Kathryn E. Plant

Intergenic transcription in the human beta-globin gene cluster.

ABSTRACT Our previous studies on nascent transcription across the human β-globin gene cluster revealed the presence of intergenic transcripts in addition to the expected genic transcripts. We now show that transcription into the β-globin locus control region (LCR) begins within an ERV9 endogenous retroviral long terminal repeat upstream of DNase I hypersensitive site 5. However, in a transgenic mouse, which has the human β-globin LCR but lacks the ERV9 LTR, transcription begins upstream of the transgenic locus. We postulate that in this transgenic mouse nearby endogenous mouse promoters are activated by the LCR. Intergenic transcription is also detected across the whole transgenic globin gene locus independently of the stage of erythroid development. Intergenic transcription in the β-globin cluster is erythroid specific; however, it can be induced in nonerythroid cells by several means: by transinduction with a plasmid transcribing part of the cluster, by exogenous addition of transcription factors, and by treatment with the histone deacetylase inhibitor trichostatin A.

Up-regulation of the glutathione S-transferase system in human liver by polycyclic aromatic hydrocarbons; comparison with rat liver and lung

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the alpha-class selective 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.

Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Gγ-globin gene

ABSTRACT The human γ-globin genes form part of a 5-kb tandem duplication within the β-globin gene cluster on chromosome 11. Despite a high degree of identity between the two genes, we show that while the upstream Gγ-globin gene terminates transcription efficiently, termination in the Aγ gene is inefficient. This is primarily due to the different strengths of the poly(A) signals of the two genes; Gγ-globin has a functionally stronger poly(A) signal than the Aγ gene. The probable cause of this difference in poly(A) efficiency characteristics lies with a number of base changes which reduce the G/U content of the GU/U-rich region of the Aγ poly(A) signal relative to that of Gγ. The 3′ flanking regions of the two γ-globin genes have similar abilities to promote transcription termination. We found no evidence to suggest a cotranscriptional cleavage event, such as that seen in the human β-globin gene, occurs in either γ-globin 3′ flank. Instead we find evidence that the 3′ flank of the Gγ-globin gene contains multiple weak pause elements which, combined with the strong poly(A) signal the gene possesses, are likely to cause gradual termination across the 3′ flank.

The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1.

The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimers or Parkinsonism.

Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence.

A PXR-mediated negative feedback loop attenuates the expression of CYP3A in response to the PXR agonist pregnenalone-16α-carbonitrile.

The nuclear receptor superfamily of ligand-activated transcription factors plays a central role in the regulation of cellular responses to chemical challenge. Nuclear receptors are activated by a wide range of both endogenous and exogenous chemicals, and their target genes include those involved in the metabolism and transport of the activating chemical. Such target gene activation, thus, acts to remove the stimulating xenobiotic or to maintain homeostatic levels of endogenous chemicals. Given the dual nature of this system it is important to understand how these two roles are balanced, such that xenobiotics are efficiently removed while not impacting negatively on homeostasis of endogenous chemicals. Using DNA microarray technology we have examined the transcriptome response of primary rat hepatocytes to two nuclear receptor ligands: Pregnenalone-16α-carbonitrile (PCN), a xenobiotic PXR agonist, and lithocholic acid, an endogenous mixed PXR/VDR/FXR agonist. We demonstrate that despite differences in the profile of activated nuclear receptors, transcriptome responses for these two ligands are broadly similar at lower concentrations, indicating a conserved general response. However, as concentrations of stimulating ligand rises, the transcriptome responses diverge, reflecting a need for specific responses to the two stimulating chemicals. Finally, we demonstrate a novel feed-back loop for PXR, whereby ligand-activation of PXR suppresses transcription of the PXR gene, acting to attenuate PXR protein expression levels at higher ligand concentrations. Through in silico simulation we demonstrate that this feed-back loop is an important factor to prevent hyperexpression of PXR target genes such as CYP3A and confirm these findings in vitro. This novel insight into the regulation of the PXR-mediated regulatory signal networks provides a potential mechanistic rationale for the robustness in steroid homeostasis within the cell.

Differential response of human and rat epoxide hydrolase to polycyclic aromatic hydrocarbon exposure: Studies using precision-cut tissue slices

The potential of polycyclic aromatic hydrocarbons (PAHs) to modulate microsomal epoxide hydrolase activity, determined using benzo[a]pyrene 5-oxide as substrate, in human liver, was evaluated and compared to rat liver. Precision-cut liver slices prepared from fresh human liver were incubated with six structurally diverse PAHs, at a range of concentrations, for 24h. Of the six PAHs studied, benzo[a]pyrene, dibenzo[a,h]anthracene and fluoranthene gave rise to a statistically significant increase in epoxide hydrolase activity, which was accompanied by a concomitant increase in epoxide hydrolase protein levels determined by immunoblotting. The other PAHs studied, namely dibenzo[a,l]pyrene, benzo[b]fluoranthene and 1-methylphenanthrene, influenced neither activity nor enzyme protein levels. When rat slices were incubated under identical conditions, only benzo[a]pyrene and dibenzo[a,h]anthracene elevated epoxide hydrolase activity, which was, once again accompanied by a rise in protein levels. At the mRNA level, however, all six PAHs caused an increase, albeit to different extent. In rat, epoxide hydroxylase activity in lung slices was much lower than in liver slices. In lung slices, epoxide hydrolase activity was elevated following exposure to benzo[a]pyrene and dibenzo[a,l]pyrene and, to a lesser extent, 1-methylphenanthrene; similar observations were made at the protein level. At both activity and protein levels extent of induction was far more pronounced in the lung compared with the liver. It is concluded that epoxide hydrolase activity is an inducible enzyme by PAHs, in both human and rat liver, but induction potential by individual PAHs varies enormously, depending on the nature of the compound involved. Marked tissue differences in the nature of PAHs stimulating activity in rat lung and liver were noted. Although in the rat basal lung epoxide hydrolase activity is much lower than liver, it is more markedly inducible by PAHs.

Inhibition of prenyltransferase activity by statins in both liver and muscle cell lines is not causative of cytotoxicity

As inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, statins are an important first-line treatment for hypercholesterolemia. However, a recognized side-effect of statin therapy is myopathy, which in severe cases can present as potentially fatal rhabdomyolysis. This represents an important impediment to successful statin therapy, and despite decades of research the molecular mechanisms underlying this side-effect remain unclear. Current evidence supports a role for reduced levels of mevalonate pathway intermediates, with the most accepted hypothesis being a reduction in isoprenoids formation, leading to faulty post-translational modifications of membrane-associated proteins. We have undertaken a comprehensive analysis of the impact of nine statins on two human cell lines; Huh7 hepatoma and RD rhabdomyosarcoma. In both cell lines, concentration-dependent inhibition of prenylation was observed for cerivastatin and simvastatin, which could be rescued with the pathway intermediate mevalonate; in general, muscle cells were more sensitive to this effect, as measured by the levels of unprenylated Rap1A, a marker for prenylation by geranylgeranyl transferase I. Concentration-dependent toxicity was observed in both cell lines, with muscle cells again being more sensitive. Importantly, there was no correlation between inhibition of prenylation and cell toxicity, suggesting they are not causally linked. The lack of a causal relationship was confirmed by the absence of cytotoxicity in all cell lines following exposure to specific inhibitors of geranylgeranyl transferases I and II, and farnesyl transferase. As such, we provide strong evidence against the commonly accepted hypothesis linking inhibition of prenylation and statin-mediated toxicity, with the two processes likely to be simultaneous but independent.

Phenethyl isothiocyanate, a naturally occurring phytochemical, is an antagonist of the aryl hydrocarbon receptor

SCOPE The aryl hydrocarbon (Ah) receptor is a ligand-activated transcription factor that is activated by many carcinogens, and its target gene products play a major role in tumour development, so that antagonists of the Ah receptor represent potential chemopreventive agents. METHODS AND RESULTS Experimental evidence is presented herein that phenethyl isothiocyanate (PEITC), a phytochemical present in cruciferous vegetables, is such an antagonist. PEITC was a very weak ligand to the Ah receptor, as assessed using the chemical-activated luciferase expression (CALUX) assay, and a poor inducer of CYP1A1 mRNA levels when incubated in precision-cut rat liver slices for 24 h. It antagonised effectively, however, the interaction of benzo[a]pyrene to the receptor, being capable of preventing its binding as well as displacing it from the receptor. Moreover, PEITC suppressed in concentration-dependent manner the benzo[a]pyrene-mediated rise in rat hepatic CYP1A1 mRNA levels in rat slices. Finally, PEITC antagonised the benzo[a]pyrene-mediated increase in the O-deethylation of ethoxyresorufin in both rat and human precision-cut liver slices. CONCLUSION It is concluded that PEITC is an effective antagonist of the Ah receptor in rat and human liver, and this potential may contribute to its established chemopreventive activity.

The naturally occurring aliphatic isothiocyanates sulforaphane and erucin are weak agonists but potent non-competitive antagonists of the aryl hydrocarbon receptor

As the Ah receptor target gene products play a critical role in chemical carcinogenesis, antagonists are considered as potential chemopreventive agents. It is demonstrated in this paper that the isothiocyanates R,S-sulforaphane and erucin are non-competitive antagonists of the aryl hydrocarbon (Ah) receptor. Both isothiocyanates were poor agonists for the receptor and elevated CYP1A1 mRNA levels only modestly when incubated with precision-cut rat liver slices. In contrast, the classical Ah receptor agonist benzo[a]pyrene was a potent inducer of CYP1A1 mRNA levels, with this effect being effectively antagonized by the two isothiocyanates. In further studies, it was demonstrated that R,S-sulforaphane could both prevent the interaction of and displace already bound benzo[a]pyrene from the Ah receptor, but no concentration dependency was observed with respect to the isothiocyanate. Both erucin and R,S-sulforaphane antagonized the benzo[a]pyrene-mediated increase in the CYP1A-mediated O-deethylation of ethoxyresorufin in rat precision-cut liver slices. Of the two isomers of R,S-sulforaphane, the naturally occurring R-isomer was more effective than the S-isomer in antagonizing the activation of the Ah receptor by benzo[a]pyrene. Antagonism of the Ah receptor may be a major contributor to the established chemoprevention of aliphatic isothiocyanates.