Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole.

Individual variation in drug metabolism is a major cause of unpredictable side effects during therapy. Drug metabolism is controlled by a class of orphan nuclear receptors (NRs), which regulate expression of genes such as CYP (cytochrome)3A4 and MDR-1 (multi-drug resistance-1), that are involved in this process. We have found that xenobiotic-mediated induction of CYP3A4 and MDR-1 gene transcription was inhibited by ketoconazole, a commonly used antifungal drug. Ketoconazole mediated its effect by inhibiting the activation of NRs, human pregnenolone X receptor and constitutive androstene receptor, involved in regulation of CYP3A4 and MDR-1. The effect of ketoconazole was specific to the group of NRs that control xenobiotic metabolism. Ketoconazole disrupted the interaction of the xenobiotic receptor PXR with the co-activator steroid receptor co-activator-1. Ketoconazole treatment resulted in delayed metabolism of tribromoethanol anesthetic in mice, which was correlated to the inhibition of PXR activation and downmodulation of cyp3a11 and mdr-1 genes and proteins. These studies demonstrate for the first time that ketoconazole represses the coordinated activation of genes involved in drug metabolism, by blocking activation of a specific subset of NRs. Our results suggest that ketoconazole can be used as a pan-antagonist of NRs involved in xenobiotic metabolism in vivo, which may lead to novel strategies that improve drug effect and tolerance.

Targeting cyclin D1, a downstream effector of INI1/hSNF5, in rhabdoid tumors.

Rhabdoid tumors (RTs) are aggressive and currently incurable pediatric malignancies. INI1/hSNF5 is a tumor suppressor biallelically inactivated in RTs. Our previous studies have indicated that cyclin D1 is a key downstream target of INI1/hSNF5 and genesis and/or survival of RTs in vivo is critically dependent on the presence of cyclin D1. In this report, we have tested the hypothesis that therapeutic targeting of cyclin D1 is an effective means of treating RTs. We found that RNA interference of cyclin D1 in rhabdoid cells was sufficient to induce G1 arrest and apoptosis. Furthermore, we found that pharmacological intervention with low micromolar concentrations of N-(4-hydroxyphenyl)retinamide (4-HPR), which downmodulates cyclin D1, induced G1 arrest and apoptosis in rhabdoid cell lines. 4-HPR in combination with 4-hydroxy-tamoxifen (4OH-Tam), synergistically inhibited survival as well as anchorage-dependent and -independent growth of rhabdoid cells and caused synergistic induction of cell cycle arrest and apoptosis. 4-HPR and tamoxifen exhibited synergistic growth inhibition of RTs in xenograft models in vivo. The effects of combination of drugs were correlated to the depletion of cyclin D1 levels both in in vitro and in vivo tumor models. These results demonstrate that 4-HPR and tamoxifen are effective chemotherapeutic agents for RTs. We propose that downmodulation of cyclin D1 is a novel and effective therapeutic strategy for RTs.

Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications.

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.

Inhibition of HIV-1 virion production by a transdominant mutant of integrase interactor 1.

Integase interactor 1 (INI1), also known as hSNF5, is a protein that interacts with HIV-1 integrase. We report here that a cytoplasmically localized fragment of INI1 (S6; aa183-294) containing the minimal integrase-interaction domain potently inhibits HIV-1 particle production and replication. Mutations in S6 or integrase that disrupt integrase-INI1 interaction abrogated the inhibitory effect. An integrase-deficient HIV-1 transcomplemented with integrase fused to Vpr was not affected by S6. INI1 was specifically incorporated into virions and was required for efficient HIV-1 particle production. These results indicate that INI1 is required for late events in the viral life cycle, and that ectopic expression of S6 inhibits HIV-1 replication in a transdominant manner via its specific interaction with integrase within the context of Gag-Pol, providing a novel strategy to control HIV-1 replication.

Isolation and characterization of an oligomerization-negative mutant of HIV-1 integrase.

The yeast two-hybrid method was used to screen mutagenized DNAs to isolate a variant of the human immunodeficiency virus type 1 integrase (IN) that does not interact with the wild-type IN. The responsible mutation, leading to a single amino acid change (V260E) in the C-terminal domain of IN, blocks IN-IN multimerization but has only small effect on binding to a host interacting protein, INI1 (hSNF5). Binding studies in vitro confirmed the defect in multimerization of the mutant IN. Biochemical analyses of the mutant IN enzyme expressed in bacteria detected only subtle changes in its properties, suggesting that the yeast system is a sensitive reporter of correct IN conformation. Mutant virus carrying the V260E substitution was blocked in replication at the time of DNA integration, consistent with IN multimerization being important for its activity in vivo.

Retroviral vectors for liver-directed gene therapy.

Retroviruses are popular gene therapy vectors because they stably integrate the DNA copy of their genome into the host chromosome during their replication cycle. The widely used murine retroviral vector systems have two components: the transfer vector for the transgene carries all the cis-acting elements necessary for the replication and efficient integration of the viral DNA; and the packaging cell line produces all the trans-acting proteins necessary for both structural and catalytic functions of the virus. Advances in design of retroviral vectors have resulted in greater degree of biosafety, expanded host range, and increased stability of the virus particles. Retroviral vectors have been widely used in the ex vivo gene therapy protocols to correct the liver diseases in a wide variety of species. In a limited number of applications, in vivo gene therapy has been achieved after the liver cells have been stimulated to regenerate. One major limitation of murine retroviral vectors is their inability to infect nondividing cells. This problem has been overcome by deriving vectors from lentiviruses (a class of retroviruses) that have the ability to infect both dividing and nondividing cells. The lentiviral vectors are derived from human immunodeficiency virus type 1 (HIV-1). Initial studies using lentiviral vectors for gene delivery to the liver in vivo show promising results. A highly crippled version of lentivirus has been generated by using producer cells in which the trans-acting components are expressed by several different coding elements and vectors that incorporate features of self-inactivation. These improvements should ensure biosafety of lentiviruses and make them useful in efficient delivery of therapeutic genes to nondividing differentiated tissues such as the liver.

Interaction of E1 and hSNF5 proteins stimulates replication of human papillomavirus DNA.

Mammalian viruses often use components of the host's cellular DNA replication machinery to carry out replication of their genomes, which enables these viruses to be used as tools for characterizing factors that are involved in cellular DNA replication. The human papillomavirus (HPV) E1 protein is essential for replication of the virus DNA. Here we identify the cellular factor that participates in viral DNA replication by using a two-hybrid assay in the yeast Saccharomyces cerevisiae and E1 protein as bait. Using this assay, we isolated Inil/hSNF5, a component of the SWI/SNF complex which facilitates transcription by altering the structure of chromatin. In vitro binding and immunoprecipitation confirmed that E1 interacts directly with Ini1/hSNF5. Transient DNA-replication assay revealed that HPV DNA replication is stimulated in a dose-dependent manner by addition of Ini1/hSNF5, and that Ini1/hSNF5 antisense RNA blocks the replication of HPV DNA. Amino-acid substitution at residues that are conserved among E1 proteins prevented the E1-Ini1/hSNF5 interaction and reduced DNA replication of HPV in vivo. Our results indicate that Ini1/hSNF5 is required for the efficient replication of papillomavirus DNA and is therefore needed, either alone or in complex with SWI/SNF complex, for mammalian DNA replication as well.

c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function.

Chromatin organization plays a key role in the regulation of gene expression. The evolutionarily conserved SWI/SNF complex is one of several multiprotein complexes that activate transcription by remodelling chromatin in an ATP-dependent manner. SWI2/SNF2 is an ATPase whose homologues, BRG1 and hBRM, mediate cell-cycle arrest; the SNF5 homologue, INI1/hSNF5, appears to be a tumour suppressor. A search for INI1-interacting proteins using the two-hybrid system led to the isolation of c-MYC, a transactivator. The c-MYC-INI1 interaction was observed both in vitro and in vivo. The c-MYC basic helix-loop-helix (bHLH) and leucine zipper (Zip) domains and the INI1 repeat 1 (Rpt1) region were required for this interaction. c-MYC-mediated transactivation was inhibited by a deletion fragment of INI1 and the ATPase mutant of BRG1/hSNF2 in a dominant-negative manner contingent upon the presence of the c-MYC bHLH-Zip domain. Our results suggest that the SWI/SNF complex is necessary for c-MYC-mediated transactivation and that the c-MYC-INI1 interaction helps recruit the complex.

Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex.

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN in trans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.

Structure-function analysis of integrase interactor 1/hSNF5L1 reveals differential properties of two repeat motifs present in the highly conserved region.

Retroviral integrase (IN) catalyzes the integration of retroviral cDNA into host chromosome. Ini1 (integrase interactor 1) is a host protein that specifically binds and stimulates in vitro joining activity of HIV-1 IN. Ini1 has homology to yeast transcription factor SNF5 and is a component of the analogous mammalian SWI/SNF complex that can remodel chromatin. Little is known about the function of Ini1 in mammalian cells. To gain insight into the functional domains of Ini1, and to understand the details of protein-protein interactions of IN and Ini1, a structure-function analysis of Ini1 was initiated. By means of the yeast two-hybrid system, the minimal IN binding domain of Ini1 was characterized. One of the two repeat motifs present in the highly conserved regions of Ini1 was found necessary and sufficient to bind to IN in yeast as well as in vitro. Because IN binds to only one of the two repeat motifs in this conserved region of Ini1, it appears that the IN-Ini1 interaction is very specific and functionally significant. Characterization of DNA-binding properties of Ini1 revealed that Ini1 can bind to plasmid DNA, binding more readily to supercoiled DNA than to the relaxed circular DNA. The minimal domain for DNA binding was localized to a region upstream of repeat 1. The DNA binding activity of Ini1 is not required for its ability to interact with IN. The finding that the two repeat motifs of Ini1 display differential binding to HIV-1 IN and that this discrete component of mammalian SWI/SNF complex binds to DNA will help understand the role of Ini1 in HIV-1 integration and in cellular process.

Purification and biochemical heterogeneity of the mammalian SWI-SNF complex.

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type.

Epstein-Barr virus nuclear protein 2 (EBNA2) binds to a component of the human SNF-SWI complex, hSNF5/Ini1.

Epstein-Barr nuclear antigen 2 (EBNA2), one of the six viral nuclear proteins expressed in latently infected B lymphocytes, is essential to the immortalization of B cells by Epstein-Barr virus (EBV). EBNA2 promotes transcriptional transactivation of viral and cellular genes by acting as an adapter molecule that binds to cellular sequence-specific DNA-binding proteins, JK recombination signal-binding protein (RBP-JK), and PU.1 and engages multiple members of the RNA polymerase II transcription complex. In the present study, we show that EBNA2 also interacts with hSNF5/Ini1, the human homolog of the yeast transcription factor SNF5. Gel filtration fractionation of partially purified EBV-positive lymphocyte nuclear extracts shows that a fraction of EBNA2 coelutes with both hSNF5/Ini1 and BRG1, a human homolog of SWI/SNF2, in the high-molecular-mass region (1.5 to 2.0 MDa) of a Superose 6 chromatogram. An affinity-purified rabbit antibody directed against hSNF5/Ini1 coimmunoprecipitates EBNA2 from this high-molecular-mass nuclear protein fraction, demonstrating that EBNA2 and hSNF5/Ini1 interact in vivo. This interaction is restricted to a subpopulation of phosphorylated viral EBNA2. Deletion mutation analysis of EBNA2 shows that the proline-rich aminoterminal end and a domain within the divergent region of EBNA2 mediate EBNA2-hSNF5/Ini1 interaction. Since the SNF-SWI complex participates in gene regulation through the alteration of nucleosome configuration and may be a component of the RNA polymerase II holoenzyme, the EBNA2-hSNF5/Ini1 interaction supports the hypothesis that EBNA2 facilitates transcriptional transactivation by acting as a transcription adapter molecule. We postulate that EBNA2 engages the hSNF-SWI complex to generate an open chromatin conformation at the EBNA2-responsive target genes, thereby potentiating the function of the RBP-JK-EBNA2-polymerase II transcription complex.

The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex.

During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin.

Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5.

Upon entry into a host cell, retroviruses direct the reverse transcription of the viral RNA genome and the establishment of an integrated proviral DNA. The retroviral integrase protein (IN) is responsible for the insertion of the viral DNA into host chromosomal targets. The two-hybrid system was used to identify a human gene product that binds tightly to the human immunodeficiency virus-type 1 (HIV-1) integrase in vitro and stimulates its DNA-joining activity. The sequence of the gene suggests that the protein is a human homolog of yeast SNF5, a transcriptional activator required for high-level expression of many genes. The gene, termed INI1 (for integrase interactor 1), may encode a nuclear factor that promotes integration and targets incoming viral DNA to active genes.

Inhibition of transcriptional regulator Yin-Yang-1 by association with c-Myc.

Yin-Yang-1 (YY1) regulates the transcription of many genes, including the oncogenes c-fos and c-myc. Depending on the context, YY1 acts as a transcriptional repressor, a transcriptional activator, or a transcriptional initiator. The yeast two-hybrid system was used to screen a human complementary DNA (cDNA) library for proteins that associate with YY1, and a c-myc cDNA was isolated. Affinity chromatography confirmed that YY1 associates with c-Myc but not with Max. In cotransfections, c-Myc inhibits both the repressor and the activator functions of YY1, which suggests that one way c-Myc acts is by modulating the activity of YY1.

Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system.

The retroviral integrase protein (IN) is responsible for catalyzing a concerted integration reaction in which the two termini of linear viral DNA are joined to host DNA. To probe the potential for IN to form protein multimers, we used the yeast two-hybrid system. The coexpression of a GAL4 DNA binding domain-IN fusion and a GAL4 activation domain-IN fusion together resulted in the successful activation of a GAL4-responsive LacZ reporter gene. The system was used to examine a variety of IN deletion mutants. The results suggest that the central region of the protein is necessary for multimerization and that the N-terminal zinc finger region is not important.

Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B.

Retroviral Gag protein is capable of directing the assembly of virion particles independent of other retroviral elements and plays an important role early in the infection of a cell. Using the GAL4 two hybrid system, we screened a cDNA expression library and identified two host proteins, cyclophillins (CyPs) A and B, which interact specifically with the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein Pr55gag. Glutathione S-transferase-CyP fusion proteins bind tightly to Pr55gag in vitro, as well as to the HIV-1 capsid protein p24. Cyclosporin A efficiently disrupts the Gag-CyPA interaction and less efficiently disrupts the Gag-CyPB interaction. The Gag-CyP interaction may be important for the HIV-1 life cycle and may be relevant to the pathology caused by this immunosuppressive virus.

Insertional mutagenesis and illegitimate recombination in mycobacteria.

Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man. Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria. To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis and bacille Calmette-Guérin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis. A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors. Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M. tuberculosis and BCG.