Distal Promoter Element Research Articles

Mechanism of prostaglandin E2-induced transcriptional up-regulation of Oncostatin-M by CREB and Sp1.

Oncostatin-M (OSM) is a pleotropic cytokine belonging to the interleukin-6 family. Differential expression of OSM in response to varying stimuli and exhibiting repertoire of functions in different cells renders it challenging to study the mechanism of its expression. Prostaglandin E2 (PGE2) transcriptionally increased osm levels. In silico studies of ∼1 kb upstream of osm promoter region yielded the presence of CRE (cyclic AMP response element)-like sites at the distal end (CREosm). Deletion and point mutation of CREosm clearly indicated that this region imparted an important role in PGE2-mediated transcription. Nuclear protein(s) from PGE2-treated U937 cells, bound to this region, was identified as CRE-binding protein (CREB). CREB was phosphorylated on treatment and was found to be directly associated with CREosm The presence of cofactors p300 and CREB-binding protein in the complex was confirmed. A marked decrease in CREB phosphorylation, binding and transcriptional inhibition on treatment with PKA (protein kinase A) inhibitor, H89 (N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-soquinolinesulfonamide), revealed the role of phosphorylated CREB in osm transcription. Additionally, other nuclear protein(s) were specifically associated with the proximal GC region (GCosm) post PGE2 treatment, later confirmed to be specificity protein 1 (Sp1). Interestingly, Sp1 bound to the proximal osm promoter was found to be associated with phospho-CREB-p300 complex bound to the distal osm promoter. Knockdown of Sp1 abrogated the expression and functionality of OSM. Thus, the present study conclusively proves that these transcription factors, bound at the distal and proximal promoter elements are found to associate with each other in a DNA-dependent manner and both are responsible for the PGE2-mediated transcriptional up-regulation of Oncostatin-M.

Regulation of Hypothalamic Corticotropin-Releasing Hormone Transcription by Elevated Glucocorticoids

Negative glucocorticoid feedback is essential for preventing the deleterious effects of excessive hypothalamic pituitary adrenal axis axis activation, with an important target being CRH transcription in the hypothalamic paraventricular nucleus. The aim of these studies was to determine whether glucocorticoids repress CRH transcription directly in CRH neurons, by examining glucocorticoid effects on glucocorticoid receptor (GR)-CRH promoter interaction and the activation of proteins required for CRH transcription. Immunoprecipitation of hypothalamic chromatin from intact or adrenalectomized rats subjected to either stress or corticosterone injections showed minor association of the proximal CRH promoter with the GR compared with that with phospho-CREB (pCREB). In contrast, the Period-1 (Per1, a glucocorticoid-responsive gene) promoter markedly recruited GR. Stress increased pCREB recruitment by the CRH but not the Per1 promoter, irrespective of circulating glucocorticoids. In vitro, corticosterone pretreatment (30 minutes or 18 hours) only slightly inhibited basal and forskolin-stimulated CRH heteronuclear RNA in primary hypothalamic neuronal cultures and CRH promoter activity in hypothalamic 4B cells. In 4B cells, 30 minutes or 18 hours of corticosterone exposure had no effect on forskolin-induced nuclear accumulation of the recognized CRH transcriptional regulators, pCREB and transducer of regulated CREB activity 2. The data show that inhibition of CRH transcription by physiological glucocorticoids in vitro is minor and that direct interaction of GR with DNA in the proximal CRH promoter may not be a major mechanism of CRH gene repression. Although GR interaction with distal promoter elements may have a role, the data suggest that transcriptional repression of CRH by glucocorticoids involves protein-protein interactions and/or modulation of afferent inputs to the hypothalamic paraventricular nucleus.

Gonadotrope-Specific Expression and Regulation of Ovine Follicle Stimulating Hormone Beta: Transgenic and Adenoviral Approaches Using Primary Murine Gonadotropes

The beta subunit of follicle stimulating hormone (FSHB) is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between −172/−1 bp of the ovine FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH). Additional distal promoter sequence between −4741/−750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between −1866/−750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained −2871 bp, −750 bp or −232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml) and inhibited by GnRH (100 nM) in the presence of activin (except −232FSHBLuc). However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin) was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between −2871/−750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between −750 bp and −232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences between −1866/−750 bp of the ovine FSHB promoter that are also required for tissue/cell specific expression in vivo.

Hmgb1 can facilitate activation of the matrilin-1 gene promoter by Sox9 and L-Sox5/Sox6 in early steps of chondrogenesis

The architectural high mobility group box 1 (Hmgb1) protein acts as both a nuclear and an extracellular regulator of various biological processes, including skeletogenesis. Here we report its contribution to the evolutionarily conserved, distinctive regulation of the matrilin-1 gene (Matn1) expression in amniotes. We previously demonstrated that uniquely assembled proximal promoter elements restrict Matn1 expression to specific growth plate cartilage zones by allowing varying doses of L-Sox5/Sox6 and Nfi proteins to fine-tune their Sox9-mediated transactivation. Here, we dissected the regulatory mechanisms underlying the activity of a conserved distal promoter element 1. We show that this element carries three Sox-binding sites, works as an enhancer in vivo, and allows promoter activation by the Sox5/6/9 chondrogenic trio. In early steps of chondrogenesis, declining Hmgb1 expression overlaps with the onset of Sox9 expression. Unlike repression in late steps, Hmgb1 overexpression in early chondrogenesis increases Matn1 promoter activation by the Sox trio, and forced Hmgb1 expression in COS-7 cells facilitates induction of Matn1 expression by the Sox trio. The conserved Matn1 control elements bind Hmgb1 and SOX9 with opposite efficiency in vitro. They show higher HMGB1 than SOX trio occupancy in established chondrogenic cell lines, and HMGB1 silencing greatly increases MATN1 and COL2A1 expression. Together, these data thus suggest a model whereby Hmgb1 helps recruit the Sox trio to the Matn1 promoter and thereby facilitates activation of the gene in early chondrogenesis. We anticipate that Hmgb1 may similarly affect transcription of other cartilage-specific genes.

The CpG island shore of the GLT‐1 gene acts as a methylation‐sensitive enhancer

Astrocytic lineage commitment and brain region-dependent specialization of glia are partly ascribed to epigenetic processes. Clearance of glutamate is an essential task, which astrocytes assume in a temporal-spatial fashion by distinct glutamate transporter expression. Glutamate transporter subtype 1 (GLT-1) is predominant in cortex (CTX), while it plays an inferior role in cerebellum (CER). Here, we set out to identify regulatory elements that could account for the differences in brain region-specific activity as well as response to dexamethasone (DEX) or epigenetic factors. We found a distal promoter element at the shore of the CpG island exhibiting enhancer function in response to DEX in reporter gene assays. This shore region showed slight enrichment in repressive trimethyl-histone H3 (Lys27) and under-representation of acetyl-histone H4 (H4ac) marks in DEX nonresponsive CER astrocytes as determined by chromatin immunoprecipitation. In addition, CpG sites of the shore region displayed higher methylation in CER than in CTX cells. Targeted in vitro methylation of CpG sites within the shore abrogated the stimulatory effects of DEX. Interestingly, the shore was characterized by a pronounced epigenetic plasticity in CTX cells since DEX exposure elicited an increase of H4ac in CTX in comparison to DEX nonresponsive CER. The transcriptional activity of this region was also affected by histone deacetylase inhibitors in a methylation- and brain region-dependent manner. Together, our study highlights the impact of an epigenetically adaptive DNA element of the GLT-1 promoter being decisive for brain region-specific activity and reactivity.

In Vitro and Ex Vivo Analysis of CHRNA3 and CHRNA5 Haplotype Expression

Genome-wide association studies implicate variations in CHRNA5 and CHRNA3 as being associated with nicotine addiction (NA). Multiple common haplotypes (“risk”, “mixed” and “protective”) exist in Europeans; however, high linkage disequilibrium between variations in CHRNA5 and CHRNA3 makes assigning causative allele(s) for NA difficult through genotyping experiments alone. We investigated whether CHRNA5 or CHRNA3 promoter haplotypes, associated previously with NA, might influence allelic expression levels. For in vitro analyses, promoter haplotypes were sub-cloned into a luciferase reporter vector. When assessed in BE(2)-C cells, luciferase expression was equivalent among CHRNA3 haplotypes, but the combination of deletion at rs3841324 and variation at rs503464 decreased CHRNA5 promoter-derived luciferase activity, possibly due to loss of an SP-1 and other site(s). Variation within the CHRNA5 5’UTR at rs55853698 and rs55781567 also altered luciferase expression in BE(2)-C cells. Allelic expression imbalance (AEI) from the “risk” or “protective” haplotypes was assessed in post-mortem brain tissue from individuals heterozygous at coding polymorphisms in CHRNA3 (rs1051730) or CHRNA5 (rs16969968). In most cases, equivalent allelic expression was observed; however, one individual showed CHRNA5 AEI that favored the “protective” allele and that was concordant with heterozygosity at polymorphisms ∼13.5 kb upstream of the CHRNA5 transcription start site. Putative enhancer activity from these distal promoter elements was assessed using heterologous promoter constructs. We observed no differences in promoter activity from the two distal promoter haplotypes examined, but found that the distal promoter region strongly repressed transcription. We conclude that CHRNA5 promoter variants may affect relative risk for NA in some heterozygous individuals.

C-Myc activates BRCA1 gene expression through distal promoter elements in breast cancer cells

BackgroundThe BRCA1 gene plays an important role in the maintenance of genomic stability. BRCA1 inactivation contributes to breast cancer tumorigenesis. An increasing number of transcription factors have been shown to regulate BRCA1 expression. c-Myc can act as a transcriptional activator, regulating up to 15% of all genes in the human genome and results from a high throughput screen suggest that BRCA1 is one of its targets. In this report, we used cultured breast cancer cells to examine the mechanisms of transcriptional activation of BRCA1 by c-Myc.Methodsc-Myc was depleted using c-Myc-specific siRNAs in cultured breast cancer cells. BRCA1 mRNA expression and BRCA1 protein expression were determined by quantitative RT-PCR and western blot, respectively and BRCA1 promoter activities were examined under these conditions. DNA sequence analysis was conducted to search for high similarity to E boxes in the BRCA1 promoter region. The association of c-Myc with the BRCA1 promoter in vivo was tested by a chromatin immunoprecipitation assay. We investigated the function of the c-Myc binding site in the BRCA1 promoter region by a promoter assay with nucleotide substitutions in the putative E boxes. BRCA1-dependent DNA repair activities were measured by a GFP-reporter assay.ResultsDepletion of c-Myc was found to be correlated with reduced expression levels of BRCA1 mRNA and BRCA1 protein. Depletion of c-Myc decreased BRCA1 promoter activity, while ectopically expressed c-Myc increased BRCA1 promoter activity. In the distal BRCA1 promoter, DNA sequence analysis revealed two tandem clusters with high similarity, and each cluster contained a possible c-Myc binding site. c-Myc bound to these regions in vivo. Nucleotide substitutions in the c-Myc binding sites in these regions abrogated c-Myc-dependent promoter activation. Furthermore, breast cancer cells with reduced BRCA1 expression due to depletion of c-Myc exhibited impaired DNA repair activity.ConclusionsThe distal BRCA1 promoter region is associated with c-Myc and contributes to BRCA1 gene activation.

Contribution of a Nuclear Factor-κB Binding Site to Human Angiotensinogen Promoter Activity in Renal Proximal Tubular Cells

Intrarenal angiotensinogen (AGT) is expressed highly in renal proximal tubular cells (RPTCs) and contributes to the regulation of intrarenal angiotensin II levels. Inhibition of nuclear factor (NF)-κB suppressed human (h)AGT expression in human RPTCs. However, the presence and localization of an NF-κB binding site in the hAGT promoter region have not been determined. Therefore, this study was performed to demonstrate that an NF-κB binding site in the hAGT promoter region contributes to hAGT promoter activity in human RPTCs. The hAGT promoter region was cloned from -4358 to +122 and deletion analysis was performed. A possible NF-κB binding site was removed from the hAGT promoter region (M1) and mutated (M2). Human RPTCs were transfected, and hAGT promoter activity was determined by luciferase assay. The identity of DNA binding proteins from binding assays were determined by Western blot. Progressive 5'-end deletions demonstrated removal of a distal promoter element in hAGT_-2414/+122 reduced promoter activity (0.61 ± 0.12, ratio to hAGT_-4358/+122). Inhibition of NF-κB suppressed promoter activity in hAGT_-4358/+122 (0.51 ± 0.14, ratio to control) and hAGT_-3681/+122 (0.48 ± 0.06, ratio to control) but not in the construct without the NF-κB binding site. Promoter activity was reduced in the domain mutants M1 (0.57 ± 0.08, ratio to hAGT_-4358/+122) and M2 (0.61 ± 0.16, ratio to hAGT_-4358/+122). DNA binding levels of NF-κB protein were reduced in M1. These data demonstrate the functional importance of an NF-κB binding site in the hAGT promoter region, which contributes to hAGT promoter activity in human RPTCs.

A Novel Transcriptional Mechanism of Cell Type–Specific Regulation of Vascular Gene Expression by Glucose

Vascular diabetic complications are associated with abnormal extracellular matrix and dysfunction of vascular cells, which later result in aberrant angiogenesis and development of atherosclerotic lesions. The tissue and cell specificity of the effects of high glucose are well recognized, but the underlying cell type-specific molecular mechanisms controlled by glucose are still unclear. We sought to identify cell type-specific mechanisms by which high glucose regulates transcription of genes in vascular cells. Thrombospondin-1 is a potent antiangiogenic protein associated with development of several diabetic complications and regulated by high glucose in multiple cell types. We report that distinct cell type-specific mechanisms regulate thrombospondin-1 gene (THBS1) transcription in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in response to high glucose: although a proximal fragment of 280 nucleotides is sufficient to drive transcription in ECs, THBS1 was regulated cooperatively by interaction between proximal (-272 to -275) and distal (-1016 to -1019) promoter elements in VSMCs. Transcription factors activated by high glucose in VSMCs were cell type-specific. The formation of a single complex interacting with both distal and proximal glucose-responsive elements of THBS1 promoter in VSMCs was confirmed using gel-shift assays, binding sequence decoy oligomers, and specific mutant promoter fragments. Transcriptional response of vascular cells to high glucose is cell type-specific and involves activation of distinct transcription factors, providing a basis for tissue-specific changes of vasculature in diabetics.

ZEB1 and CtBP form a repressive complex at a distal promoter element of the BCL6 locus

BCL6 is essential for normal antibody responses and is highly expressed in germinal centre B-cells. Constitutive expression due to chromosomal translocations or mutations of cis-acting regulatory elements contributes to diffuse large B-cell lymphoma. BCL6 expression is therefore tightly regulated in a lineage- and developmental-stage-specific manner, and disruption of normal controls can contribute to lymphomagenesis. In order to discover potential cis-acting control regions we carried out DNase I-hypersensitive site mapping. Gel-shift assays and chromatin immunoprecipitation of the core region of a hypersensitive site 4.4 kb upstream of BCL6 transcription initiation (HSS-4.4) showed an E-box element-binding ZEB1 (zinc finger E-boxbinding homeobox 1) and the co-repressor CtBP (C-terminal binding protein). As compared with peripheral blood B-cells, ZEB1, a two-handed zinc finger transcriptional repressor, is expressed at relatively low levels in germinal centre cells, whereas BCL6 has the opposite pattern of expression. Transfection of ZEB1 cDNA caused a reduction in BCL6 expression and a mutated ZEB1, incapable of binding CtBP, lacked this effect. siRNA (small interfering RNA)-mediated knockdown of ZEB1 or CtBP produced an increase in BCL6 mRNA. We propose that HSS-4.4 is a distal promoter element binding a repressive complex consisting of ZEB1 and CtBP. CtBP is ubiquitously expressed and the results of the present study suggest that regulation of ZEB1 is required for control of BCL6 expression.

A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

The transcription factor c-Myc enhances KIR gene transcription through direct binding to an upstream distal promoter element

AbstractThe killer cell immunoglobulin-like receptor (KIR) repertoire of natural killer (NK) cells determines their ability to detect infected or transformed target cells. Although epigenetic mechanisms play a role in KIR gene expression, work in the mouse suggests that other regulatory elements may be involved at specific stages of NK-cell development. Here we report the effects of the transcription factor c-Myc on KIR expression. c-Myc directly binds to, and promotes transcription from, a distal element identified upstream of most KIR genes. Binding of endogenous c-Myc to the distal promoter element is significantly enhanced upon interleukin-15 (IL-15) stimulation in peripheral blood NK cells and correlates with an increase in KIR transcription. In addition, the overexpression of c-Myc during NK-cell development promotes transcription from the distal promoter element and contributes to the overall transcription of multiple KIR genes. Our data demonstrate the significance of the 5′ promoter element upstream of the conventional KIR promoter region and support a model whereby IL-15 stimulates c-Myc binding at the distal KIR promoter during NK-cell development to promote KIR transcription. This finding provides a direct link between NK-cell activation signals and KIR expression required for acquisition of effector function during NK-cell education.

Molecular Heterogeneity of β-Thalassemia in Algeria: How to Face Up to a Major Health Problem

This study concerns the molecular characterization of β-thalassemia (β-thal) alleles in 210 chromosomes. In the studied population, mutations were detected in 98% of the β-thalassemic chromosomes. Twenty-one molecular defects have been found, where the five dominant mutations, IVS-I-110 (G>A), nonsense mutation at codon 39 (C>T), the frameshift codon (FSC) 6 (−A), IVS-I-1 (G>A), and IVS-I-6 (T>C), account for 80% of the independent chromosomes. Among the remaining alleles, 16 different mutations were identified, half of them being described for the first time in Algeria. These include the −101 (C>T) and the −90 (C>T) mutations in the distal and proximal promoter elements, respectively, the FSC 8 (−AA), IVS-I-5 (G>T), IVS-I-128 (T>G), FSC 47 (+A), IVS-II-1 (G>A), and the substitution in the polyadenylation signal (poly A) site AATAAA>AATGAA. Haplotype analyses on rare variants were performed. The possible origin of these mutations either by founder effect or by migrations is discussed, and raises the question of an adequate strategy to be used adapted to socio-economical status.

Antisense Transcripts Negatively Regulate Transcription of Multiple Variegated Killer Immunoglobulin-Like Receptor (KIR) Genes

Natural killer cells are CD3 negative large granular lymphocytes that lyse virally infected and malignantly transformed targets. NK cell functions are regulated by an array of inhibitory and activating receptors, including members of the killer immunoglobulin-like receptor (KIR) family. Human KIR genes are expressed in a variegated and clonally restricted manner on the surface of mature NK cells. While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides within the promoter region proximal to the transcriptional start site, the mechanisms that regulate variegated KIR expression are largely unknown. Our goal is to uncover the genetic mechanisms governing KIR transcriptional regulation. We have recently identified an active distal promoter element approximately 1 Kb upstream of the transcriptional start site. This region contains c-MYC binding sites, and KIR expression is increased when MYC is overexpressed in developing NK cells or when c-MYC is physiologically increased by IL-15 activation. Bi-directional promoter activity is found within the previously characterized proximal promoter of all KIR genes. Therefore, double-stranded RNA with homology to the KIR promoter can be generated within this non-coding region. The generation of double-stranded RNA has recently been shown to contribute to promoter DNA methylation of the p15 gene in mammalian cells through a Dicer-independent mechanism. Using quantitative real-time PCR, we found that purified peripheral blood CD56+/KIR3DL1− NK cells express 5-fold more KIR3DL1 antisense transcript than purified CD56+/KIR3DL1+ cells. Therefore, the transcriptional activation of the KIR3DL1 gene correlates with a significant decrease in KIR3DL1 antisense expression. To explore the possibility that KIR antisense transcripts can silence KIR expression, we over-expressed the KIR3DL1 antisense transcript in CD34+ hematopoietic precursor cells and differentiated these cells into NK cells in vitro. After 21 days in culture, we assayed KIR3DL1 mRNA levels using quantitative real-time PCR. KIR3DL1 expression was reduced 4-fold compared to eGFP control cultures, further supporting the hypothesis that antisense transcripts negatively regulate KIR expression. Importantly, expression of the KIR2DL4 gene, which does not contain a promoter with significant homology to KIR3DL1 promoter, was not affected by KIR3DL1 antisense overexpression. This provides the first evidence for gene silencing through double-stranded RNA in the immune system and may be the key mechanism for the establishment of DNA promoter methylation within the KIR locus. Understanding the mechanisms of KIR expression, a requisite for NK cell education/licensing, may allow us to understand the acquisition of effector function and manipulate it for therapeutic benefit.

Developmental and Environmental Signals Induce Distinct Histone Acetylation Profiles on Distal and Proximal Promoter Elements of theC4-PepcGene in Maize

The maize C(4)-Pepc gene is expressed in an organ- and cell-type-specific manner, inducible by light and modulated by nutrient availability and the metabolic state of the cell. We studied the contribution of histone acetylation at five lysine residues to the integration of these signals into a graduated promoter response. In roots and coleoptiles, where the gene is constitutively inactive, three of the five lysines were acetylated and the modifications showed unique patterns with respect to their distribution on the gene. A similar pattern was observed in etiolated leaves, where the gene is poised for activation by light. Here, illumination selectively induced the acetylation of histone H4 lysine 5 and histone H3 lysine 9 in both the promoter and the transcribed region, again with unique distribution patterns. Induction was independent of transcription and fully reversible in the dark. Nitrate and hexose availability modulated acetylation of all five lysines restricted to a distal promoter region, whereas proximal promoter acetylation was highly resistant to these stimuli. Our data suggest that light induction of acetylation is controlled by regulating HDAC activity, whereas metabolic signals regulate HAT activity. Acetylation turnover rates were high in the distal promoter and the transcribed regions, but low on the proximal promoter. On the basis of these results, we propose a model with three levels of stimulus-induced histone modifications that collectively adjust promoter activity. The results support a charge neutralization model for the distal promoter and a stimulus-mediated, but transcription-independent, histone acetylation pattern on the core promoter, which might be part of a more complex histone code.

Exploring the contribution of distal P4 promoter elements to the oncoselectivity of Minute Virus of Mice

Minute Virus of Mice (MVM) shares inherent oncotropic properties with other members of the genus Parvovirus. Two elements responsible, at least in part, for this oncoselectivity have been mapped to an Ets1 binding site adjacent to the P4 TATA box of the initiating promoter, P4, and to a more distal cyclic AMP responsive element (CRE), located within the telomeric hairpin stem. Here the CRE overlaps one half-site for the binding of parvoviral initiation factor (PIF), which is essential for viral DNA replication. We used a degenerate oligonucleotide selection approach to show that CRE binding protein (CREB) selects the sequence ACGTCAC within this context, rather than its more generally accepted palindromic TGACGTCA recognition site. We have developed strategies for manipulating these sequences directly within the left-end palindrome of the MVM infectious clone and used them to clone mutants whose CRE either matches the symmetric consensus sequence or is scrambled, or in which the PIF binding site is incrementally weakened with respect to the CRE. The panel of mutants were tested for fitness relative to wildtype in normal murine fibroblasts A9 or transformed human fibroblasts 324 K, through multiple rounds of growth in co-infected cultures, using a differential real-time quantitative PCR assay. We confirmed that inactivating the CRE substantially abrogates oncoselectivity, but found that improving its fit to the palindromic consensus is somewhat debilitating in either cell type. We also confirmed that reducing the PIF half-site spacing by one basepair enhances oncoselectivity, but found that a further basepair deletion significantly reduces this effect.

Identification of distal KIR promoters and transcripts

A more complete understanding of the transcriptional control of the human and murine class I MHC receptors will help to shed light on the mechanism of selective, stochastic, gene activation that operates in these gene families. Studies of the murine Ly49 class I MHC receptor genes have revealed an important role for distal transcripts originating upstream of the proximal promoter. To date, there have been no reports of distal promoters within the functionally analogous human KIR family of class I MHC receptors. In the current study, reverse transcriptase-polymerase chain reaction (RT-PCR) and RNase protection assays were used to reveal the presence of distal KIR transcripts initiating upstream of the previously characterized proximal KIR promoter. The intergenic promoter elements detected were associated with repetitive elements of the Alu and L1 families. Unlike the proximal KIR promoter, the distal promoter regions were not NK cell-specific. KIR genes expressed in a variegated manner produced a low level of distal transcripts containing a large 5' untranslated region. In contrast, the highly expressed KIR2DL4 gene possessed a higher level of spliced distal transcripts that were capable of producing KIR2DL4 protein. The identification of distal KIR promoter elements suggests that intergenic transcripts may influence the expression of KIR genes.

Regulation of the Rhox5 Homeobox Gene in Primary Granulosa Cells: Preovulatory Expression and Dependence on SP1/SP3 and GABP1

Homeobox genes encode transcription factors that regulate embryonic development and postnatal events. Rhox5 (previously called Pem), the founding member of a homeobox gene cluster that we recently identified on the X chromosome, is selectively expressed in granulosa cells in the ovary and other somatic-cell types in other reproductive organs. In this report, we investigate its regulation in granulosa cells in the rat ovary. We found that Rhox5 expression in the ovary is governed by the Rhox5 distal promoter and is expressed at least as early as Day 5 postpartum. Rhox5 mRNA levels are regulated during the ovarian cycle, peaking before ovulation. Deletion analysis revealed a 25-nt element essential for distal promoter transcription in primary granulosa cells. This distal promoter element contains two ETS and one SP1 transcription-factor family binding sites that mutagenesis analysis indicated were essential for high-level transcription. This element was both necessary and sufficient for transcription, because it activated transcription when placed upstream of a heterologous minimal promoter. Cold competition and electrophoretic mobility shift assay studies demonstrated that SP1, SP3, and the ETS family transcription factor GABP bound this element. Dominant-negative forms of GABP and SP3 repressed distal promoter expression in primary rat granulosa, showing that these factors are crucial for Rhox5 expression. Cotransfection of dominant-negative mutants indicated that Rhox5 expression in granulosa cells is regulated by the c-Jun N-terminal protein kinase (JNK, MAPK8) and RAS pathways, which are known to be upstream of ETS family transcription factors. The discovery that Rhox5 expression in granulosa cells is regulated by MAPK pathways and ETS and SP1 family members provides an opportunity to understand how these regulatory pathways and factors collaborate to regulate gene expression during the ovarian cycle.

The mitochondrial RNA polymerase contributes critically to promoter specificity in mammalian cells

Initiation of transcription in mammalian mitochondria depends on three proteins: mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We show here that the recombinant mouse and human transcription machineries are unable to initiate transcription in vitro from the heterologous light-strand promoter (LSP) of mitochondrial DNA. This species specificity is dependent on the interaction of TFAM and POLRMT with specific distal and proximal promoter elements. A sequence element localized from position -1 to -2 relative to the transcription start site in LSP functionally interacts with POLRMT. The POLRMT/TFB2M heterodimer is unable to interact with promoter elements and initiate even abortive transcription in the absence of TFAM. TFAM is thus an integral part of the mammalian transcription machinery, and we propose that TFAM induces a structural change of the promoter that is required for POLRMT-dependent promoter recognition.

The bovine iris–ciliary epithelium expresses components of rod phototransduction

Earlier studies have documented that the iris in lower vertebrates is photosensitive. In the present work, we examined whether the bovine iris which exhibits a common embryonic origin with the ocular ciliary epithelium and the neural retina, expresses components of phototransduction. By Northern blot and RT–PCR amplification we detected in the iris, rhodopsin, rhodopsin kinase and arrestin transcripts and DNA products, respectively, of the same size as in the retina. By Western blot, antibodies to rhodopsin, rhodopsin kinase and arrestin detected low levels of protein with similar molecular masses as in the retina. Transient transfections of bovine iris cells in vitro with rhodopsin promoter-luciferase-reporter constructs (p130-Luc, p176-Luc, 1225-Luc and p2000-Luc) containing proximal and distal promoter elements led to a significant stimulation of promoter activity over the basal activity. In particular, the construct p225-Luc containing proximal promoter elements upstream of the transcription start site (−225 to +70 bp) led to 3.1-fold stimulation of activity over p176-Luc, 2.1-fold over p130 or p2000-Luc and 190-fold over the basal activity. These results suggested that the bovine iris cells contain factors that could either stimulate or attenuate rhodopsin transcription. The data also supported the view that components associated with non-visual phototransduction are expressed in extraretinal sites including the ciliary epithelium and the iris.