Phenotypic consequences of lung-specific inducible expression of FGF-3.

Members of the fibroblast growth factor (FGF) family play a critical role in embryonic lung development and adult lung physiology. The in vivo investigation of the role FGFs play in the adult lung has been hampered because the constitutive pulmonary expression of these factors often has deleterious effects and frequently results in neonatal lethality. To circumvent these shortcomings, we expressed FGF-3 in the lungs under the control of the progesterone antagonist-responsive binary transgenic system. Four binary transgenic lines were obtained that showed ligand-dependent induction of FGF-3 with induced levels of FGF-3 expression dependent on the levels of expression of the GLp65 regulator as well as the dose of the progesterone antagonist, RU486, administered. FGF-3 expression in the adult mouse lung resulted in two phenotypes depending on the levels of induction of FGF-3. Low levels of FGF-3 expression resulted in massive free alveolar macrophage infiltration. High levels of FGF-3 expression resulted in diffuse alveolar type II cell hyperplasia. Both phenotypes were reversible after the withdrawal of RU486. This system will be a valuable means of investigating the diverse roles of FGFs in the adult lung.

Adenovirus-mediated regulable target gene expression in vivo.

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.

A regulatory system for target gene expression.

Temporally-regulated expression of endogenous genes is a desirable goal in stable cell line and transgenic animal systems, as well as in clinical gene therapy. Protocols for introducing genes into stable cell lines and experimental animals are often unsatisfactory due to the constitutive expression of such transgenes. To circumvent this problem we have demonstrated specific and temporally regulable expression of a target gene in vivo effected by a chimeric regulator in response to an orally-administered, non-toxic chemical. This regulatory system utilizes a chimeric regulator GLVP, consisting of a mutated human progesterone receptor ligand binding domain (PRLBD-delta) fused to the yeast GAL4 DNA binding domain (DBD) and the HSV VP16 transcriptional activation domain and whose activity is solely regulable by non-physiological doses of RU486 but not by progesterone or other endogenous progestins. Replacing the activation domain of the chimeric regulator with a transcriptional repression domain results in inducible repression of target gene expression in vitro. Our regulatory system functions in transient and stable transfections as well as in transgenic animals, and will have a wide variety of potential applications.

Steroid receptor coactivator-1 is a histone acetyltransferase.

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. The steroid-receptor coactivator SRC-1 is a coactivator for many members of the steroid-hormone receptor superfamily of ligand-inducible transcription factors. Here we show that SRC-1 possesses intrinsic histone acetyltransferase activity and that it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates.

Steroid receptor induction of gene transcription: a two-step model.

Coactivators, such as steroid receptor coactivator 1 (SRC-1A) and CREB (cAMP response element binding protein)-binding protein (CBP), are required for efficient steroid receptor transactivation. Using an in vitro transcription assay, we found that progesterone receptor (PR)-driven transcription is inhibited by a dominant negative PR ligand-binding domain-interacting region of SRC-1A, indicating that SRC-1A is required for actual transcriptional processes. In addition, these coactivators also possess intrinsic histone acetyltransferase (HAT) activity and bind to each other and another HAT, p300/CBP-associated factor. Here we show that the human PR also interacts with p300/CBP-associated factor in vitro. Recruitment of multiple HATs to target promoters suggests an important role for chromatin remodeling in transcriptional activation of genes by steroid receptors. In transient transfection assays, we found that addition of a histone deacetylase inhibitor, trichostatin A, strongly potentiated PR-driven transcription. In contrast, directing histone deacetylase-1 (HD1) to a promoter using the GAL4 DNA binding domain inhibited transcription. Furthermore, PR transactivation was repressed by recruiting HD1 into the PR-DNA complex by fusing HD1 to a PR ligand-binding domain-interacting portion of SRC-1. Collectively, these results suggest that targeted histone acetylation by recruited HAT cofactors and histone deacetylation are important factors affecting PR transactivation. Recruitment of coactivators and HATs by the liganded PR in vivo may result in (i) remodeling of transcriptionally repressed chromatin to facilitate assembly and (ii) enhanced stabilization of the preinitiation complex by the activation functions of coactivators and the liganded PR itself.