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J P Whitlock

Publications and source records attributed to J P Whitlock.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cytochromes P450 5: induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription.

The induction of microsomal cytochrome P4501A1 by polycyclic aromatic hydrocarbons represents an interesting response by which mammalian cells adapt to xenobiotic exposure. Enzyme induction reflects increased transcription of the corresponding CYP1A1 gene. Analyses of the induction mechanism using genetic, biochemical, and molecular biological approaches have revealed a novel transcriptional regulatory pathway that involves ligand-dependent heterodimerization between two basic helix-loop-helix proteins (the Ah receptor and Arnt), interaction of the heterodimer with a xenobiotic-responsive enhancer, transmission of the induction signal from the enhancer to the CYP1A1 promoter, and alterations in chromatin structure. Current techniques permit examination of the induction mechanism in intact cells and analyses of the CYP1A1 gene in its native chromosomal configuration. Such experiments generate new insights into the control of mammalian transcription that are of relatively broad interest.

Animals↗
PubMed1996

Dioxin-induced CYP1A1 transcription in vivo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure.

We have analyzed the dioxin-inducible transcriptional control mechanism for the mouse CYP1A1 gene in its native chromosomal context. Our genetic and biochemical studies indicate that a C-terminal segment of the aromatic hydrocarbon receptor (AhR) contains latent transactivation capability and communicates the induction signal from enhancer to promoter. Thus, transactivation and enhancer-promoter communication may be congruent functions of AhR. Both functions require heterodimerization between AhR and the AhR nuclear translocator (Arnt). Our findings also indicate that heterodimerization activates AhR's latent transactivation function and silences that of Arnt. Furthermore, removal of Arnt's transactivation domain does not affect dioxin-induced CYP1A1 transcription in vivo. In addition, our studies demonstrate that dioxin-induced changes in chromatin structure occur by different mechanisms at the CYP1A1 enhancer and promoter and that events at an enhancer can be experimentally dissociated from events at the cognate promoter during mechanistic analyses of mammalian transcription in vivo.

Animals↗
PubMed1996

The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin.

The aromatic hydrocarbon receptor (AhR) has been defined and characterized according to its ability to mediate biological responses to exogenous ligands, such as the synthetic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand(s) for AhR is unknown, and we know relatively little about AhR function in the absence of TCDD. Here, we have exploited the availability of AhR-defective (AhR-D) mouse hepatoma (Hepa 1c1c7) cells to analyze AhR's effects under conditions in which TCDD is not present. Our results reveal that AhR-D cells exhibit a different morphology, decreased albumin synthesis, and a prolonged doubling time compared with wild-type cells. Introduction of AhR cDNA into AhR-D cells by stable transfection alters these characteristics such that the cells resemble wild-type cells. Conversely, introduction of antisense AhR cDNA into wild-type cells changes their phenotype such that they resemble AhR-D cells. Fluorescence microscopy reveals that AhR-D cells do not exhibit an increased rate of death. Flow cytometric and biochemical analyses imply that the slowed growth rate of AhR-D cells reflects prolongation of G1. Our findings reveal a potential link between AhR and the G1 phase of the Hepa 1c1c7 cell cycle. These effects of AhR occur in the absence of TCDD. We speculate that they represent responses to an endogenous AhR ligand in Hepa 1c1c7 cells.

Animals↗
PubMed1995

Transcriptional activation by the mouse Ah receptor. Interplay between multiple stimulatory and inhibitory functions.

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates cellular responses to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We cloned AhR cDNA from C57BL/6 mouse liver and verified by transfection that it encodes a functional protein. Analyses of deletion mutants indicate that the carboxyl half of AhR contains several types of transactivation domain, which function independently of domains that mediate TCDD recognition, DNA binding, and heterodimerization with the Ah receptor nuclear translocator (Arnt) protein. The transactivation domains function independently of each other, display different levels of activity, and act synergistically when linked. In addition, AhR contains an 82-amino acid domain that inhibits transactivation. The inhibitory domain displays specificity, in that it blocks the transactivating functions of AhR and Arnt, but not that of the herpes simplex protein VP16. The inhibitory activity depends upon the cell type in which AhR is expressed, implying that a cell-specific protein mediates the effect.

Animals↗
PubMed1995

Dioxin induces localized, graded changes in chromatin structure: implications for Cyp1A1 gene transcription.

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) induces Cyp1A1 gene transcription, a process that requires two basic helix-loop-helix regulatory proteins, the aromatic hydrocarbon receptor (AhR) and the aromatic hydrocarbon receptor nuclear translocator (Arnt). We have used a ligation-mediated PCR technique to analyze dioxin-induced changes in protein-DNA interactions and chromatin structure of the Cyp1A1 enhancer-promoter in its native chromosomal setting. Dioxin-induced binding of the AhR/Arnt heteromer to enhancer chromatin is associated with a localized (about 200 bp) alteration in chromatin structure that is manifested by increased accessibility of the DNA; these changes probably reflect direct disruption of a nucleosome by AhR/Arnt. Dioxin induces analogous AhR/Arnt-dependent changes in chromatin structure and accessibility at the Cyp1A1 promoter. However, the changes at the promoter must occur by a different, more indirect mechanism, because they are induced from a distance and do not reflect a local effect of AhR/Arnt binding. Dose-response experiments indicate that the changes in chromatin structure at the enhancer and promoter are graded and mirror the graded induction of Cyp1A1 transcription by dioxin. We discuss these results in terms of a TCDD-induced shift in an equilibrium between nucleosomal and nonnucleosomal chromatin configurations.

Animals↗
PubMed1994

Transcriptional activation function of the mouse Ah receptor nuclear translocator.

We cloned from mouse hepatoma cells a cDNA which encodes the Ah receptor nuclear translocator (Arnt). Sequence comparisons reveal 89% nucleotide and 92% amino acid identity between mouse and human Arnt. Transfection of the cDNA into Arnt-defective mouse hepatoma cells fully restores their responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), indicating that the cDNA encodes a functional Arnt protein. Transfection of the cDNA into wild type mouse hepatoma cells increases the magnitude, but not the sensitivity, of the transcriptional response to TCDD. Analyses of mutants indicate that Arnt has a modular organization. The unit that mediates both heterodimerization with the liganded Ah receptor and DNA recognition is functionally distinct from the unit that mediates transcriptional activation. A 96-amino acid, C-terminal domain of Arnt, which includes a glutamine-rich region, confers transcriptional activation capability upon the protein.

Amino Acid Sequence↗
PubMed1994

Dioxin-dependent, DNA sequence-specific binding of a multiprotein complex containing the Ah receptor.

Fractionation of rat liver cytosol, using as an affinity reagent the DNA recognition sequence for the liganded aromatic hydrocarbon (Ah) receptor, enriches for proteins that are about 110, 106, 98/96, 57, and 54 kDa in size. The proteins display 2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent, DNA sequence-specific binding that is characteristic of the liganded Ah receptor. Immunological studies imply: 1. That the 98/96-kDa protein is the Ah receptor nuclear translocator (Arnt); 2. That the 110- and 106-kDa proteins are not immunologically related either to each other or to Arnt; and 3. That the 110-, the 106-kDa, and the Arnt proteins are members of a multicomponent protein complex. cDNA cloning studies indicate that the 106-kDa protein is the rat Ah receptor. The N-terminal 384 amino acids of the rat receptor show substantial sequence homology to the mouse and human Ah receptor. The sequence conservation across species imputes functional importance to this region of the receptor. In contrast, the remainder of the protein is substantially less well conserved among rat, mouse, and human. The tissue distribution of Ah receptor mRNA is consistent with previous studies of the distribution of the receptor protein. Our findings demonstrate the use of DNA recognition site chromatography for purification of the Ah receptor and imply that the ligand receptor binds to DNA as part of a multiprotein complex.

Amino Acid Sequence↗
PubMed1994

DNA sequence requirements for Ah receptor/Arnt recognition determined by in vitro transcription.

The enhancer of the mouse cytochrome P450 cyp1a1 gene, which contains six binding sites (A-F) for the Ah receptor (AhR), has been a useful model system for studying the mechanism of AhR-mediated activation of transcription. In the presence of ligand, AhR interacts with its dimerization partner, Arnt, and the heteromeric complex is able to bind DNA. In the present study, we test the effects of single base pair substitutions of site D on the ability of the AhR/Arnt heteromer to recognize this response element using an in vitro transcription system. Synthetic oligodeoxyribonucleotides corresponding to the wild-type sequence of site D, or single base pair mutations of that sequence, were used to compete for AhR/Arnt binding with the transcription template. Using this competition assay, the sequence of the core recognition motif 5'-GCGTG-3' was shown to be critical for AhR/Arnt binding, and the importance of the position and orientation of the G:C and A:T base pairs of this sequence was determined.

Amino Acid Sequence↗
PubMed1993

Protein-DNA interactions at a dioxin-responsive enhancer. Analysis of six bona fide DNA-binding sites for the liganded Ah receptor.

The DNA upstream of the dioxin-inducible CYP1A1 gene contains six distinct sites to which the liganded Ah receptor binds in intact mouse hepatoma cells. Here, we have analyzed these six bona fide receptor-binding sites in order to study the relationships between DNA sequence, receptor binding, and dioxin responsiveness. Gel retardation studies reveal that the sites vary by about 7-fold in their relative affinities for the liganded receptor. Within this range, there is no obvious association between the strength of receptor binding and the degree of dioxin responsiveness, as measured in transfection experiments. In fact, one site binds the receptor well but fails to respond to dioxin. This observation implies that the receptor-DNA binding event per se is not sufficient to confer dioxin responsiveness upon a linked gene. Comparison of the DNA sequences of the six receptor-binding sites permits the derivation of a "functional consensus" recognition sequence, which is more extended in length than the "core"-binding sequence previously described. In corroboration of these results, protein-DNA cross-linking studies indicate that the liganded receptor contacts base pairs beyond the core sequence. Our observations also indicate that the liganded receptor can tolerate limited sequence heterogeneity at its DNA-binding site and still elicit a response to dioxin. This finding might reflect corresponding heterogeneity in the amino acid sequence of the liganded receptor's DNA-binding domain.

Animals↗
PubMed1993

Mechanism of dioxin action: receptor-enhancer interactions in intact cells.

We have used a ligation-mediated polymerase chain reaction technique to analyze protein-DNA interactions at a dioxin-responsive enhancer upstream of the CYP1A1 gene in intact mouse hepatoma cells. In its inactive state, the enhancer binds few, if any, proteins within the major DNA groove in vivo. Thus, the inactive enhancer is relatively inaccessible to DNA-binding proteins. Exposure of cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to the binding of the liganded Ah receptor at six sites within the major DNA groove of the enhancer. The receptor-enhancer interactions occur rapidly and do not require ongoing transcription, consistent with their role in regulating CYP1A1 gene expression. The liganded receptor, which is a heteromer composed of at least two basic helix-loop-helix proteins, is probably the only DNA-binding transcription factor necessary to activate the enhancer in vivo. The small size and irregular distribution of receptor binding sites suggest that chromatin structure imposes substantial steric constraints upon the function of the receptor-enhancer system in intact cells.

Animals↗
PubMed1992

Transcription-dependent and transcription-independent nucleosome disruption induced by dioxin.

In mouse hepatoma cells, both the regulatory and the transcribed regions of the cyp1a1 gene assume a nucleosomal configuration when the gene is silent; two nucleosomes occupy specific sites at the transcriptional promoter. Activation of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is accompanied by changes in chromatin structure, which depend upon a functional aromatic hydrocarbon (Ah) receptor. In the transcribed region of the gene, nucleosome disruption occurs as a consequence of RNA elongation. In contrast, at the promoter, loss of positioned nucleosome sis independent of transcription and represents an event in the mechanism by which the liganded Ah receptor enhances transcriptional initiation.

Animals↗
PubMed1992

Superinduction of CYP1A1 transcription by cycloheximide. Role of the DNA binding site for the liganded Ah receptor.

Nuclear run-off experiments reveal that four distinct DNA domains, each of which contains a binding site for the liganded Ah receptor, can mediate the superinduction of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin plus cycloheximide. Superinduction requires substantial inhibition of protein synthesis by cycloheximide. Gel retardation analyses of nuclear extracts and methylation protection studies in intact cells reveal no evidence for cycloheximide-sensitive protein-DNA interactions that might mediate the superinduction response. These observations suggest the existence of a labile protein(s) that acts to inhibit the function of the dioxin-responsive receptor/enhancer regulatory system via protein-protein interactions.

Base Sequence↗
PubMed1992

Mechanism of dioxin action: Ah receptor-mediated increase in promoter accessibility in vivo.

We have analyzed dioxin-inducible, Ah receptor-dependent changes in protein-DNA interactions at the CYP1A1 transcriptional promoter in intact mouse hepatoma cells. Our findings indicate that in uninduced cells, the promoter is inaccessible to its cognate binding proteins, which are known to be expressed constitutively. Dioxin induces, in Ah receptor-dependent fashion, an increase in promoter accessibility, which occurs rapidly and does not require ongoing transcription of the CYP1A1 gene. The change in promoter accessibility is not due to an altered pattern of cytosine methylation at the promoter; it probably reflects a 2,3,7,8-tetrachlorodibenzo-p-dioxin- induced change in the chromatin structure. These findings provide new insight into the mechanism of dioxin action and contribute to a better understanding of the regulation of inducible gene transcription in mammalian cells.

Animals↗
PubMed1992

Protein-DNA interactions at a dioxin-responsive enhancer. Mutational analysis of the DNA-binding site for the liganded Ah receptor.

The liganded Ah receptor activates transcription by binding to a specific DNA-recognition motif within a dioxin-responsive enhancer upstream of the CYP1A1 gene. Analyses of mutant enhancers by gel retardation reveal that each base pair within the domain 5'CGTG(GCAC)3' is essential to the receptor-enhancer interaction. The three base pairs immediately flanking each end of the essential domain contribute less strongly to receptor binding. Analyses of enhancer function by transfection reveal that a mutation in the essential domain, which abolishes receptor-DNA binding, obliterates enhancer function. Mutations outside the essential domain, which diminish, but do not abolish, receptor-DNA binding, also obliterate enhancer function. Additionally, one mutation adjacent to the essential binding motif does not affect receptor-DNA binding, but destroys enhancer activity. These findings imply that transcriptional enhancement by the dioxin-responsive system cannot be predicted solely by the strength of the receptor-enhancer interaction.

Base Sequence↗
PubMed1991

Analysis of CYP1A1 promoter function by transcription in vitro.

We have analyzed the function of the CYP1A1 promoter using in vitro transcription. Using nuclear extracts from HeLa cells, we found that transcription from the promoter in vitro was constitutive. Transcription in vitro was not increased by prior exposure of the HeLa cells to the inducer 2,3,7, 8-tetrachlorodibenzo-p-dioxin nor by the inclusion of a dioxin-responsive enhancer in the DNA template. Analyses of mutants revealed that a TATAAA sequence and two recognition motifs for CCAAT box transcription factor/nuclear factor I contributed to the constitutive activity of the promoter in vitro.

Base Sequence↗