Werner's syndrome helicase participates in transcription of phenobarbital-inducible CYP2B genes in rat and mouse liver.

Werner's syndrome (WS) is a rare human autosomal recessive segmental progeroid syndrome clinically characterized by atherosclerosis, cancer, osteoporosis, type 2 diabetes mellitus and ocular cataracts. The WRN gene codes for a RecQ helicase which is present in many tissues. Although the exact functions of the WRN protein remain unclear, accumulating evidence suggests that it participates in DNA repair, replication, recombination and telomere maintenance. It has also been proposed that WRN participates in RNA polymerase II-dependent transcription. However no promoter directly targeted by WRN has yet been identified. In this work, we report mammalian genes that are WRN targets. The rat CYP2B2 gene and its closely related mouse homolog, Cyp2b10, are both strongly induced in liver by phenobarbital. We found that there is phenobarbital-dependent recruitment of WRN to the promoter of the CYP2B2 gene as demonstrated by chromatin immunoprecipitation analysis. Mice homozygous for a Wrn mutation deleting part of the helicase domain showed a decrease in basal and phenobarbital-induced CYP2B10 mRNA levels compared to wild type animals. The phenobarbital-induced level of CYP2B10 protein was also reduced in the mutant mice. Electrophoretic mobility shift assays showed that WRN can participate in the formation of a complex with a specific sequence within the CYP2B2 basal promoter. Hence, there is a WRN binding site in a region of DNA sequence to which WRN is recruited in vivo. Taken together, these results suggest that WRN participates in transcription of CYP2B genes in liver and identifies the first physical interaction between a specific promoter sequence and WRN.

The CYP2B2 5' flank contains a complex glucocorticoid response unit.

Rat CYP2B1 and CYP2B2 and mouse CYP2B10 are dramatically induced by phenobarbital (PB) in liver. PB responsiveness requires the constitutive androstane receptor (CAR). However, dexamethasone treatment can also induce CYP2B genes in both rat and mouse liver. Three regions have been shown to be involved in conferring dexamethasone responsiveness on CYP2B2 reporter constructs. They are the PB response unit, a functional glucocorticoid response element at -1.3kb in the 5' flank and a weak element in the basal promoter. We report here the identification, by deletion analysis of the CYP2B2 5' flank, of new glucocorticoid response elements or accessory factor sites. Moreover, we show that CAR acts as an accessory factor in the dexamethasone response in vivo of CYP2B10 protein in mice, by increasing both the basal and induced levels. We propose a model to explain the dexamethasone responsiveness of the CYP2B2 gene in which induction is mediated by a complex glucocorticoid response unit.

The CYP2B2 phenobarbital response unit contains binding sites for hepatocyte nuclear factor 4, PBX-PREP1, the thyroid hormone receptor beta and the liver X receptor.

A 163 bp enhancer in the CYP2B2 5' flank confers PB (phenobarbital) inducibility and constitutes a PBRU (PB response unit). The PBRU contains several transcription factor binding sites, including NR1, NR2 and NR3, which are direct repeats separated by 4 bp of the nuclear receptor consensus half-site AGGTCA, as well as an ER (everted repeat) separated by 7 bp (ER-7). Constitutive androstane receptor (CAR)-RXR (retinoic X receptor) heterodimers are known to bind to NR1, NR2 and NR3. Electrophoretic mobility-shift analysis using nuclear extracts from livers of untreated or PB-treated rats revealed binding of several other proteins to different PBRU elements. Using supershift analysis and in vitro coupled transcription and translation, the proteins present in four retarded complexes were identified as TRbeta (thyroid hormone receptor beta), LXR (liver X receptor), HNF-4 (hepatocyte nuclear factor 4) and heterodimers of PBX-PREP1 (pre-B cell homoeobox-Pbx regulatory protein 1). LXR-RXR heterodimers bound to NR3 and TRbeta bound to NR3, NR1 and ER-7, whereas the PBX-PREP1 site is contained within NR2. The HNF-4 site overlaps with NR1. A mutation described previously, GRE1m1, which decreases PB responsiveness, increased the affinity of this site for HNF-4. The PBRU also contains a site for nuclear factor 1. The PBRU thus contains a plethora of transcription factor binding sites. The profiles of transcription factor binding to NR1 and NR3 were quite similar, although strikingly different from, and more complex than, that of NR2. This parallels the functional differences in conferring PB responsiveness between NR1 and NR3 on the one hand, and NR2 on the other.