Chloramphenicol Acetyltransferase Reporter Assays Research Articles

Expression of brain-derived neurotrophic factor is regulated by the Wnt signaling pathway

Brain-derived neurotrophic factor (BDNF) is a well characterized neurotrophin that mediates a wide variety of activities in the central nervous system, including neuronal differentiation, neuroprotection, and synaptic plasticity. The canonical Wnt signaling pathway is a critical regulator of embryonic development and homeostasis in adult tissues. Our group and others recently demonstrated that Wnt signaling induces BDNF expression in neurons and glia. However, the precise relationship between BDNF and Wnt signaling pathways is not understood. Here, we investigated Wnt signaling regulation of BDNF at the transcriptional level using a combination of bioinformatics and molecular analyses. Analysis of the BDNF gene promoter identified seven binding motifs for Wnt-dependent TCF/LEF transcription factors. Furthermore, specific BDNF promoters were induced by the Wnt3a ligand using chloramphenicol acetyl transferase reporter assays and a dominant-negative TCF4 gene reduced Wnt3a-mediated induction. Finally, Wnt3a induced expression of BDNF and other members of the BDNF signaling pathway in glia cells. Therefore, these data indicate that BDNF is a direct target of Wnt signaling, which provides a new insight into the interaction between two essential signaling pathways.

Proteasomal Degradation Is Transcriptionally Controlled by TCF11 via an ERAD-Dependent Feedback Loop

Coordinated regulation of the ubiquitin-proteasome system (UPS) is crucial for the cell to adjust its protein degradation capacity to changing proteolytic requirements. We have shown previously that mammalian cells upregulate proteasome gene expression in response to proteasome inhibition. Here, we report the identification of the transcription factor TCF11 (long isoform of Nrf1) as a key regulator for 26S proteasome formation in human cells to compensate for reduced proteolytic activity. Under noninducing conditions, TCF11 resides in the endoplasmic reticulum (ER) membrane. There, TCF11 is targeted to ER-associated protein degradation requiring the E3 ubiquitin ligase HRD1 and the AAA ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding to antioxidant response elements in their promoter regions. Thus, we uncovered the transcriptional control loop regulating human proteasome-dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition.

Role of glyceraldehyde-3-phosphate dehydrogenase binding to hepatitis B virus posttranscriptional regulatory element in regulating expression of HBV surface antigen

The hepatitis B virus (HBV) posttranscriptional regulatory element (HPRE) has been demonstrated to facilitate the cytoplasmic localization of unspliced transcripts. One cellular protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to combine with this element. However, its function on HPRE remains unclear. Here, we show that recombinant GAPDH protein binds to HPRE RNA in vitro in streptavidin pull-down assays. Functional analysis demonstrated that GAPDH inhibited HPRE function in a pDM138-HPRE chloramphenicol acetyltransferase reporter assay system. Overexpression of GAPDH depressed the expression of HBs antigen, as detected both in cells transiently expressing HBs-HPRE and in HepG2.2.15 cells. These data indicate that GAPDH may be involved in the posttranscriptional regulation of HBV, which is critical for the life cycle of HBV.

Comparative genomic analysis of the HNF-4α transcription factor gene

Hepatocyte nuclear factor-4α (HNF-4α), the gene for the maturity-onset diabetes of the young type 1 (MODY1) form of type 2 diabetes mellitus (T2DM), is within the T2DM-linked region on chromosome 20q12-q13.1 and consequently, is a positional candidate gene for T2DM. Mutations in the coding region of HNF-4α are rare in diabetes affected subjects. Altered regulation of HNF-4α gene expression, controlled by distant enhancer sequences, may contribute to the development of type 2 diabetes. Comparative sequence analysis was performed between 13 kb of genomic DNA 5 ′ to the P1 promoter sequences of the human, mouse, and rat HNF-4α coding sequences. Three regions, located at −10.5 kb (295 bp in length), −6.25 kb (421 bp in length), and −5.36 kb (263 bp in length), have significant sequence identity between the species. These three regions were functionally characterized using the chloramphenicol acetyltransferase (CAT) reporter assay, in which the conserved 5 ′ regions of mouse HNF-4α were cloned in front of the herpes simplex virus thymidine kinase promoter driving transcription of the CAT gene. A fragment containing the 421 bp conserved region significantly increased CAT activity in differentiated rat hepatoma cells (13.7- ± 1.9-fold control), while only a modest increase in CAT activity was observed in pancreatic cells (2.5- ± 0.9-fold control; 1.6- ± 0.1-fold control) and dedifferentiated hepatoma cells (1.7- ± 0.4-fold control). The remaining two conserved regions increased CAT activity minimally in pancreatic (1.1- ± 0.1-fold control to 1.9- ± 0.1-fold control) and hepatic (1.6- ± 0.5-fold control to 2.3- ± 0.4-fold control) cell lines. Denaturing high-performance liquid chromatography (DHPLC) was used to search for sequence variants in DNA from 259 T2DM individuals. Two single nucleotide polymorphisms (SNPs) were identified, both of which increased CAT activity in the insulinoma cell lines in the CAT reporter assay (1.4-fold increase over wild-type; 1.7-fold increase over wild-type). These results suggest that comparative sequence analysis can efficiently identify regulatory elements and that sequence variants in regulatory elements of HNF-4α can contribute to altered HNF-4α gene expression.

Analysis of the initiator tRNA genes from a slow- and a fast-growing Mycobacterium.

Initiation of protein synthesis is a major post-transcriptional regulatory step in gene expression. The initiator tRNA gene from Mycobacterium smegmatis, a fast-growing mycobacterium, was characterized and compared with its counterpart from Mycobacterium tuberculosis, a slow-growing mycobacterium. In both mycobacteria, the functional initiator tRNA genes were found in a single copy. Unlike the M. tuberculosis initiator tRNA, the CCA end of the M. smegmatis initiator is not encoded in the gene, and it is most likely added post-transcriptionally. Transcription start site mapping allowed accurate assignment of the hexameric -10 and -35 promoter elements for both genes. These elements of the M. smegmatis initiator tRNA gene contain single nucleotide changes compared to their respective counterparts in the M. tuberculosis gene. Chloramphenicol acetyl transferase reporter assays suggested that the promoter of the initiator tRNA gene from M. smegmatis is twice as strong as that of M. tuberculosis, irrespective of whether the assays were performed in the fast-growing homologous host (M. smegmatis) or the slow-growing heterologous host (M. tuberculosis). Characterization of the M. smegmatis metU promoter, in this study, provides a valuable tool for the expression of genes in mycobacteria.

Quantification of p53 expression in the nervous system

The transcription factor p53 is a short lived protein that is thought to be associated with cellular proliferation and apoptosis. In the current study, we present a protocol to measure p53 expression across both the central and peripheral nervous systems of transgenic and parental mice using the enzyme linked immuno-substrate assay (ELISA), chloramphenicol acetyl transferase reporter assay (CAT) and immunohistochemistry approaches. The profiles of the ELISA tissue data of CD1 mice were compared to the CAT assay data of the p53-promoter-driven CAT gene transgenic mice. Subsequently, high resolution immunohistochemical analysis of positive tissues in both mouse strains were evaluated. As the p53 protein is apparently subject to high turnover, the comparison of the more stable CAT data to the pan p53 ELISA assay should effectively complement each other in identifying which nervous system structures express p53. ELISA analysis alone could give ambiguous data. Immunohistochemical studies confirmed and further defined p53 expression in several regions of the nervous system. Significantly, p53 promoter-driven CAT expression was visualized in the Purkinje cells of the cerebellum and in the cornea as well as in the retina of the eye. This approach for the analysis of very short half-life proteins in the nervous system should be transferable to the study of other proteins.

Role of the Initiator Element in the Regulation of the Melanoma Cell Adhesion Molecule Gene

The melanoma cell adhesion molecule is a membrane glycoprotein whose expression is associated with tumor progression and the development of metastatic potential. The mechanisms for upregulation of the melanoma cell adhesion molecule during melanoma progression are still poorly understood. In this study, we show further evidence that melanoma cell adhesion molecule expression is tightly regulated at the transcriptional level. Using a combination of chloramphenicol acetyl transferase reporter assays and DNA mobility shift experiments, we investigated the role played by three putative melanoma cell adhesion molecule regulatory elements, namely the initiator sequence, the SCA element, and the ASp element. The SCA and the ASp boxes can potentially interact with the transcription factors Sp1 and AP-2. Sp1 binding to both sites was confirmed, but only the SCA sequence could form a complex with AP-2. AP-2-driven downregulation of the melanoma cell adhesion molecule promoter, however, did not depend only on a functional SCA element. The pyrimidine-rich CTCACTTG initiator, which overlaps the RNA start site, was essential for promoter function and was shown to interact with proteins related to basic helix-loop-helix transcription factors. Binding in nonmetastatic melanoma cells was induced by cAMP. In metastatic cells, however, binding was constitutive, but could be markedly decreased upon treatment with phorbol esters. As melanoma cell adhesion molecule expression is modulated by cAMP and phorbol ester signaling, these results suggest that the initiator is the central element that mediates cAMP and phorbol ester sensitivity and initiates melanoma cell adhesion molecule overexpression in melanomas.

Use of the ornithine decarboxylase promoter to achieve N-MYC-mediated overexpression of a rabbit carboxylesterase to sensitize neuroblastoma cells to CPT-11.

Overexpression of specific transcription factors by tumor cells can be exploited to regulate expression of proteins that induce apoptosis or activate prodrugs, thereby producing tumor-selective toxicity. A majority of advanced-stage neuroblastomas overexpress the transcription factor N-MYC, and this overexpression is associated with poor prognosis. This study describes regulation of expression by N-MYC, via the ornithine decarboxylase (ODC) promoter, of a rabbit liver carboxylesterase (CE) that activates the prodrug CPT-11. Chloramphenicol acetyltransferase reporter assays and CE activity assays in transiently transfected neuroblastoma cell lines (SJNB-1, SJNB-4, NB-1691) and rhabdomyosarcoma cell lines (JR1neo20, JR1Nmyc6, JR1Nmyc9) support this approach as a potential method for sensitizing tumor cells to CPT-11. Clonogenic assays with IMR32 human neuroblastoma cells which express N-MYC and that had been stably transfected with a plasmid containing an ODC promoter/CE cassette corroborated results of enzyme activity assays. Specifically, IMR32.ODC.CE cells expressed approximately eightfold more CE activity than IMR32.CMV.neo cells; and 5 microM CPT-11 reduced the clonogenic potential of IMR32.ODC.CE cells to zero, while 50 microM CPT-11 was required to produce the same effect with IMR32.CMV.neo cells. Current experiments focus on adenoviral delivery of an ODC promoter/CE cDNA cassette for potential virus-directed enzyme prodrug therapy applications.

Dox-Dependent SIVmac with Tetracycline-Inducible Promoter in the U3 Promoter Region

An attenuated live vaccine is a candidate in developing vaccines against human immunodeficiency virus type 1 (HIV-1). The study using macaques and simian immunodeficiency virus (SIVmac) showed an attenuated virus to be more effective than any other vaccine candidate. However, development of a safer vaccine is required for clinical application. In this study, we constructed pSIVmac Δ nef with tetracycline inducible promoter (pTet) and tried to control viral expression in a drug-dependent manner. Promoter/enhancer motifs in the U3 region of the long terminal repeats (LTRs) were serially deleted and replaced with pTet. In mutant LTRs, which lack NF-κB and Sp1 binding sites, TATA box motifs, and the 5′ half of the U3 region, promoter activity was stringently controlled by doxycycline (Dox). Their activities were similar to or higher than that of wild-type LTR in the presence of Dox, based on the transient chloramphenicol acetyltransferase reporter assay. Three of these mutant LTRs were introduced into the pSIVmac239 Δ nef genome. Viral protein from these viruses was efficiently expressed in a Dox-dependent manner after transfection to a HeLa cell, which expresses reverse tetracycline transactivator (rtTA). The 2-LTR-form viral DNA of these viruses could be detected in M8166 cells that had been infected with supernatants from the transfected rtTA HeLa cell. These results suggest that pSIVmac Δ nef containing mutant LTRs can proceed through one viral replication cycle consisting of transcription, formation of viral particles, infection to cells, and reverse transcription. Although continuous replication of these Dox-dependent viruses requires a supply of rtTA as a constituent for the pTet-On viral genome, the successful replacement of the original promoter with a drug-dependent promoter suggests a new possibility for developing a safer attenuated live virus.

Identification of epithelial and myeloid-specific DNA elements regulating MRP14 gene transcription.

Macrophage migration inhibition factor-related protein 14 (MRP14) is a Ca2+-binding protein of the S-100 family highly abundant in myelomonocytic and epithelial cells. The expression pattern is restricted to myeloid and epithelial cells and therefore the MRP14 gene is ideally suited to study the regulation of gene expression in these cells. We characterized the human MRP14 promoter by using the chloramphenicol acetyltransferase reporter assay system. The analysis was performed in epithelial (TR146) and myeloid (HL-60) cells, and we were successful in mapping positive and negative regulatory elements. The region -114/-419 contains strong myeloid-specific regulatory elements, whereas the domain -600/-1000 enhances the MRP14 transcription in epithelial cells. The sequence -462/-600 accommodates a regulatory element that enhances the promoter activity in both myeloid and epithelial cells. Regions -114/-419 and -1000/-4500 reduce the expression of MRP14 only in epithelial cells, whereas the domains -419/-462 and -4500/-6500 contain down-regulating elements in both, epithelial and myeloid cells. The presented data demonstrate that transcription of the human MRP14 gene is regulated in a complex manner enabling the precise control of the MRP14 level in epithelial and myeloid cells.

Molecular Mechanisms of Promoter Regulation of the gp34 Gene That Is Trans-activated by an Oncoprotein Tax of Human T Cell Leukemia Virus Type I

We investigated the molecular mechanism of transcriptional activation of the gp34 gene by the Tax oncoprotein of human T cell leukemia virus type I (HTLV-I). gp34 is a type II transmembrane molecule belonging to the tumor necrosis factor family and is constitutively expressed on HTLV-I-producing cells but not normal resting T cells. The transcriptional regulatory region of the gp34 gene was activated by HTLV-I Tax in the human T cell line Jurkat, in which endogenous gp34 is induced by Tax. Sequence analysis demonstrated that two NF-kappaB-like elements (1 and 2) were present in the regulatory region. Both NF-kappaB-like elements were able to bind to NF-kappaB or its related factor(s) in a Tax-dependent manner. Chloramphenicol acetyltransferase assays indicated that NF-kappaB-like element 1 was Tax-responsive, although the activity was lower than that the native promoter. NF-kappaB-like element 2 elevated promoter activity when combined with NF-kappaB-like element 1, indicating cooperative function of the elements for maximum promoter function. Unlike typical NF-kappaB elements, the NF-kappaB-like elements in gp34 were not activated by treatment of Jurkat cells with phorbol ester despite induction of the NF-kappaB-like binding activity. Chloramphenicol acetyltransferase reporter assays using the region upstream of the NF-kappaB-like elements identified an upstream region that reduced transcription from cognate and noncognate core promoters in a Tax-independent manner. Our results imply complex regulation of expression of the gp34 gene and suggest implication of gp34 in proliferation of HTLV-I infected T cells.

Hormone Regulation of Bovine Leukemia Virus via the Long Terminal Repeat

The hormone regulation of viruses has been of great interest since the discovery of glucocorticoid stimulation of mouse mammary tumor virus via a hormone response element in the viral long terminal repeat (LTR) promoter region. This report describes the investigation of the hormone responsiveness of bovine leukemia virus (BLV), an oncogenic retrovirus that infects dairy and beef cattle worldwide. It is a member of the human T cell leukemia (HTLV)/BLV group of retroviruses, which encode a protein, Tax, that is essential for regulating transcription of their own proviruses and for transforming host cells. We investigated the responsiveness of BLV to the hormones 17β-estradiol, progesterone, prolactin, insulin, and dexamethasone, a potent glucocorticoid. Only dexamethasone, in combination with insulin or insulin/prolactin, consistently stimulated BLV expression, as measured by reverse transcriptase activity, RNA blot hybridization (Northern blots), and CAT (chloramphenicol acetyltransferase) reporter assays of cell lines transiently or stably transfected with the BLV LTR. This effect required the presence of glucocorticoid receptors and Tax. This is the first report of hormone responsiveness in a virus of the HTLV/BLV group.

Molding a peptide into an RNA site by in vivo peptide evolution.

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single alpha-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage lambda N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial alpha-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an "RNA world."

Agonistic Effect of Tamoxifen Is Dependent on Cell Type, ERE-Promoter Context, and Estrogen Receptor Subtype: Functional Difference between Estrogen Receptors α and β

To investigate the functional differences between estrogen receptor (ER) α and β subtypes, we studied the expression and the transcription stimulating activities of these receptors. RT-PCR has demonstrated that ERα is expressed at a high level in MCF-7 cells derived from human breast cancer. Both ERα and ERβ were expressed at a lower level in HOS-TE85 and Saos2 cells derived from human osteosarcoma. Chloramphenicol acetyltransferase reporter assay detected the transcriptional activation by the endogenous receptor only in MCF-7 cells. Agonistic effect of tamoxifen was observed as strong as that of 17β-estradiol on ERE activation in MCF-7 cells at the concentration of 10−7M when ERE-containing reporter is constructed with β-globin promoter. The effect of tamoxifen was not apparent when the reporter was constructed with thymidine kinase promoter, suggesting that the differential gene activation between tamoxifen and estrogen may take place depending upon ERE-promoter context. Agonistic activity of tamoxifen was also detected in COS-7 and Saos-2 cells, but not in HEC-1 cells derived from human endometrial carcinoma via exogenously expressed ER. Interestingly, this effect was ERα specific. Thus, we demonstrate that agonistic effect of tamoxifen depends on the cell type, ERE-promoter context, and ER subtype. These parameters would explain at least a part of the tissue specific effects of antiestrogensin vivo.

Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells.

O6-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O6 of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer+) and -deficient (Mer-) cells, correlated with the endogenous MGMT activity in Mer+ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5'-CTGGGTCGC-3') for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer+ cells tested but was apparently restricted to the cytoplasm of Mer- cells. We conclude that the MEBP-enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer+ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer- cells.

Transcriptional Regulation of the Gene Coding for Human Protein C

The promoter for the gene coding for human protein C has been characterized as to nucleotide sequences that regulate the synthesis of mRNA. The major transcription start site was found 65 nucleotides upstream from the first intron/exon boundary along with two minor sites. Functional characterization of 1528 base pairs at the 5'-end of the gene was then carried out by chloramphenicol acetyltransferase reporter assays, protection from DNase I digestion, and electrophoretic mobility shift assays employing HepG2 and HeLa cells. One of the upstream regions (nucleotides -25 to +9) contained binding sites for at least two different transcription factors, including a hepatic nuclear factor 1-binding site (-10 to +9) and two overlapping and oppositely oriented hepatic nuclear factor 3-binding sites (-25 to -11). A second major region (PCE1) (+12 to +30) appeared to be a unique, liver-specific regulatory sequence. An Sp1-binding site in exon I (+58 to +65) was also recognized by cotransfection experiments with an Sp1 expression plasmid. Specific mutations in these promoter elements reduced transcriptional activity and abolished the binding of hepatic nuclear proteins. Finally, a strong silencer element (PCS1) (between -162 and -82) and two possible liver-specific enhancer regions (PCE2 and PCE3), which interact coordinately with the promoter elements, were also found (between -1462 and -162).

Characterization of the promoter region of human steriod sulfatase: A gene which escapes X inactivation

The human X-linked steroid sulfatase gene (STS) was among the first genes shown to escape X inactivation. At least fourteen genes regulated in this fashion have now been recognized. They are dispersed into several regions of the X chromosome and may be controlled in a locus specific manner. Studies of the promoters of these genes could provide insights into the mechanism of X inactivation, however little information of this nature is currently available. For this reason we examined 5' flanking sequences of the human STS gene for promoter function. Four transcription start sites scattered over a 50bp region were identified. Functional domains of this TATA-less and GC poor promoter were identified by study of a series of terminal and internal deletions. A putative promoter sequence was identified which by itself exhibits little or no basal activity. However when combined with upstream regulatory elements, this segment showed weak but reproducible activity in a CAT (chloramphenicol acetyltransferase) reporter assay. Several regulatory domains acting as enhancers and repressors were subsequently identified. The relationship of this 5' sequence to the ability of the STS gene to escape X-inactivation is discussed.