Engineering of monotransregulators.

Abstract

<p>(<b>A</b>) Schematic of ERα. 595 amino-acid long ERα is a modular protein that contains an amino-terminally located ligand-independent activation function, A/B, domain followed by the DNA binding, C, domain. The hinge, D, domain encompasses a nuclear localization signal and connects the C domain to the carboxyl-terminus, E/F, domain. E/F is a multi-functional domain containing ligand binding, dimerization and ligand-dependent activation functions. ERα binds to specific DNA sequences, so-called estrogen responsive elements (EREs), derivatives of the consensus GGTCAnnnTGACC wherein ‘n’ denotes three non-specific nucleotides. (<b>B</b>) The engineered monomeric ERE binding module CDC is composed of two C domains joined with the D domain. (<b>C</b>) The cDNA of the activation domain (AD) of p65 and VP16 proteins or of the repression domain (RD) KRAB of KOX-1 of and/or SID of Mad1 was genetically joined to the 5′ and 3′ ends, respectively, of the CDC-cDNA or the ERE binding defective CDC_EBD to generate the monotransregulators. The constructs also contain sequences encoding the Flag and 6xHis epitopes at the amino and carboxyl-termini, respectively.</p