PI Promoter Research Articles

Regulation of Safracin Biosynthesis and Transport in Pseudomonas poae PMA22.

Pseudomonas poae PMA22 produces safracins, a family of compounds with potent broad-spectrum anti-bacterial and anti-tumor activities. The safracins' biosynthetic gene cluster (BGC sac) consists of 11 ORFs organized in two divergent operons (sacABCDEFGHK and sacIJ) that are controlled by Pa and Pi promoters. Contiguous to the BGC sac, we have located a gene that encodes a putative global regulator of the LysR family annotated as MexT that was originally described as a transcriptional activator of the MexEF-OprN multidrug efflux pump in Pseudomonas. Through both in vitro and in vivo experiments, we have demonstrated the involvement of the dual regulatory system MexT-MexS on the BGC sac expression acting as an activator and a repressor, respectively. The MexEF-OprN transport system of PMA22, also controlled by MexT, was shown to play a fundamental role in the metabolism of safracin. The overexpression of mexEF-oprN in PMA22 resulted in fourfold higher production levels of safracin. These results illustrate how a pleiotropic regulatory system can be critical to optimizing the production of tailored secondary metabolites, not only through direct interaction with the BGC promoters, but also by controlling their transport.

Gram-Negative Bacterial Endotoxin LPS Induces NeuGc Loss through Ets1-Dependent Downregulation of Intestine-Specific pcmah Transcript in Porcine Intestinal Cells.

N-glycolylneuraminic acid (NeuGc), a non-human sialic acid derivative synthesized by cytidine-5′-monophospho-N-acetylneuraminic acid hydroxylase (CMAH), plays a crucial role in mediating infections by certain pathogens. Although it has been postulated that NeuGc biosynthesis and CMAH expression are downregulated during microbial infection, the underlying mechanisms remain unclear. The present study showed that exposure to lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, leads to loss of NeuGc biosynthesis in pig small intestinal I2I-2I cells. This LPS-induced NeuGc loss was accompanied by decreased CMAH transcript levels, especially intestine-specific 5′pcmah-1. Furthermore, LPS suppressed the activity of the Pi promoter responsible for 5′pcmah-1 by inhibiting DNA binding of Est1. These findings provide insight into the regulatory mechanisms of Neu5Gc biosynthesis during pathogenic infectious events, which may represent a host defense mechanism that protects the self against pathogenic bacterial infections even in non-sanitary environments.

Intestine specific regulation of pig cytidine-5\u2032-monophospho-N-acetylneuraminic acid hydroxylase gene for N-glycolylneuraminic acid biosynthesis

N-glycolylneuraminic acid (Neu5Gc), a generic form of sialic acid, is enzymatically synthesized by cytidine-5′-monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Although expression of pig CMAH gene pcmah encoding CMAH has been reported to be regulated by pathogenic infection and developmental processes, little is known about the mechanisms underlying the regulation of pcmah gene expression. The objective of this study was to determine mechanism(s) involved in intestine specific regulation of pcmah gene by identifying several cis-acting elements and nuclear transcription factors that could directly interact with these cis-acting elements. We identified intestine specific promoter region (Pi) of pcmah gene located at upstream regions of the 5′flanking region of exon 1a and found that the promoter region is responsible for the transcriptional regulation of 5′pcmah-1. Based on reporter assays using serially constructed luciferase genes with each deleted promoter, we demonstrated that the Pi promoter activity was more active in intestinal IPI-2I cells than that in kidney PK15 cells, corresponding to both mRNA expression patterns in the two cell lines. In addition, we found that Sp1 transcription factor was necessary for basal activity of Pi promoter and that Ets-1 contributed to intestine-specific activity of Pi promoter. This study helps us understand transcriptional regulation of pcmah in the intestine of pig tissues. It also allows us to consider potential roles of Neu5Gc in interaction with environmental factors present in the intestinal tissue during pathogenic infection and developmental process.

Discovery and Structural Characterization of G-quadruplex DNA in Human Acetyl-CoA Carboxylase Gene Promoters: Its Role in Transcriptional Regulation and as a Therapeutic Target for Human Disease.

Accumulating evidence suggests that G-quadruplexes play vital roles in gene expression, DNA replication, and recombination. Three distinct promoters (PI, PII, and PIII) regulate human acetyl-CoA carboxylase 1 (ACC1) gene expression. In this study, we asked whether the G-rich sequences within the human ACC1 (PI and PII) promoters can form G-quadruplex structures and regulate normal DNA transactions. Using multiple complementary methods, we show that G-rich sequences of PI and PII promoters form intramolecular G-quadruplex structures and then establish unambiguously the topologies of these structures. Importantly, G-quadruplex formation in ACC1 gene promoter region blocks DNA replication and suppresses transcription, and this effect was further augmented by G-quadruplex stabilizing ligands. Altogether, these results are consistent with the notion that G-quadruplex structures exist within the human ACC1 gene promoter region, whose activity can be suppressed by G-quadruplex stabilizing ligands, thereby revealing a novel regulatory mechanism of ACC1 gene expression and as a possible therapeutic target.

Molecular basis of non-mutational derepression of ramA in Klebsiella pneumoniae.

The ram locus, consisting of the romA-ramA genes, is repressed by the tetracycline-type regulator RamR, where regulation is abolished due to loss-of-function mutations within the protein or ligand interactions. The aim of this study was to determine whether the phenothiazines (chlorpromazine and thioridazine) directly interact with RamR to derepress ramA expression. Quantitative real-time PCR analyses were performed to determine expression levels of the romA-ramA genes after exposure to the phenothiazines. Electrophoretic mobility shift assays (EMSAs) and in vitro transcription experiments were performed to show direct binding to and repression by RamR. Direct binding of the RamR protein to the phenothiazines was measured by fluorescence spectroscopy experiments and molecular docking models were generated using the RamR crystal structure. Exposure to either chlorpromazine or thioridazine resulted in the up-regulation of the romA-ramA genes. EMSAs and in vitro transcription experiments demonstrated that both agents reduce/abolish binding and enhance transcription of the target PI promoter upstream of the ramR-romA genes in Klebsiella pneumoniae compared with RamR alone. Fluorescence spectroscopy measurements demonstrated that RamR directly binds both chlorpromazine and thioridazine with micromolar affinity. Molecular docking analyses using the RamR crystal structure demonstrated that the phenothiazines interact with RamR protein through contacts described for other ligands, in addition to forming unique strong polar interactions at positions D152 and K63. These data demonstrate that phenothiazines can modulate loci linked to the microbe-drug response where RamR is an intracellular target for the phenothiazines, thus resulting in a transient non-mutational derepression of ramA concentrations.

Role of PU.1 in MHC class II expression through transcriptional regulation of class II transactivator pI in dendritic cells

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family. We hypothesized that PU.1 is involved in MHC class II expression in dendritic cells (DCs). The role of PU.1 in MHC class II expression in DCs was analyzed. Transcriptional regulation of the DC-specific pI promoter of the class II transactivator (CIITA) gene and subsequent MHC class II expression was investigated by using PU.1 small interfering RNA (siRNA) and reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays. PU.1 siRNA introduction suppressed MHC class II expression, allogeneic and syngeneic T-cell activation activities of bone marrow-derived DCs (BMDCs) with reduction of CIITA mRNA driven by the DC-specific promoter pI, and MHC class II mRNA. The chromatin immunoprecipitation assay showed constitutive binding of PU.1 to the pI region in BMDCs, whereas acetylation of histone H3 on pI was suppressed by LPS stimulation in parallel with shutdown of CIITA transcription. PU.1 transactivated the pI promoter through cis-elements at -47/-44 and -30/-27 in a reporter assay and to which PU.1 directly bound in an electrophoretic mobility shift assay. Acetylation of histones H3 and H4 on pI was reduced in PU.1 siRNA-introduced BMDCs. Knockdown of interferon regulatory factor 4 or 8, which is a heterodimer partner of PU.1, by siRNA did not affect pI-driven CIITA transcription or MHC class II expression. PU.1 basally transactivates the CIITA pI promoter in DCs by functioning as a monomeric transcription factor and by affecting histone modification, resulting in the subsequent expression and function of MHC class II.

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.

Spotlight

Pandey et al., pp. 2520–2533 Hypermethylation of the gluthathione-S-transferase pi (GSTP1) promoter is detected in over 90% of invasive prostate cancer biopsies. Reversing the process of DNA methylation using DNA methyltransferase inhibitors exerts anti-cancer effects. However, this process can also result in the demethylation–and by extension the activation–of prometastatic genes, causing genomic instability, increased cancer aggressiveness and cellular toxicity. Green tea polyphenols (GTPs) have various anti-cancer effects. One study showed that GTPs are able to inhibit DNA methyltransferase and reactivate methylation-silenced genes in cancer cells. In this report, Pandey et al. investigate whether GTPs offer an alternative way of reactivating GSTP1 without causing the pathological or toxic effects that standard methyltransferase inhibitors such as 5-Aza-dC do. Using prostate cancer cell lines, GTP was shown to inhibit DNA methyltransferase activity and reactivate GSTP1 expression in a dose- and time-dependent manner. The authors showed that treatment of prostate cancer cells with GTP for 7 days resulted in the demethylation of the proximal GSTP1 promoter. Exposure to GTP also caused a drop in methyl-binding protein expression–proteins which are known to contribute to hypermethylated gene silencing. Notably, GTP promoted chromatin stability by inhibiting histone deacetylase activity. The most striking finding in this study was that GTP's demethylation effect was specific to GSTP1. Unlike 5-Aza-dC, it did not appear to cause the genome-wide demethylatation that is associated with inadvertent activation of prometastatic genes and cell toxicity. These experiments show that GTPs can exert two positive epigenetic effects: promoting specific DNA demethylation and chromatin stability without excessive toxicity. GTPs offer the promise of superior in vivo anti-cancer effects relative to the methyltransferase inhibitor 5-Aza-dC and warrant further study. Zhang et al., pp. 2534–2541 The early stages of metastasis are difficult to characterize using histological methods. In this report Zhang et al., employ a small animal imaging system (OV100) and fluorescence molecular tomography imager (VisEn) in vivo to study early metastasis in the GFP-PC-3 prostate cancer mouse model, which mirrors metastasis in patients. Using the OV100 imaging system, extensive GFP-PC-3 cells were visualized in the liver vasculature of live tumor-bearing mice through an abdominal skin flap. No tumor foci could be found in extravascular tissue. Far less extensive intravascular metastasis was found in the lungs of tumor-bearing mice. Matrix metalloproteinases (MMP) activity was tracked in both organs using a fluorescence probe. Z-stack images showed that GFP-PC-3 cancer cells produce active MMP in vivo. Additional VisEn imaging offered picomolar sensitivity and spatial resolution measurements: tumor-bearing mice showed 3 times higher MMP activity than control animals and the signal was stronger in the liver than in the lung. Dissected liver and lungs from tumor-bearing mice confirmed the in vivo observations. Metastasized liver cells were found in small and large vessels as clusters of up to 100 cells. No extravasation was observed 3 weeks after primary tumor inoculation. In lung tissue, smaller clusters (10 cells or less) were seen mainly in the pulmonary micro-vessels although extravascular metastasis was observed in the hilar region. This study demonstrates that PC-3 cells gather in the blood vessels of secondary organs to proliferate and develop into metastatic cells. This combined in vivo imaging technique offers a new opportunity to study the intravascular microenvironment and process of metastasis in vivo. Bastos et al., pp. 2700–2715 National public health strategies for cancer vary greatly. On page 2700, Bastos et al. present a comprehensive view of 2008 cancer control structures in 27 European Union member countries in addition to Norway, Iceland and Switzerland. The report draws on a number of sources and offers useful definitions of the provisions described. Just over half of the countries had cancer plans in place. Cancer registries are much more common; only Greece and Luxembourg didn't have a population-based registry and the majority of registries offer national coverage. Seventeen of the countries had a breast cancer screening program but participant rates varied from 25 to 85%. Participation rates were more extreme for cervical cancer screening programs, ranging from 10 to 80%. Newer programs coming on stream include HPV vaccination, colorectal cancer screening and smoking restrictions but implementation is still patchy across Europe. The authors stress that good cancer control structures do not automatically guarantee quality of care. This report clearly demonstrates the heterogeneity in cancer structures across Europe and offers the opportunity for inter-country co-operation and sharing of best practice.

Alterations in tumor biomarker GSTP gene methylation patterns induced by prenatal exposure to PFOS

The adverse environmental exposure in early life may have adverse effects on animals through epigenetic aspects. The current study examined the possibility of early epigenetic alteration in PFOS-exposed rat liver. Pregnant Sprague–Dawley (SD) rats were exposed to perfluorooctane sulfonate (PFOS) at doses of 0.1, 0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2 to 21. The dams were allowed to give birth and liver samples from weaned (postnatal day 21) offspring rats were analyzed for PFOS content, relative liver weight, global DNA methylation, methylation of LINE-1 regulatory region, tumor suppressor gene glutathione S-transferase pi (GSTP) and p16 promoter methylation level, as well as related genes expression level. In PFOS-exposed weaned rats, compared to the control, global DNA methylation and methylation of LINE-1 regulatory region decreased significantly only in the 2.0 mg/kg/d group. Up to 30% of critical CpG sites (+79, 81 and 84) in GSTP promoter region were methylated in the livers of PFOS-treated rats, while p16 promoter methylation was not affected. In addition, the up-regulated expression of GSTP was observed and this increase was associated with its main pathway of transcription regulation: Keap1–Nrf2/MafK. Thus, early-induced changes in critical cytosines within the GSTP gene promoter region may be a biomarker of hepatic PFOS burden, though their direct role in PFOS-induced hepatotoxicity, including its potential carcinogenic action, needs further research.

Abstract 5000: Epigenetic de-silencing of Glutathione s-transferase pi by tumor suppressive maspin

Abstract Abstract: Loss of expression of the GST pi isoform due to DNA hypermethylation-mediated epigenetic silencing is one of the most common molecular events in precancerous and cancerous lesions of human prostate. Our previous studies demonstrated that tumor suppressive maspin, an endogenous inhibitor of histone deacetylase 1, could up-regulated GST activity and reduced reactive oxygen species (ROS) generation in response to oxidative stress treatment. We confirmed that maspin restored GST pi expression in maspin transfected prostate cancer cell line DU145. Conversely, maspin knocking down by siRNA in PC3 cells led to reduced GST pi expression. We further investigated the possibility that the epigenetic regulation by DNA-methylation and histone deacetylation were coupled dynamic processes that HDAC inhibition by maspin alone was sufficient to reverse the DNA-methylation silencing effects. Using methylation-specific polymerase chain reaction (M-PCR), DNA demethylation in GST pi promoter was detected only in maspin-expressing cells. Chromatin immunoprecipitation (ChIP) assay showed that a significant higher level of acetylated histone 3 was associated with the GST pi promoter DNA complex. In the same DNA complex from maspin transfected cells, less DNA methylation transferase (DNMT1) and methyl-CpG-binding domain (MBD2/3) proteins were detected as compared to that from the mock control cells. It was noted that in DU145-derived transfected cells, maspin simultaneously restored GST pi expression and sensitized tumor cells to drug-induced apoptosis, while overexprssion of GST pi alone in LNCaP prostate cancer cells conferred resistance to oxidative stress in cell death, HIF-1α induction, and phosphorylation of histone 2A.X. These data suggest that, as an epithelial-specific HDAC1 inhibitor, maspin may function to maintain the epithelial tissue homeostasis through balancing the GST pi-dependent detoxification and HDAC1 inhibition-dependent apoptosis sensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5000.

AGHR polymorphism and its associations with carcass traits in Harrwoo cattle

The growth hormone receptor (GHR) is a membrane transmitter for the growth hormone signal transduction pathway that regulates various metabolic activities, including cell growth and expressions of cytokine genes. The presence or absence of a genetic polymorphism for the LINE-1 retroposon in the PI promoter, which specifically regulates theGHR gene expression in the liver, was screened by a novel detection method and examined for its relationships with carcass traits in Hanwoo cattle. Han woo cattle had taurine type LINE-1 present (alleleI) as well as incidine type LINE-1 absent (alleleA) promoter sequences. Three genotypes,I/I, I/A andA/A, showed frequencies of 49.1, 36.7 and 14.2%, respectively. The effects of allele A were significant on mean differences for final weight, eye muscle area, marbling score and fat color (p 0.05). Most 30-month old Hanwoo steers bearing the LINE-1 absent promoter had whiter fat color, heavier live weight and higher marbling score, reflecting intramuscular fat deposition inM. longissimus dorsi, compared to animals bearing a LINE-1 present promoter. This suggests that aGHR polymorphism could be a potential genetic marker for improving beef production of Hanwoo cattle.

Circulating, soluble forms of major histocompatability complex antigens are not exosome‐associated

In vitro studies have shown that soluble MHC (sMHC) released by cell lines is bound to nano-vesicles termed exosomes. It is thought that exosomes may represent the major reservoir of sMHC class I and II molecules in biological fluids. However, most studies have been confined to in vitro assays performed with cell lines. We show here that sMHC in the serum or plasma differs from exosome-bound sMHC in five ways: In contrast to exosome-associated sMHC, circulating sMHC is of low density, has a low apparent molecular mass (40-300 kDa) and is not detergent-labile. Moreover, the majority of MHC class II isoforms and MHC class I in blood are not physically linked and circulating HLA-DR is accessible to an antibody specific for the HLA-DR alpha-chain intracellular epitope, which is masked by its association with cellular or exosomal membranes. Finally, utilizing transcriptional activator of murine MHC class II (C2ta) promoter-mutant mice, we showed that the release of sMHC class II into the circulation is dependent on the C2ta pI promoter, but not pIII or pIV. This suggests that myeloid dendritic cells and/or macrophages, which preferentially use promoter pI of the C2ta gene, produce most of the sMHC class II found in the circulation.

Soy Protein Suppresses Gene Expression of Acetyl-CoA Carboxylase Alpha from Promoter PI in Rat Liver

Dietary soy protein isolate (SPI) reduces hepatic lipogenesis by suppressing gene expression of lipogenic enzymes, including acetyl-CoA carboxylase (ACC). In order to elucidate the mechanism of this regulation, the effect of dietary SPI on promoter (PI and PII) specific gene expression of ACC alpha was investigated. Rats were fed experimental diets containing SPI or casein as a nitrogen source. SPI feeding decreased the hepatic contents of total ACC mRNA as well as triglyceride (TG) content, but dietary SPI affected the amount of sterol-regulatory element binding protein (SREBP)-1 mRNA and protein very little. The amount of ACC mRNA transcribed from PII promoter containing SRE was not significantly affected by dietary protein, while a significant decrease in PI-generated ACC mRNA content was observed in rats fed the SPI diet. These data suggest that SPI feeding decreased the hepatic contents of ACC alpha mRNA mainly by regulating PI promoter via a nuclear factor(s) other than SREBP-1.

SeqA-mediated stimulation of a promoter activity by facilitating functions of a transcription activator.

It was demonstrated recently that the SeqA protein, a main negative regulator of Escherichia coli chromosome replication initiation, is also a specific transcription factor. SeqA specifically activates the bacteriophage lambda pR promoter while revealing no significant effect on the activity of another lambda promoter, pL. Here, we demonstrate that lysogenization by bacteriophage lambda is impaired in E. coli seqA mutants. Genetic analysis demonstrated that CII-mediated activation of the phage pI and paQ promoters, which are required for efficient lysogenization, is less efficient in the absence of seqA function. This was confirmed in in vitro transcription assays. Interestingly, SeqA stimulated CII-dependent transcription from pI and paQ when it was added to the reaction mixture before CII, although having little effect if added after a preincubation of CII with the DNA template. This SeqA-mediated stimulation was absolutely dependent on DNA methylation, as no effects of this protein were observed when using unmethylated DNA templates. Also, no effects of SeqA on transcription from pI and paQ were observed in the absence of CII. Binding of SeqA to templates containing the tested promoters occurs at GATC sequences located downstream of promoters, as revealed by electron microscopic studies. In contrast to pI and paQ, the activity of the third CII-dependent promoter, pE, devoid of neighbouring downstream GATC sequences, was not affected by SeqA both in vivo and in vitro. We conclude that SeqA stimulates transcription from pI and paQ promoters in co-operation with CII by facilitating functions of this transcription activator, most probably by allowing more efficient binding of CII to the promoter region.

Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter

The ADAR1 gene encodes an RNA-specific adenosine deaminase that alters the functional activity of both viral and cellular RNAs by posttranscriptional adenosine-to-inosine RNA editing. The interferon (IFN) responsive PI promoter of the ADAR1 gene possesses an IFN-stimulated response element (ISRE) responsible for IFN-inducibility, as well as an adjacent upstream sequence, designated kinase conserved sequence-like (KCS-ℓ) element. The KCS-ℓ element is similar to the 15-bp KCS element so far unique to the human and mouse RNA-dependent PKR kinase gene promoters. The KCS element of the PKR kinase ( PKR) promoter is essential for both basal and IFN-inducible PKR promoter activity. We have now examined the functional properties of the KCS-ℓ element of the ADAR1 PI promoter. Electrophoretic mobility shift assays (EMSAs) detected constitutively expressed nuclear proteins that bound selectively to the ADAR1 KCS-ℓ element. Competition EMSA and antibody supershift assays indicated that ADAR1 KCS-ℓ-binding proteins shared some properties with PKR KCS-binding proteins. However, transient transfection analyses performed with ADAR1 PI promoter constructs possessing deletion and substitution mutant forms of the KCS-ℓ element revealed that the ADAR1 KCS-ℓ element was not essential for either basal or IFN-inducible promoter activity. Substitution of the ADAR1 KCS-ℓ element with the PKR KCS element increased both basal and inducible ADAR1 PI promoter activity. These results suggest that the KCS-ℓ element of the ADAR1 PI promoter is not functionally equivalent to the KCS element of the PKR promoter.

Methylation of α-type embryonic globin gene απrepresses transcription in primary erythroid cells

The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.

Glucose Regulation of the Acetyl-CoA Carboxylase Promoter PI in Rat Hepatocytes

The rat acetyl-CoA carboxylase (ACC) alpha gene is transcribed from two promoters, denoted PI and PII, that direct regulated expression in a tissue-specific manner. Induction of ACC, the rate-controlling enzyme of fatty acid biosynthesis, occurs in the liver in response to feeding of a high carbohydrate, low fat diet, conditions that favor enhanced lipogenesis. This induction is mainly due to increases in PI promoter activity. We have used primary cultured hepatocytes from the rat to investigate glucose regulation of ACC expression. Glucose and insulin synergistically activated expression of ACC mRNAs transcribed from the PI promoter with little or no effect on PII mRNAs. Glucose treatment stimulated PI promoter activity in transfection assays and a glucose-regulated element was identified (-126/-102), homologous to those previously described in other responsive genes, including l-type pyruvate kinase, S(14) and fatty acid synthase. Mutation of this element eliminated the response to glucose. This region of the ACC PI promoter was able to bind a liver nuclear factor designated ChoRF that interacts with other conserved glucose-regulated elements. This ACC PI element is also capable of conferring a strong response to glucose when linked to a heterologous promoter. We conclude that induction of ACC gene expression under lipogenic conditions in hepatocytes is mediated in part by the activation of a glucose-regulated transcription factor, ChoRF, which stimulates transcription from the PI promoter. Similar mechanisms operate on related genes permitting the coordinate induction of the lipogenic pathway.

Genomic structure and transcriptional regulation of human Galbeta1,3GalNAc alpha2,3-sialyltransferase (hST3Gal I) gene.

Previous studies have shown that hST3Gal I mRNA is overexpressed in colorectal cancer tissues and primary breast carcinoma compared with nonmalignant or benign tissue, suggesting that the transcriptional regulation of hST3Gal I gene is altered during malignant transformation. We report transcriptional regulation of the hST3Gal I gene in colon adenocarcinoma and leukemia cell lines. To determine the genomic structure of the 5'-untranslated region, we cloned and identified the 5'-untranslated region of hST3Gal I from a human genome library. The 5'-untranslated region was found to be divided into three exons, namely, exons Y, X, and C1. The transcription initiation sites map at -1035 bp from the translation initiation site. Our results indicate that the transcriptional regulation of hST3Gal I depends on the pI promoter that exists 5'-upstream of exon Y in these cell lines. The results of luciferase assay suggest that the nt -304 to -145 region is important for transcriptional activity of hST3Gal I gene in both cell lines. The nt -304 to -145 region contains two sequences similar to the Sp1 recognition elements (GC-box) and one USF binding site. The results of site-directed mutagenesis indicated that the Sp1 binding sites and USF binding site of the pI promoter are involved in the transcription of hST3Gal I mRNA. However, the triple mutant of these sites still exhibits about 50% transcriptional activity, suggesting that there are other transcription factors involved in the transcription of hST3Gal I mRNA. These results suggest that these factors may play a critical role in the up-regulation of the hST3Gal I gene during malignant transformation.

Regulation of Bacteriophage λ Development by Guanosine 5′-Diphosphate-3′-diphosphate

On infection of its host, Escherichia coli, bacteriophage λ can follow one of two alternative developmental pathways: lytic or lysogenic. Here we demonstrate that the “lysis-versus-lysogenization” decision is influenced by guanosine tetraphosphate (ppGpp), a nucleotide that is synthesized in E. coli cells in response to amino acid or carbon source starvation. We found that the efficiency of lysogenization is the highest at ppGpp concentrations somewhat higher than the basal level; too low and too high levels of ppGpp result in less efficient lysogenization. Maintenance of the already integrated λ prophage and phage lytic development were not significantly influenced in the host lacking ppGpp. We found that the level of HflB/FtsH protease, responsible for degradation of the CII protein, an activator of “lysogenic” promoters, depends on ppGpp concentration. The highest levels of HflB/FtsH was found in bacteria lacking ppGpp and in cells bearing increased concentrations of this nucleotide. Using lacZ fusions, we investigated the influence of ppGpp on activities of λ promoters important at the stage of the lysis-versus-lysogenization decision. We found that each promoter is regulated differentially in response to the abundance of ppGpp. Moreover, our results suggest that the cAMP level may influence ppGpp concentration in cells. The mechanism of the ppGpp-mediated control of λ development at the stage of the lysis-versus-lysogenization decision may be explained on the basis of differential influence of guanosine tetraphosphate on activities of pL, pR, pE, pI, and paQ promoters and by dependence of HflB/FtsH protease level on ppGpp concentration.

Nus A is involved in transcriptional termination on λ tI

The transcriptional terminator tI generates the 3'end of the integrase ( int) gene transcript that is read from the λ PI promoter in lambda phage. We have studied the factors that affect transcription termination in vitro and in vivo at the λ tI terminator. In vitro transcriptional studies showed that tI is about 80% efficient in the presence of purified NusA protein, whereas it is only about 50% efficient in its absence. In vivo studies, where the readthrough transcript of λ tI was measured by quantitative dot blot analysis, gave about 80% efficiency in wild-type strains, but only 60% in the nusA1 mutant strain at non-permissive temperatures. These results support the idea that termination at λ tI in vivo involves interaction with the NusA factor.