Catharine L. Smith

TRANSCRIPTIONAL REGULATION OF MAMMALIAN GENES IN VIVO : A TALE OF TWO TEMPLATES

The past two decades have brought a major evolution in our understanding of promoter structure, transcription factors, and mechanisms by which transcriptional initiation is regulated in eukaryotes. Multiple approaches have been used to establish current models for transcriptional regulation, including the development of in vitro transcription systems using either naked DNA or reconstituted chromatin, genetic analysis of gene regulation in yeast and Drosophila, and in vivo analysis of either endogenous cellular genes or transiently transfected, exogenous gene promoters. It is now clear that chromatin structure, once considered to be transparent to the process of transcription, plays an important role in the regulation of gene expression (reviewed in Ref. 1). Since all cellular genes are packaged into ordered chromatin structures, an understanding of the mechanisms by which nucleoprotein structure influences transcription activation is necessary for a complete paradigm of gene regulation in higher eukaryotes. Studies on regulation of endogenous mammalian genes are challenging due to the lack of genetic techniques available in yeast and Drosophila systems, which allow targeted insertion of promoters and gene disruption. Structural analysis of integrated gene promoters in mammalian cells requires the time-consuming generation of multiple stable cell lines or pools, in which the integrated genes would be subject to position effects from surrounding chromatin. Therefore, the identification and characterization of factors involved in mammalian gene expression have been addressed primarily through the use of transient transfection assays. In this approach, exogenous plasmid DNA, usually promoter/reporter constructs and transcription factor expression vectors, is introduced into cultured cells and expressed transiently, without replication or integration into the cellular genome. A variety of transfection methods has been utilized, including calcium phosphate precipitation (2), DEAE-dextran (3), electroporation (4, 5), and liposomemediated transfer (6). These studies have resulted in an abundance of information about promoter structure (i.e. identification of cisacting elements), transcription factor structure and function, and the characterization of transcription factor interactions that are part of various cellular regulatory pathways. However, in light of the accumulating evidence indicating a role for chromatin structure in transcription, it is appropriate to question whether transiently transfected gene promoters are adequate models for transcriptional regulatory mechanisms active on endogenous genes in ordered, replicated chromatin.

Inhibition of MMTV transcription by HDAC inhibitors occurs independent of changes in chromatin remodeling and increased histone acetylation

Increased histone acetylation has been associated with activated gene transcription and decreased acetylation with repression. However, there is a growing number of genes known, which are downregulated by histone deacetylase (HDAC) inhibitors through unknown mechanisms. This study examines the mechanism by which the mouse mammary tumor virus (MMTV) promoter is repressed by the HDAC inhibitor, trichostatin A (TSA). We find that this repression is transcriptional in nature and that it occurs in the presence and absence of glucocorticoids. TSA decreases MMTV transcription at a rapid rate, reaching maximum in 30–60 min. In contrast with previous reports, the repression does not correlate with an inhibition of glucocorticoid-induced nuclease hypersensitivity or NF1-binding at the MMTV promoter. Surprisingly, TSA does not induce sizable increases in histone acetylation at the MMTV promoter nor does it inhibit histone deacetylation, which accompanies deactivation of the glucocorticoid-activated MMTV promoter. Repression of MMTV transcription by TSA does not depend on the chromatin organization of the promoter because a transiently transfected MMTV promoter construct with a disorganized nucleoprotein structure was also repressed by TSA treatment. Mutational analysis of the MMTV promoter indicates that repression by TSA is mediated through the TATA box region. These results suggest a novel mechanism that involves acetylation of nonhistone proteins necessary for basal transcription.

Differential Activity of Progesterone and Glucocorticoid Receptors on Mouse Mammary Tumor Virus Templates Differing in Chromatin Structure

In vivo, transcription factors interact with promoters having complex nucleoprotein structures. The transiently expressed progesterone receptor (PR) efficiently activates a transfected mouse mammary tumor virus (MMTV) promoter but is a poor activator of the MMTV promoter when it acquires an ordered chromatin structure as an endogenous, replicating gene. We show that the deficiency in PR activity is not due to insufficient expression of either B or A isoforms or competition between the two types of MMTV templates. Rather, this deficiency reflects an inability to induce the chromatin remodeling event that is required for activation of the replicated MMTV template. To determine whether this characteristic is common to transiently expressed steroid receptors or specific to the PR, we examined the activity of transiently expressed glucocorticoid (GR) receptor. Unlike the PR, the transiently expressed GR is an effective activator of both MMTV templates and efficiently induces the necessary chromatin remodeling event at the replicated template. These results indicate that the GR and PR have unique requirements for activation of promoters with ordered chromatin structure. These differences may provide a mechanism for establishing target gene specificity in vivo for steroid receptors that recognize and bind to identical DNA sequences.

Nucleoprotein structure influences the response of the mouse mammary tumor virus promoter to activation of the cyclic AMP signalling pathway.

Recent studies have provided evidence of crosstalk between steroid receptors and cyclic AMP (cAMP) signalling pathways in the regulation of gene expression. A synergism between intracellular phosphorylation inducers and either glucocorticoids or progestins has been shown to occur during activation of the mouse mammary tumor virus (MMTV) promoter. We have investigated the effect of 8-Br-cAMP and okadaic acid, modulators of cellular kinases and phosphatases, on the hormone-induced activation of the MMTV promoter in two forms: a transiently transfected template with a disorganized, accessible nucleoprotein structure and a stably replicating template with an ordered, inaccessible nucleoprotein structure. Both okadaic acid and 8-Br-cAMP synergize significantly with either glucocorticoids or progestins in activating the transiently transfected MMTV template. In contrast, 8-Br-cAMP, but not okadaic acid, is antagonistic to hormone-induced activation of the stably replicating MMTV template. Nuclear run-on experiments demonstrate that this inhibition is a transcriptional effect on both hormone-induced transcription and basal transcription. Surprisingly, 8-Br-cAMP does not inhibit glucocorticoid-induced changes in restriction enzyme access and nuclear factor 1 binding. However, association of a complex with the TATA box region is inhibited in the presence of 8-Br-cAMP. Thus, cAMP treatment interferes with the initiation process but does not inhibit interaction of the receptor with the template. Since the replicated, ordered MMTV templates and the transfected, disorganized templates show opposite responses to 8-Br-cAMP treatment, we conclude that chromatin structure can influence the response of a promoter to activation of the cAMP signalling pathway.

A Ligand Binding Domain Mutation in the Mouse Glucocorticoid Receptor Functionally Links Chromatin Remodeling and Transcription Initiation

ABSTRACT We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter. A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2. The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal structure, though significantly less well than the wild-type GR. With an integrated, replicated promoter, which is assembled in an ordered nucleosomal array, the mutant GR does not activate transcription, and it fails to induce chromatin remodeling of the MMTV LTR promoter, as indicated by nuclease accessibility assays. Together, these findings support a two-step model for the transition of a nucleosome-assembled, repressed promoter to its transcriptionally active, derepressed form. In addition, we find that the C-terminal GR mutation is dominant over the transcription activation function of the N-terminal GR activation domain. These findings suggest that the primary activation function of the C-terminal activation domain is different from the function of the N-terminal activation domain and that it is required for derepression of the chromatin-repressed MMTV promoter.

A composite intronic element directs dynamic binding of the progesterone receptor and GATA-2

The progesterone receptor (PR) plays a pivotal role in proper development and function of the mammary gland and has also been implicated in mammary tumorigenesis. PR is a ligand-activated transcription factor; however, relatively, little is known about its mechanisms of action at endogenous target promoters. The aim of our study was to identify a natural PR-responsive gene and investigate its transcriptional regulation in the mammary microenvironment. Our experiments revealed FKBP5 as a direct target of the PR, because it exhibited a rapid activation by progestin that was cycloheximide independent and correlated with recruitment of RNA polymerase II to the promoter. Site-directed mutagenesis and chromatin immunoprecipitation assays showed that progestin responsiveness is mediated through a composite element in the first intron, to which the PR binds concomitantly with GATA-2. Mutational analysis of the element revealed that the GATA-2 site is essential for progestin activation. Direct binding of PR to DNA contributes to the efficiency of activation but is not sufficient, suggesting that the receptor makes important protein-protein interactions as part of its mechanism of action at the FKBP5 promoter. Using chromatin immunoprecipitation assays we also determined that the intronic region is in communication with the promoter, probably via DNA looping. Time course analysis revealed a cyclical pattern of PR recruitment to the FKBP5 gene but a persistent recruitment to the mouse mammary tumor virus promoter, indicating that receptor cycling is a gene-specific phenomenon rather than a characteristic of the receptor itself. Our study offers new insight in the nature of PR-regulated transcription in mammary cancer cells.

Glucocorticoid Receptor Domain Requirements for Chromatin Remodeling and Transcriptional Activation of the Mouse Mammary Tumor Virus Promoter in Different Nucleoprotein Contexts

The glucocorticoid receptor (GR) contains several activation domains, τ1 (AF-1), τ2, and AF-2, which were initially defined using transiently transfected reporter constructs. Using domain mutations in the context of full-length GR, this study defines those domains required for activation of the mouse mammary tumor virus (MMTV) promoter in two distinct nucleoprotein configurations. A transiently transfected MMTV template with a disorganized, accessible chromatin structure was largely dependent on the AF-2 domain for activation. In contrast, activation of an MMTV template in organized, replicated chromatin requires both domains but has a relatively larger dependence on the τ1 domain. Domain requirements for GR-induced chromatin remodeling of the latter template were also investigated. Mutation of the AF-2 helix 12 domain partially inhibits the induction of nuclease hypersensitivity, but the inhibition was relieved in the absence of τ1, suggesting the occurrence of an important interaction between the two domains. Further mutational analysis indicates that GR-induced chromatin remodeling requires the ligand-binding domain in the region of helix 3. Our study shows that the GR activation surfaces required for transcriptional modulation of a target promoter were determined in part by its chromatin structure. Within a particular cellular environment the GR appears to possess a significant degree of versatility in the mechanism by which it activates a target promoter.

Intranuclear trafficking and gene targeting by members of the steroid/nuclear receptor superfamily.

Upon binding to regulatory elements in mammalian chromosomes, steroid receptors induce specific transitions in the nucleoprotein structure of the template. These transitions reflect, in part, the reorganization of chromatin structure to permit interaction of secondary factors with target sequences in promoter regulatory regions. Steroid receptors represent a class of transcriptional activators that are able to interact with repressed nucleoprotein templates and recruit necessary activities for chromatin remodeling. The ligand-induced movement of nuclear receptors from inactive states, either in the cytoplasm or in the nucleus, to productive interactions with chromatin is complex and likely reflects the interaction with multiple protein complexes and subcellular structures. Regulation of gene expression by nuclear receptors is thus mediated through the subcellular distribution of inactive receptors, the redistribution of activated receptor complexes to appropriate nuclear domains, the reorganization of chromatin structures for interaction with soluble components of the nucleoplasm, and direct protein-protein contacts between receptors and the basal transcription apparatus.

Class i lysine deacetylases facilitate glucocorticoid-induced transcription

Background: KDACis impair GR transactivation of the MMTV promoter, but their impact on cellular target genes is unknown. Results: KDACi or KDAC depletion suppresses transactivation of about 50% of GR target genes. Conclusion: KDAC1 is required for efficient GR transactivation in a gene-selective fashion. Significance: Because KDACs facilitate GR transactivation, clinical KDACi use may have a major impact on GR signaling. Nuclear receptors use lysine acetyltransferases and lysine deacetylases (KDACs) in regulating transcription through histone acetylation. Lysine acetyltransferases interact with steroid receptors upon binding of an agonist and are recruited to target genes. KDACs have been shown to interact with steroid receptors upon binding to an antagonist. We have shown previously that KDAC inhibitors (KDACis) potently repress the mouse mammary tumor virus promoter through transcriptional mechanisms and impair the ability of the glucocorticoid receptor (GR) to activate it, suggesting that KDACs can play a positive role in GR transactivation. In the current study, we extended this analysis to the entire GR transcriptome and found that the KDACi valproic acid impairs the ability of agonist-bound GR to activate about 50% of its target genes. This inhibition is largely due to impaired transcription rather than defective GR processing and was also observed using a structurally distinct KDACi. Depletion of KDAC1 expression mimicked the effects of KDACi in over half of the genes found to be impaired in GR transactivation. Simultaneous depletion of KDACs 1 and 2 caused full or partial impairment of several more GR target genes. Altogether we found that Class I KDAC activity facilitates GR-mediated activation at a sizable fraction of GR-activated target genes and that KDAC1 alone or in coordination with KDAC2 is required for efficient GR transactivation at many of these target genes. Finally, our work demonstrates that KDACi exposure has a significant impact on GR signaling and thus has ramifications for the clinical use of these drugs.

The effect of sulforaphane on histone deacetylase activity in keratinocytes: Differences between in vitro and in vivo analyses

Sulforaphane is a natural product found in broccoli, which is known to exert many different molecular effects in the cell, including inhibition of histone deacetylase (HDAC) enzymes. Here, we examine for the first time the potential for sulforaphane to inhibit HDACs in HaCaT keratinocytes and compare our results with those found using HCT116 colon cancer cells. Significant inhibition of HDAC activity in HCT116 nuclear extracts required prolonged exposure to sulforaphane in the presence of serum. Under the same conditions HaCaT nuclear extracts did not exhibit reduced HDAC activity with sulforaphane treatment. Both cell types displayed down‐regulation of HDAC protein levels by sulforaphane treatment. Despite these reductions in HDAC family member protein levels, acetylation of marker proteins (acetylated Histone H3, H4, and tubulin) was decreased by sulforaphane treatment. Time‐course analysis revealed that HDAC6, HDAC3, and acetylated histone H3 protein levels are significantly inhibited as early as 6 h into sulforaphane treatment. Transcript levels of HDAC6 are also suppressed after 48 h of treatment. These results suggest that HDAC activity noted in nuclear extracts is not always translated as expected to target protein acetylation patterns, despite dramatic inhibition of some HDAC protein levels. In addition, our data suggest that keratinocytes are at least partially resistant to the nuclear HDAC inhibitory effects of sulforaphane, which is exhibited in HCT116 and other cells.