Modulation of TAT Gene Induction by Glucocorticoids Involves a Neutralizing Sequence David A. Jackson*, Clayton D. Collier$, Hisaji Oshima} and S. Stoney Simons Jr.* Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, MD, U.S.A. Recent studies have indicated that two elements in addition to the glucocorticoid response element (GRE) are involved in the induction of the endogenous TAT gene in Fu5-5 rat hepatoma cells. The ®rst is the 21 bp glucocorticoid modulatory element (GME) at À3648 bp, which causes reporter con- structs to display both a left shift in the dose±response curve for glucocorticoids and increased per- centages of agonist activity for antiglucocorticoids. The second is a negative element at À3340 to À3050 that blocks the action of the GME. This last observation raised the question of how GME ac- tivity can be expressed in Fu5-5 cells in the intact TAT gene that contains both the GME and the negative element. The present study identi®es a third element, a ``neutralizing'' sequence, that re- stores the activity of the GME even when otherwise inactivated by the negative element. This neu- tralizing sequence was located within the region surrounding the GREs of the TAT gene but is separate from the GREs. The activity of the individual GME and negative elements was found to depend upon spacing. However, in combination with the natural GRE, the native TAT gene spacing of the GME and negative elements was able to reproduce the activity of the intact gene. Thus, a total of three additional elements (an activator, a negative element, and a neutralizer) appear to co- operate with the GREs in glucocorticoid induction of the TAT gene in Fu5-5 cells. While such a grouping of elements may be novel among steroid regulated genes, it is a not uncommon occurrence for the transcriptional control of other genes. # 1998 Elsevier Science Ltd. All rights reserved. J. Steroid Biochem. Molec. Biol., Vol. 66, No. 3, pp. 79±91, 1998 INTRODUCTION Glucocorticoid induction of the rat liver tyrosine ami- notransferase (TAT) gene has proved a useful model for steroid-induced gene expression for several reasons. First, it is a biologically relevant response. It was also one of the ®rst systems to show a correlation between the steroid binding of receptors and the whole cell induction of a protein [1]. TAT induction is a primary effect of the receptor±steroid complex in that the induction of enzyme requires mRNA synthesis and is down regulated by steroid withdrawal [2]. Finally, the TAT gene was shown to contain speci®c DNA sequences, called glucocorti- coid response elements (GREs), which are steroid- inducible enhancers. GREs are obligatory com- ponents of the ternary DNA±receptor±steroid com- plexes that are thought to interact with the transcriptional machinery to increase the rate of TAT gene transcription [3, 4]. Over the last few years, however, the picture has become much more complicated. While the TAT GRE is still a ``simple GRE'' in that receptor±steroid complex binding to the isolated GRE is capable of inducing transcription without the help of other cis- acting elements or transcription factors[5], differences with other GREs have emerged and other TAT gene elements have been found to participate in the induc- tion process. Thus, the TAT GREs are much further upstream from the start of transcription than for most other steroid hormone responsive elements J. Steroid Biochem. Molec. Biol. Vol. 66, No. 3, pp. 79±91, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-0760/98 $19.00 + 0.00 PII: S0960-0760(98)00048-X *Current address: Bldg. 6, Room B1-08, NIDDK/LCDB, NIH, Bethesda, MD 20892, U.S.A. $Current address: Digene, 2301-B Broadbirch Drive, Silver Spring, MD 20904, U.S.A. }Current address: Department of Medicine, Fujita Health University School of Medicine, Toyoake, Aichi 470-11, Japan. *Correspondence to S. S. Simons, Jr. Tel. 496 6796; Fax: 402 3572; e-mail: steroids@helix.nih.gov Received 8 Dec. 1997; accepted 9 Feb. 1998. 79