BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10alpha in activated transcription.

The functions of most of the 12 subunits of the RNA polymerase II (Pol II) enzyme are unknown. In this study, we demonstrate that two of the subunits, hRPB2 and hRPB10alpha, mediate the regulated stimulation of transcription. We find that the transcriptional coactivator BRCA1 interacts directly with the core Pol II complex in vitro. We tested whether single subunits from Pol II would compete with the intact Pol II complex to inhibit transcription stimulated by BRCA1. Excess purified Pol II subunits hRPB2 or hRPB10alpha blocked BRCA1- and VP16-dependent transcriptional activation in vitro with minimal effect on basal transcription. No other Pol II subunits tested inhibited activated transcription in these assays. Furthermore, hRPB10alpha, but not hRPB2, blocked Sp1-dependent activation.

Critical structural elements and multitarget protein interactions of the transcriptional activator AF-1 of hepatocyte nuclear factor 4.

The nuclear receptor hepatocyte nuclear factor 4 (HNF-4) is an important regulator of several genes involved in diverse metabolic and developmental pathways. Mutations in the HNF-4A gene are responsible for the maturity-onset diabetes of the young type 1. Recently, we showed that the 24 N-terminal residues of HNF-4 function as an acidic transcriptional activator, termed AF-1 (Hadzopoulou-Cladaras, M., Kistanova, E., Evagelopoulou, C., Zeng, S. , Cladaras C., and Ladias, J. A. A. (1997) J. Biol. Chem. 272, 539-550). To identify the critical residues for this activator, we performed an extensive genetic analysis using site-directed mutagenesis. We showed that the aromatic and bulky hydrophobic residues Tyr6, Tyr14, Phe19, Lys10, and Lys17 are essential for AF-1 function. To a lesser degree, five acidic residues are also important for optimal activity. Positional changes of Tyr6 and Tyr14 reduced AF-1 activity, underscoring the importance of primary structure for this activator. Our analysis also indicated that AF-1 is bipartite, consisting of two modules that synergize to activate transcription. More important, AF-1 shares common structural motifs and molecular targets with the activators of the tumor suppressor protein p53 and NF-kappaB-p65, suggesting similar mechanisms of action. Remarkably, AF-1 interacted specifically with multiple transcriptional targets, including the TATA-binding protein; the TATA-binding protein-associated factors TAFII31 and TAFII80; transcription factor IIB; transcription factor IIH-p62; and the coactivators cAMP-responsive element-binding protein-binding protein, ADA2, and PC4. The interaction of AF-1 with proteins that regulate distinct steps of transcription may provide a mechanism for synergistic activation of gene expression by AF-1.

Genetic polymorphism of cytochrome P4502E1 and risk of alcoholic liver disease in Caucasians.

Genetic factors may be of importance in determining inter-individual susceptibility to alcoholic liver disease (ALD). Among the candidate genes which have been considered to be important are those which code for enzymes involved in alcohol metabolism. Cytochrome P4502E1 (CYP2E1) metabolizes alcohol to acetaldehyde and the hydroxyethyl radical, and is also inducible by alcohol. A Rsa I restriction fragment length polymorphism (RFLP) in the 5'-flanking region of the CYP2E1 gene has been identified by other investigators, studies showing that the mutant allele (termed c2) shows greater transcriptional activity, higher protein levels and increased activity compared with the wild-type allele (c1). We have used PCR-RFLP analysis to determine whether the frequency of these alleles differed in 95 Caucasian patients with ALD compared with 205 control subjects (comprising 58 alcoholics with no liver disease, 47 patients with non-alcoholic liver disease and 100 healthy volunteers). In controls, the frequency (0.024) of the c2 allele was similar to that previously reported in other Caucasian populations. The c2 allele frequency in patients with ALD (0.1), however, was significantly (p = 0.0003; odds ratio (OR) 4.5, 95% CI 1.9-10.9) higher than in control subjects. The findings indicate that Caucasians carrying the Rsa I c2 allele of the CYP2E1 gene may be at higher risk of developing ALD if they abuse alcohol.

Genetic analysis of microsomal epoxide hydrolase in patients with carbamazepine hypersensitivity.

Carbamazepine therapy is occasionally complicated by hypersensitivity reactions, the mechanism of which is poorly understood. It has been suggested that affected individuals may have a genetically-determined defect of microsomal epoxide hydrolase. The aim of this study was to determine whether a single genetic mutation or pattern of mutations could be used to predict individual susceptibility to carbamazepine-hypersensitivity. DNA was isolated from 10 carbamazepine-hypersensitive patients and 10 healthy volunteers. The patients had developed various forms of toxicity with carbamazepine, including toxic epidermal necrolysis, Stevens-Johnson syndrome, hepatitis and pneumonitis. The technique of polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) was used to screen for mutations in all nine exons of the microsomal epoxide hydrolase gene. Any new mutations detected by this method were characterised by direct sequencing of the DNA. In addition, in the most severely affected patient, we sequenced all nine exons of the gene. There was a higher frequency of mutations in the hypersensitive group when compared with the controls, but there was no consistent mutation (or pattern of mutations) in the microsomal epoxide hydrolase gene which was common to the hypersensitive group. DNA sequencing of all nine exons of the microsomal epoxide hydrolase gene from the most severely affected patient showed the sequence to be "wild-type," when compared to the previously published sequences. The results of this study suggest that a single mutation within the coding region of the microsomal epoxide hydrolase gene cannot be the sole determinant of the predisposition to carbamazepine hypersensitivity.

Glutathione S-transferase mu genotype (GSTM1*0) in Alzheimer's patients with tacrine transaminitis.

1. Tacrine (1,2,3,4-tetrahydro-9-aminoacridine) which is used in Alzheimer's disease, causes elevation of liver transaminases ('tacrine transaminitis') in 40-50% of patients. This may be related to the formation of a chemically reactive metabolite from tacrine, which can be detoxified in vitro by glutathione. 2. Glutathione-S-transferase mu (GSTM1), a detoxication enzyme, is polymorphically expressed being absent in about 50% of patients. Its role in the detoxication of the reactive metabolite of tacrine is not known. 3. The frequency of the enzyme deficiency (GSTM1*0) has been investigated in patients with tacrine transaminitis using polymerase chain reaction (PCR) to determine whether the GSTM1 status can be used as an absolute predictive factor for susceptibility to tacrine transaminitis. 4. The frequency of the GSTM1*0 genotype in patients with tacrine transaminitis (n = 33; 45.5%) was not significantly different from that in patients treated with tacrine without liver dysfunction (n = 37; 43%), and when compared with all the controls used in the study (n = 167; 56%). 5. The frequency of the GSTM1*0 genotype in patients with Alzheimer's disease (n = 79; 46%) was not significantly different from that in healthy volunteers (n = 121; 59.5%). 6. Our results indicate that the GSTM1 status cannot be used clinically to predict individual susceptibility to tacrine transaminitis, and that patients with the GSTM1*0 genotype are unlikely to have an increased risk of tacrine-induced liver damage. Furthermore, the GSTM1 status was not associated with Alzheimer's disease.

Lymphocyte microsomal epoxide hydrolase in patients on carbamazepine therapy.

1. In order to determine whether carbamazepine is an inducer of lymphocyte microsomal epoxide hydrolase, the activity of the enzyme has been measured in the lymphocytes of 40 patients on continuous drug therapy using [3H]-cis stilbene oxide as a substrate. 2. Induction of the cytochrome P450 isoform, CYP3A, has been assessed in the same patients by measurement of the 24 h urinary excretion of 6 beta-hydroxycortisol by radioimmunoassay. The urinary concentrations of carbamazepine and its two metabolites, the 10,11-epoxide and trans-dihydrodiol, have also been measured by h.p.l.c. 3. The 24 h urinary 6 beta-hydroxycortisol excretion in the patients increased with the dose of carbamazepine (r = 0.57, P < 0.001) indicating induction of CYP3A. 4. The total amount of trans-dihydrodiol excreted in the urine increased with the dose of carbamazepine, and it was the most abundant urinary metabolite in all patients and at all dose-levels. There was no relationship between the dose of carbamazepine and the diol to epoxide ratio (r = 0.01, NS). 5. Lymphocyte microsomal epoxide hydrolase activity was marginally, but significantly (P = 0.02) higher in the patients (28.4 pmol diol min-1 mg-1 protein) than in drug-free controls (23.4 pmol diol min-1 mg-1 protein (95% CI for difference -9 to -0.8)). 6. The results indicate that at concentrations of carbamazepine which produce marked induction of hepatic CYP3A, an enzyme involved in the metabolism and bioactivation of carbamazepine, there is only a slight increase in lymphocyte microsomal epoxide hydrolase.