A REGULATORY SYSTEM FOR TARGET GENE EXPRESSION

Mark M. Burcin, Bert W. O´Malley and Sophia Y. Tsai

Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

Received 1/17/98 Accepted 2/20/98

2. INTRODUCTION

The level of intracellular proteins in an eukaryotic cell is regulated at multiple levels including transcription of mRNA, mRNA stability, splicing and protein translation, modifications and stability. In this cascade, transcriptional regulation plays a key role in production of intracellular secreted proteins. The initiation of transcription is thought to be mediated by the sequential formation of the preinitiation complex. As a first step, TFIID recognizes the TATA box followed by the binding of TFIIA and TFIIB. Subsequently, RNA polymerase and other TFII proteins enter to form the preinitiation complex thereby allowing transcription to be initiated. Binding of transcription factors to specific DNA elements influences the formation and stability of this preinitation complex (1). Steroid/thyroid hormone receptors, the largest known family of transcription factors, have been shown to bind specific DNA sequences and activate or repress gene transcription upon binding their respective hormonal ligands. These steroid hormones such as progesterone, androgens, estrogen, glucocorticoids and mineralocorticoids, secreted by endocrine cells travel via the blood stream to their target cells and enter these cells by passive diffusion. Upon binding to hormone, steroid receptors undergo a "transformation", bind specific DNA sequences and activate or repress gene transcription. Thus, the function of steroid receptors can be regulated by exogenously added ligands. The structure of nuclear receptors can be subdivided into different modular functional domains which are exchangeable between different receptors to form chimeric constructs (2). The modular structure of these receptors makes them readily applicable for designing novel transactivators.

Based on this knowledge we designed a chimeric regulatory system (3-5) containing three important modules that together constitute a regulable transcription factor: (I) A domain able to bind a specific DNA-sequence, (II) a domain that regulates the ability of the receptor to bind DNA upon administration of an exogenous chemical (ligand) and (III) a domain with the potential to activate or repress transcription. In order to regulate a specific target gene in vivo, a desirable chimeric regulator should contain the following features: The DNA-binding domain should not bind to DNA elements in the nucleus and alter endogenous gene expression. The ligand binding domain (LBD) must be stimulated by an easily administerable, nontoxic drug which retains biological activity at low concentrations without the interference of endogenous compounds. The regulation mediated by the LBD should only occur in the presence of the ligand to guarantee a finely controlled regulation without "leaky" expression of the target gene in absence of the drug. The activation- or repressor domain must be sufficiently potent to generate high expression or tight repression of the coupled target genes.

This review will give a short summary of different regulatory systems that have been developed over the last few years and will then focus on the RU486-inducible regulatory system created by our group.