The activity of the glucocorticoid receptor is regulated by SUMO conjugation to FKBP51.

FK506-binding protein 51 (FKBP51) regulates the activity of the glucocorticoid receptor (GR), and is therefore a key mediator of the biological actions of glucocorticoids. However, the understanding of the molecular mechanisms that govern its activity remains limited. Here, we uncover a novel regulatory switch for GR activity by the post-translational modification of FKBP51 with small ubiquitin-like modifier (SUMO). The major SUMO-attachment site, lysine 422, is required for FKBP51-mediated inhibition of GR activity in hippocampal neuronal cells. Importantly, impairment of SUMO conjugation to FKBP51 impacts on GR-dependent neuronal signaling and differentiation. We demonstrate that SUMO conjugation to FKBP51 is enhanced by the E3 ligase PIAS4 and by environmental stresses such as heat shock, which impact on GR-dependent transcription. SUMO conjugation to FKBP51 regulates GR hormone-binding affinity and nuclear translocation by promoting FKBP51 interaction within the GR complex. SUMOylation-deficient FKBP51 fails to interact with Hsp90 and GR thus facilitating the recruitment of the closely related protein, FKBP52, which enhances GR transcriptional activity. Moreover, we show that the modification of FKBP51 with SUMO modulates its binding to Hsp90. Our data establish SUMO conjugation as a novel regulatory mechanism in the Hsp90 cochaperone activity of FKBP51 with a functional impact on GR signaling in a neuronal context.

Beware of your Cre-Ation: lacZ expression impairs neuronal integrity and hippocampus-dependent memory.

Expression of the lacZ-sequence is a widely used reporter-tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is ß-galactosidase, an analog to the mammalian senescence-associated ß-galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub-populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus-dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper-arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression. © 2016 Wiley Periodicals, Inc.

FKBP51 inhibits GSK3ß and augments the effects of distinct psychotropic medications.

Psychotropic medications target glycogen synthase kinase 3ß (GSK3ß), but the functional integration with other factors relevant for drug efficacy is poorly understood. We discovered that the suggested psychiatric risk factor FK506 binding protein 51 (FKBP51) increases phosphorylation of GSK3ß at serine 9 (pGSK3ß(S9)). FKBP51 associates with GSK3ß mainly through its FK1 domain; furthermore, it also changes GSK3ß's heterocomplex assembly by associating with the phosphatase PP2A and the kinase cyclin-dependent kinase 5. FKBP51 acts through GSK3ß on the downstream targets Tau, ß-catenin and T-cell factor/lymphoid enhancing factor (TCF/LEF). Lithium and the antidepressant (AD) paroxetine (PAR) functionally synergize with FKBP51, as revealed by reporter gene and protein association analyses. Deletion of FKBP51 blunted the PAR- or lithium-induced increase in pGSK3ß(S9) in cells and mice and attenuated the behavioral effects of lithium treatment. Clinical improvement in depressive patients was predicted by baseline GSK3ß pathway activity and by pGSK3ß(S9) reactivity to ex vivo treatment of peripheral blood mononuclear lymphocytes with lithium or PAR. In sum, FKBP51-directed GSK3ß activity contributes to the action of psychotropic medications. Components of the FKBP51-GSK3ß pathway may be useful as biomarkers predicting AD response and as targets for the development of novel ADs.

Deciphering the spatio-temporal expression and stress regulation of Fam107B, the paralog of the resilience-promoting protein DRR1 in the mouse brain.

Understanding the molecular mechanisms that promote stress resilience might open up new therapeutic avenues to prevent stress-related disorders. We recently characterized a stress and glucocorticoid-regulated gene, down-regulated in renal cell carcinoma - DRR1 (Fam107A). DRR1 is expressed in the mouse brain; it is up-regulated by stress and glucocorticoids and modulates neuronal actin dynamics. In the adult mouse, DRR1 was shown to facilitate specific behaviors which might be protective against some of the deleterious consequences of stress exposure: in the hippocampal CA3 region, DRR1 improved cognitive performance whereas in the septum, it specifically increased social behavior. Therefore DRR1 was suggested as a candidate protein promoting stress-resilience. Fam107B (family with sequence similarity 107, member B) is the unique paralog of DRR1, and both share high sequence similarities, predicted glucocorticoid response elements, heat-shock induction and tumor suppressor properties. So far, the role of Fam107B in the central nervous system was not studied. The aim of the present investigation, therefore, was to analyze whether Fam107B and DRR1 display comparable mRNA expression patterns in the brain and whether both are modulated by stress and glucocorticoids. Spatio-temporal mapping of Fam107B mRNA expression in the embryonic and adult mouse brain, by means of in situ hybridization, showed that Fam107B was expressed during embryogenesis and in the adulthood, with particularly high and specific expression in the forming telencephalon suggestive of an involvement in corticogenesis. In the adult mouse, expression was restricted to neurogenic niches, like the dentate gyrus. In contrast to DRR1, Fam107B mRNA expression failed to be modulated by glucocorticoids and social stress in the adult mouse. In summary, Fam107B and DRR1 show different spatio-temporal expression patterns in the central nervous system, suggesting at least partially different functional roles in the brain, and where the glucocorticoid receptor (GR)-induced regulation appears to be a unique property of DRR1.

Acute stress regulation of neuroplasticity genes in mouse hippocampus CA3 area--possible novel signalling pathways.

Stress exposure can lead to the precipitation of psychiatric disorders in susceptible individuals, but the molecular underpinnings are incompletely understood. We used forced swimming in mice to reveal stress-regulated genes in the CA3 area of the hippocampus. To determine changes in the transcriptional profile 4 h and 8 h after stress exposure microarrays were used in the two mouse strains C57BL/6J and DBA/2J, which are known for their differential stress response. We discovered a surprisingly distinct set of regulated genes for each strain and followed selected ones by in situ hybridisation. Our results support the concept of a phased transcriptional reaction to stress. Moreover, we suggest novel stress-elicited pathways, which comprise a number of genes involved in the regulation of neuronal plasticity. Furthermore, we focused in particular on dihydropyrimidinase like 2, to which we provide evidence for its regulation by NeuroD, an important factor for neuronal activity-dependent dendritic morphogenesis.

Enantioconvergent synthesis by sequential asymmetric Horner-Wadsworth-Emmons and palladium-catalyzed allylic substitution reactions.

A new method for enantioconvergent synthesis has been developed. The strategy relies on the combination of an asymmetric Horner-Wadsworth-Emmons (HWE) reaction and a palladium-catalyzed allylic substitution. Different alpha-oxygen-substituted, racemic aldehydes were initially transformed by asymmetric HWE reactions into mixtures of two major alpha,beta-unsaturated esters, possessing opposite configurations at their allylic stereocenters as well as opposite alkene geometry. Subsequently, these isomeric mixtures of alkenes could be subjected to palladium-catalyzed allylic substitution reactions with carbon, nitrogen, and oxygen nucleophiles. In this latter step, the respective (E) and (Z) alkene substrate isomers were observed to react with opposite stereospecificity: the (E) alkene reacted with retention and the (Z) alkene with inversion of stereochemistry with respect to both the allylic stereocenter and the alkene geometry. Thus, a single gamma-substituted ester was obtained as the overall product, in high isomeric purity. The method was applied to a synthesis of a subunit of the iejimalides, a group of cytotoxic macrolides.

Synergistic inhibition of the glucocorticoid receptor by radicicol and benzoquinone ansamycins.

Radicicol (RAD) and the benzoquinone ansamycin geldanamycin (GA) are potential anticancer drugs known to inhibit heat shock protein 90 (hsp90) and, therefore, the activation of proteins dependent on its function such as proto-oncogenic kinases and nuclear receptors. Using the glucocorticoid receptor (GR) as a model system we analysed the effects of RAD and various benzoquinone ansamycins. All compounds efficiently abolished GR-dependent transactivation. Surprisingly, whenever one of the ansamycins was applied in combination with RAD, synergistic inhibition of GR-dependent transcription and of hormone binding of GR was observed. In contrast, combination of two ansamycins showed no synergy. These findings suggest synergism within the hsp90 dimer and may open new ways to explore hsp90 as therapeutic target.

A versatile stereocontrolled approach to chiral tetrahydrofuran and tetrahydropyran derivatives via sequential asymmetric Horner-Wadsworth-Emmons and palladium-catalyzed ring closure reactions

[reaction: see text]An approach to chiral tetrahydrofuran and tetrahydropyran derivatives is reported which is based on the sequential use of an asymmetric Horner-Wadsworth-Emmons desymmetrization of a meso-dialdehyde and a palladium-catalyzed intramolecular allylic substitution. The strategy is versatile in that either a cis- or a trans-relation between the stereocenters adjacent to the ring oxygen can be obtained.

Parallel kinetic resolution of racemic aldehydes by use of asymmetric Horner-Wadsworth-Emmons reactions

[reaction: see text] A racemic aldehyde can undergo parallel kinetic resolution (PKR) by simultaneous reaction with two different chiral phosphonates, differing either in the structure of the chiral auxiliary or in the structure of the phosphoryl group (i.e., one (E)- and one (Z)-selective reagent). This strategy allows conversion of a racemic aldehyde to two different, synthetically useful chiral products with essentially doubled material throughput and similar or improved selectivities as compared to conventional kinetic resolution.

Rifampicin is not an activator of glucocorticoid receptor.

Rifampicin, an antibiotic widely used in tuberculosis therapy, is known to exert psychotropic side effects in some patients. Recently, rifampicin has been reported to activate the glucocorticoid receptor (GR) in human hepatocytes. Because there is evidence that increased levels of glucocorticoids may induce cognitive impairment, sometimes culminating in depression, the side effects of rifampicin may result from GR activation in central nerve cells. Therefore, we used reporter gene assays to determine whether rifampicin displays glucocorticoid-like effects in human neuroblastoma SK-N-MC cells or mouse hippocampal HT22 cells. Rifampicin was unable to elicit any detectable transactivation of GR in both cell types, whereas cortisol or dexamethasone led to a potent transcriptional response. Rifampicin was also inactive in the same HepG2 cell line that was originally used to demonstrate the effect of rifampicin on GR. Moreover, rifampicin was unable to compete with dexamethasone for binding to GR. Finally, by blocking the multidrug resistance P-glycoprotein transporter (a xenobiotic extrusion pump) with verapamil or cyclosporin A, we excluded the possibility that the lack of effect by rifampicin was due to its export from the cell. Our results establish that rifampicin does not activate GR, and rule out the hypothesis that the psychotropic side effects of rifampicin treatment are a consequence of GR activation.

DNA methylation at mammalian replication origins.

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene. Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.

Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone.

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.

Identifying 5-methylcytosine and related modifications in DNA genomes.

Intense interest in the biological roles of DNA methylation, particularly in eukaryotes, has produced at least eight different methods for identifying 5-methylcytosine and related modifications in DNA genomes. However, the utility of each method depends not only on its simplicity but on its specificity, resolution, sensitivity and potential artifacts. Since these parameters affect the interpretation of data, they should be considered in any application. Therefore, we have outlined the principles and applications of each method, quantitatively evaluated their specificity,resolution and sensitivity, identified potential artifacts and suggested solutions, and discussed a paradox in the distribution of m5C in mammalian genomes that illustrates how methodological limitations can affect interpretation of data. Hopefully, the information and analysis provided here will guide new investigators entering this exciting field.

Absence of an unusual "densely methylated island" at the hamster dhfr ori-beta.

An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-beta locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G1, S, or G2 + M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines (mC) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of mC residues that complements the more commonly used hydrazine method. These methods were capable of detecting mC in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori-beta DMI was not detected in any of these cell populations using any of these methods.

Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.

ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins.

Expression of a 74-kDa nuclear factor 1 (NF1) protein is induced in mouse mammary gland involution. Involution-enhanced occupation of a twin NF1 binding element in the testosterone-repressed prostate message-2/clusterin promoter.

Testosterone repressed prostate message-2 (TRPM-2)/clusterin gene expression is rapidly induced in early involution of the mouse mammary gland, after weaning, and in the rat ventral prostate, after castration. A search for involution-enhanced DNaseI footprints in the proximal mouse TRPM-2/clusterin gene promoter led to the identification and characterization (by DNase I footprinting and EMSA) of a twin nuclear factor 1 (NF1) binding element at -356/-309, relative to the proposed transcription start site; nuclear extracts from 2-day involuting mouse mammary gland showed an enhanced footprint over the proximal NF1 element; extracts from involuting prostate showed enhanced occupancy of both NF1 binding elements. Subsequent EMSA and Western analysis led to the detection of a 74-kDa NF1 protein whose expression is triggered in early involution in the mouse mammary gland; such an induced protein is not found in the involuting rat ventral prostate. This protein was not found in lactation where three other NF1 proteins of 114, 68, and 46 kDa were detected. Reiteration of the epithelial cell apoptosis associated with early mammary gland involution, in vitro, in a primary cell culture system, triggered the appearance of the 74-kDa NF1. Overlaying the cells with laminin-rich extracellular matrix suppressed the apoptosis and the expression of the 74-kDa NF1 and, in the presence of lactogenic hormones, initiated milk protein gene expression and the expression of two of the lactation-associated NF1 proteins (68 and 46 kDa). This study, thus, identifies for the first time the occurrence of a switch in expression of different members of the family of NF1 transcription factors as mammary epithelial cells move from the differentiated to the involution/apoptotic state, and it is likely that the involution-specific 74-kDa NF1 accounts for the enhanced NF1 footprint detected on the TRPM-2/clusterin promoter with extracts of mouse mammary gland.

YY1 and NF1 both activate the human p53 promoter by alternatively binding to a composite element, and YY1 and E1A cooperate to amplify p53 promoter activity.

A novel transcription factor binding element in the human p53 gene promoter has been characterized. It lies about 100 bp upstream of the major reported start site for human p53 gene transcription. On the basis of DNase I footprinting studies, electromobility shift assay patterns, sequence specificity of binding, the binding pattern of purified transcription factors, effects of specific antibodies, and methylation interference analysis we have identified the site as a composite element which can bind both YY1 and NF1 in an independent and mutually exclusive manner. The site is conserved in the human, rat, and mouse p53 promoters. The occupancy of the site varies in a tissue-specific manner. It binds principally YY1 in nuclear extracts of rat testis and spleen and NF1 in extracts of liver and prostate. This may facilitate tissue-specific control of p53 gene expression. When HeLa cells were transiently transfected with human p53 promoter-chloramphenicol acetyltransferase reporter constructs, a mutation in this composite element which disabled YY1 and NF1 binding caused a mean 64% reduction in basal p53 promoter activity. From mutations which selectively impaired YY1 or NF1 binding and the overexpression of YY1 or NF1 in HeLa cells we concluded that both YY1 and NF1 function as activators when bound to this site. In transient cotransfections E1A could induce the activity of the p53 promoter to a high level; 12S E1A was threefold as efficient as 13S E1A in this activity, and YY1 bound to the composite element was shown to mediate 55% of this induction. Overexpressed YY1 was shown to be able to synergistically activate the p53 promoter with E1A when not specifically bound to DNA. Deletion of an N-terminal domain of E1A, known to be required for direct E1A-YY1 interaction and E1A effects mediated through transcriptional activator p300, blocked the E1A induction of p53 promoter activity.

Organization of the alpha-globin promoter and possible role of nuclear factor I in an alpha-globin-inducible and a noninducible cell line.

Nuclear factor I (NFI) was suggested to be involved in the expression of the human alpha-globin gene. Two established cell lines, which express alpha-globin differentially, were therefore compared for differences in binding of NFI at the alpha-globin promoter in vivo. HeLa cells, in which alpha-globin is repressed, show a high density promoter occupation with several proteins associated with structurally distorted DNA. Cell line K562, which is inducible for alpha-globin, surprisingly was found to be heterogeneous consisting mainly of cells (approximately 95%) unable to express alpha-globin. However, the promoter of the nonexpressing K562 cells was clearly different from that of HeLa cells, being occupied only at basal transcriptional elements. Therefore, the alpha-globin gene in these K562 cells may not be truly repressed, but in an intermediate state between repression and active transcription. The NFI site of the alpha-globin promoter appeared occupied in HeLa but free of proteins in K562 cells. All cells of both cell lines produce NFI, but the composition and DNA binding affinity of NFI species differ significantly between the two cell lines. Therefore, distinct forms of NFI may repress alpha-globin transcription in HeLa cells. However, NFI is apparently not involved in establishing the latent transcriptional state of the majority of K562 cells.

Conserved high-affinity NF-kappa B binding site in the interferon regulatory factor-1 promoter is not occupied by NF-kappa B in vivo and is transcriptionally inactive.

The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast cells, despite the simultaneous induction of IRF-1 mRNA and NF-kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.

In vivo footprinting of the IRF-1 promoter: inducible occupation of a GAS element next to a persistent structural alteration of the DNA.

GAS (gamma activated sequence) and GAS-like elements are found in a rapidly growing number of genes. Data from EMSA (electromobility shift assay) and transient transfection assays using heterologous promoter systems do not necessarily reflect transcriptional involvement and protein occupation of a binding site in vivo. This has been shown recently by in vivo footprinting of the NF-kappa B site at -40 in the interferon regulatory factor-1 (IRF-1) promoter. Here we show by in vivo footprinting using dimethylsulfate (DMS) that the GAS of the IRF-1 promoter, which also contains an overlapping putative NF-kappa B site, is occupied upon treatment with gamma-interferon (IFN gamma) but not with phorbol 12-myristate 13-acetate (PMA). Irrespective of induction, we detect a very strong DMS hypersensitivity at a guanosine just adjacent to GAS and a less persistent minor DMS hypersensitivity at a central cytosine. Our data confirm the crucial role of GAS in transcriptional activation by IFN gamma and are consistent with induced binding of p91 to GAS. In addition, our data suggest a major conformational distortion of the DNA at the GAS element of the IRF-1 promoter and that this GAS element is not involved in transcriptional activation by PMA.