreover, yeast genetics provide excellent selection traps for raising of informative mutants Strong activators (e.g. mut1) shows truncation HIS3 Gal4BD NAD TEA +++ - - - 52 134 His3 3 AT Tead1(1-134) UV [or Tead4a UV] TGTGGAATG (GT-IIC) histidine Tead4a or Tead4b UV PROMOTER Tead1(1-134) UV 3 AT Support: FAPEMIG, CNPq