Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

Histone deacetylase-dependent transcriptional repression by pRB in yeast occurs independently of interaction through the LXCXE binding cleft

We have developed a yeast model system to address transcriptional repression by the retinoblastoma protein (pRB). When fused to the DNA-binding domain of Gal4p (DB-pRB), pRB can repress transcription of reporter genes containing Gal4p binding sites; the histone deacetylase activity encoded by yeast RPD3 is required for DB-pRB repression. Mutation of the LXCXE binding cleft in pRB, a region reported to be required for histone deacetylase recruitment, does not interfere with pRB-mediated repression. From these findings based on yeast experiments, we surmise that the small pocket region of pRB must contain an additional domain that confers histone deacetylase-dependent transcriptional repression. This hypothesis was verified by experiments examining pRB-dependent histone deacetylase association in mammalian cells. In addition to RPD3, repression by pRB in yeast requires MSI1, an ortholog of RbAp48, but not SIN3 or SAP30. By comparing the genetic requirements of DB-pRB repression in yeast to those of other DB-repressor fusions, we can suggest a mechanism by which pRB recruits histone deacetylase activity.

4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor γ

The estrogen-related receptors (ERRα, ERRβ, and ERRγ) form a family of orphan nuclear receptors that share significant amino acid identity with the estrogen receptors, but for which physiologic roles remain largely unknown. By using a peptide sensor assay, we have identified the stilbenes diethylstilbestrol (DES), tamoxifen (TAM), and 4-hydroxytamoxifen (4-OHT) as high-affinity ligands for ERRγ. In direct binding assays, 4-OHT had a Kd value of 35 nM, and both DES and TAM displaced radiolabeled 4-OHT with Ki values of 870 nM. In cell-based assays, 4-OHT binding caused a dissociation of the complex between ERRγ and the steroid receptor coactivator-1, and led to an inhibition of the constitutive transcriptional activity of ERRγ. ERRα did not bind 4-OHT, but replacing a single amino acid predicted to be in the ERRα ligand-binding pocket with the corresponding ERRγ residue allowed high-affinity 4-OHT binding. These results demonstrate the existence of high-affinity ligands for the ERR family of orphan receptors, and identify 4-OHT as a molecule that can regulate the transcriptional activity of ERRγ.

Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase.

The 1.4-kb downstream region from a nitrilase gene (nitA) of an actinomycete Rhodococcus rhodochrous J1, which is industrially in use, was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis in experiments using a Rhodococcus-Escherichia coli shuttle vector pK4 in a Rhodococcus strain. Sequence analysis of the 1.4-kb region revealed the existence of an open reading frame (nitR) of 957 bp, which would encode a protein with a molecular mass of 35,100. Deletion of the central and 3'-terminal portion of nitR resulted in the complete loss of nitrilase activity, demonstrating that nitR codes for a transcriptional positive regulator in nitA expression. The deduced amino acid sequence of nitR showed similarity to a positive regulator family including XylS from Pseudomonas putida and AraC from E. coli. By Northern blot analysis, the 1.4-kb transcripts for nitA were detected in R. rhodochrous J1 cells cultured in the presence of isovaleronitrile, but not those cultured in the absence of isovaleronitrile. The transcriptional start site for nitA was mapped to a C residue located 26 bp upstream of its translational start site. Deletion analysis to define the nitA promoter region suggested the possible participation of an inverted repeat sequence, centered on base pair -52, in induction of nitA transcription.

Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.

RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration.

We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins.

Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.

Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.

A Ds insertion alters the nuclear localization of the maize transcriptional activator R.

The R-sc gene of maize is a member of the R gene family of transcriptional activators that regulate anthocyanin biosynthesis. A derivative of R-sc, r-m9 conditions a reduced level of aleurone pigmentation due to the presence of a 2.1-kb Ds insertion near the 3' end of the coding region. Excision of Ds from r-m9 leaves a 7-bp insertion in the darker but still mutant v24 derivative. Both the 7-bp insertion in v24 and the 2.1-kb Ds in r-m9 are predicted to truncate their respective R proteins proximal to the carboxyl terminus, which was shown previously to contain one of three nuclear localization sequences. We find that the reduced expression of r-m9 and v24 are not due to mRNA or protein instability, but most likely reflect the inefficient localization of truncated R proteins to the nucleus. To our knowledge this is the first example of a transposable element insertion that alters gene expression by affecting nuclear localization. In addition, our data indicate that the carboxyl terminus of the R protein is far more important than previously suspected and illustrates the utility of natural mutations for defining functional domains in proteins.

BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor.

Approximately 40% of diffuse large cell lymphoma are associated with chromosomal translocations that deregulate the expression of the BCL6 gene by juxtaposing heterologous promoters to the BCL-6 coding domain. The BCL6 gene encodes a 95-kDa protein containing six C-terminal zinc-finger motifs and an N-terminal POZ domain, suggesting that it may function as a transcription factor. By using a DNA sequence selected for its ability to bind recombinant BCL-6 in vitro, we show here that BCL-6 is present in DNA-binding complexes in nuclear extracts from various B-cell lines. In transient transfectin experiments, BCL6 can repress transcription from promoters linked to its DNA target sequence and this activity is dependent upon specific DNA-binding and the presence of an intact N-terminal half of the protein. We demonstrate that this part of the BCL6 molecule contains an autonomous transrepressor domain and that two noncontiguous regions, including the POZ motif, mediate maximum transrepressive activity. These results indicate that the BCL-6 protein can function as a sequence-specific transcriptional repressor and have implications for the role of BCL6 in normal lymphoid development and lymphomagenesis.

Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae.

The stress response promoter element (STRE) confers increased transcription to a set of genes following environmental or metabolic stress in Saccharomyces cerevisiae. A lambda gt11 library was screened to isolate clones encoding STRE-binding proteins, and one such gene was identified as MSN2, which encoded a zinc-finger transcriptional activator. Disruption of the MSN2 gene abolished an STRE-binding activity in crude extracts as judged by both gel mobility-shift and Southwestern blot experiments, and overexpression of MSN2 intensified this binding activity. Northern blot analysis demonstrated that for the known or suspected STRE-regulated genes DDR2, CTT1, HSP12, and TPS2, transcript induction was impaired following heat shock or DNA damage treatment in the msn2-disrupted strain and was constitutively activated in a strain overexpressing MSN2. Furthermore, heat shock induction of a STRE-driven reporter gene was reduced more than 6-fold in the msn2 strain relative to wild-type cells. Taken together, these data indicate that Msn2p is the transcription factor that activates STRE-regulated genes in response to stress. Whereas nearly 85% of STRE-mediated heat shock induction was MSN2 dependent, there was significant MSN2-independent expression. We present evidence that the MSN2 homolog, MSN4, can partially replace MSN2 for transcriptional activation following stress. Moreover, our data provides evidence for the involvement of additional transcription factors in the yeast multistress response.

Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae.

We quantitate the absolute levels of individual mRNAs per yeast cell by hybridizing total yeast RNA with an excess of gene-specific 32P-oligonucleotides, and digesting the resulting RNA-DNA hybrids with S1 nuclease. By comparing the his3 hybridization signal from a known amount of yeast cells to the signal generated by a known amount of his3 RNA synthesized in vitro, we determine that yeast strain KY114 growing in yeast extract/peptone/glucose medium at 30 degrees C contains seven molecules of his3 mRNA per cell. Using a galactose shut-off procedure, we determined that the half-life of his3 mRNA is approximately 11 min under these conditions. From these observations, we calculate that one his3 mRNA molecule is synthesized every 140 s. Analysis of other his3 promoter derivatives suggests that the maximal transcriptional initiation rate in yeast cells is one mRNA molecule every 6-8 s. Using his3 as an internal standard, the number of mRNA molecules per cell have been determined for ded1, trp3, rps4, and gall under a variety of growth conditions. From these results, the absolute mRNA level of any yeast gene can be determined in a single hybridization experiment. Moreover, the rate of transcriptional initiation can be determined for mRNAs whose decay rates are known.

Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma.

The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR. Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs. In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines. For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells. To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites. In this context, full-length PAX3-FKHR was also much more potent than PAX3. Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory. By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR. We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.

Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens from the same patient (n = 5). In situ hybridization showed that the neoplastic colonocytes had increased expression of PHS-2 (n = 4). Additionally, five colon cancer cell lines were shown to express high levels of PHS-2 mRNA even in the absence of a known inducer of PHS-2. To study the basis for this increased gene expression, we transfected a colon cancer cell line, HCT-116, with a reporter gene containing 2.0 kb of the 5' regulatory sequence of the PHS-2 gene. Constitutive transcription of the reporter gene was observed, whereas normal control cell lines transcribed the reporter only in response to an exogenous agonist. We conclude that PHS-2 is transcribed abnormally in human colon cancers and that this may be one mechanism by which prostaglandins or related compounds that support carcinogenesis are generated.