In vivo analysis of the model tyrosine aminotransferase gene reveals multiple sequential steps in glucocorticoid receptor action Thierry Grange* ,1 , Lucia Cappabianca 1,2,3 , MicheÁle Flavin 1,3 , Habib Sassi 1,3 and He leÁ ne Thomassin 1,3 1 Institut Jacques Monod du CNRS, UniversiteÂs Paris 6 ± 7, Tour 43, 2 Place Jussieu, 75251 Paris Cedex 05, France We are studying the mechanisms of transcriptional activation by nuclear receptors and we focus our studies on the glucocorticoid regulation of the model tyrosine aminotransferase gene. Rather than using in vitro biochemical approaches, we determine the actual events occurring in the cells. Our experimental approaches include genomic footprinting, chromatin immunoprecipi- tation, in situ hybridization and transgenic mice. Our results show that the glucocorticoid receptor uses a dynamic multistep mechanism to recruit successively accessory DNA binding proteins that assist in the activation process. Chromatin is ®rst remodelled, DNA is then demethylated, and the synthesis of an accessory factor is induced. Ecient transcription induction is ®nally achieved upon the formation of a `stable' multiprotein complex interacting with the regulatory element. We discuss: the relative contribution of histone acetyltransferases and ATP-dependent remodelling ma- chines to the chromatin remodelling event; the nature of the remodelled state; the contribution of regulated DNA demethylation to gene memory during development; the mechanisms of regulated DNA demethylation; the dynamics of protein recruitment at regulatory elements; the control of the frequency of transcription pulses and the control levels of the cell-type speci®city of the glucocorticoid response. Oncogene (2001) 20, 3028 ± 3038. Keywords: chromatin; DNA methylation; transcrip- tion; liver; nuclear receptor Introduction In the last two decades, our understanding of eucaryotic gene transcription has progressed enor- mously and a number of players involved have been identi®ed, mostly through biochemical approaches (Lemon and Tjian, 2000; for a recent review). The limit to biochemical analyses is that they are aimed at reconstituting in vitro what is believed to occur in vivo. In vivo analyses are thus essential to fuel the biochemical approaches and to establish their biologi- cal relevance. For example, the contribution of chromatin to gene regulation was overlooked for a while by transcription biochemists before in vivo approaches modi®ed the trends. Nowadays, even pioneers of the early biochemical approaches recognize that higher order chromatin structure and nuclear organization must be taken into account to make sense out of the in vitro data (e.g., Lemon and Tjian, 2000). Initially, eucaryotic gene regulation studies were aimed at understanding the regulation of a limited number of model genes but presently, the profusion of transacting factors and of proteins interacting with them (coactivators, corepressors,) has distracted many researchers from such an approach. However, it is clear that the actual regulation of any single target gene is still only barely understood. We believe that the understanding of many general mechanisms would gain much from thorough analyses of the regulation of a limited number of model genes, particularly if some of these analyses are performed in living cells. We have focused our research activity on the analysis of the regulation of one model gene by one model transcriptional regulator, namely the transcriptional activation of the rat tyrosine aminotransferase (Tat) gene by the glucocorticoid receptor (GR). The earliest study of this regulation was over 40 years ago (Lin and Knox, 1957) and much additional work has followed (Tomkins, 1971; Granner and Hargrove, 1983; SchuÈtz et al., 1986). In particular, a strong emphasis has been put on the study of this regulation in living cells using genomic footprinting (Becker et al., 1986; Reik et al., 1991; Rigaud et al., 1991; Espina s et al., 1994, 1995). Two properties of the GR make it a valuable model regulator: (1) it acts as an inducible transcription factor that can be turned on and o readily by just adding or removing its ligand from culture medium, thus allowing a dynamic analysis of GR's mode of action in living cells; (2) GR is expressed in most tissues in the animal, but regulates a set of target genes that diers in each tissue, thus allowing the study of the mechanisms governing cell-speci®c activity of a widely expressed regulator. We review herein our current understanding of the regulation of the model Tat gene by the GR, with an emphasis on past and ongoing experiments from our laboratory. Oncogene (2001) 20, 3028 ± 3038 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: T Grange 2 Current address: Dipartimento di medicina sperimentale, Universita degli Studi dell'Aquila, Via Vetoio (Coppito 2), 67010 Coppito di L'Aquila, Italy 3 These authors have been listed in alphabetical order