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Start Site (start + site)
Kinds of Start Site Selected AbstractsRequirement for ,B1-crystallin promoter of Xenopus laevis in embryonic lens development and lens regenerationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2005Nobuhiko Mizuno Regulation of the lens-specific ,B1-crystallin promoter in Xenopus laevis was investigated using transgenic larvae and tadpoles. Comparison of the promoter sequence with that of chicken ,B1-crystallin gene indicates significant sequence similarity over a span of several hundred base pairs starting from the transcriptional start site. Remarkably, PL-1 and PL-2 sequences identified in the chicken promoter as essential binding sites of MAF, Pax6 and Prox1 transcription factors were conserved. Mutations of X (Xenopus) PL-1 and XPL-2 sequences eliminated the promoter activity, indicating a conserved mechanism regulating ,B1-crystallin promoter among vertebrate species. A stepwise deletion of the promoter sequence starting from 2800 bp indicated that the proximal 260 bp directly upstream of the transcription initiation site is sufficient for eliciting lens-specific expression, but the 150 bp promoter sequence is inactive despite it containing the XPL-1 and XPL-2 sequences, suggesting the presence of an additional and essential regulatory sequence located between ,150 and ,260 bp. Activity of the ,B1-crystallin promoter during lens regeneration from cornea was examined using transgenic tadpoles and found to have the same dependence on promoter regions as in embryonic lens development, indicating that gene regulation is largely shared by the two lens-generating processes. [source] A green light-absorbing phycoerythrin is present in the high-light-adapted marine cyanobacterium Prochlorococcus sp.ENVIRONMENTAL MICROBIOLOGY, Issue 10 2005Summary In the high-light-adapted unicellular marine cyanobacterium Prochlorococcus sp. MED4 the cpeB gene is the only gene coding for a structural phycobiliprotein. The absence of any other phycoerythrin gene in the fully sequenced genome of this organism, the previous inability to detect a gene product, and the mutation of two out of four cysteine residues, normally involved in binding chromophores, suggested that MED4- cpeB might not code for a functional protein. Here, transcription of MED4- cpeB at a low level was detected and the transcriptional start site was mapped. Enrichment of the protein identified phycoerythrobilin as its sole chromophore in vivo, which was confirmed by chromophorylation assays in vitro using the recombinant protein. Phycourobilin is the major chromophore in low-light-adapted Prochlorococcus ecotypes such as strain SS120. Therefore, spectrally tuned phycoerythrins are a characteristic feature of distinct Prochlorococcus ecotypes. Further in vitro mutagenesis experiments replacing one or both cysteines C61R/C82S by arginine or serine, respectively, revealed that only Cys82 is required for chromophore binding. Thus, an unusual green light-absorbing phycoerythrin evolved in the high-light-adapted ecotypes of Prochlorococcus, which potentially serves as a photoreceptor. [source] Characterization of the mouse adenylyl cyclase type VIII gene promoter: regulation by cAMP and CREBEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2002Jennifer R. Chao Abstract Adenylyl cyclase (AC) type VIII has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. In the present study, we directly examined the role for the transcription factor CREB (cAMP response element binding protein) in regulating ACVIII expression by cloning a 5.2 kilobase region upstream of the translation start site of the mouse ACVIII gene. Analysis of this fragment revealed consensus elements for several transcription factors, including a canonical cAMP response element (CRE) in close proximity to the transcription initiation region. Next, ACVIII promoter activity was studied in two neural-derived cell lines and in primary cultures of rat striatal neurons. Activation of the cAMP pathway by forskolin treatment increased promoter activity, and a series of deletion and point mutants demonstrated that this activation is mediated specifically via the canonical CRE site. Gel shift assays confirmed that this site can bind CREB and several CREB family proteins. Further, activation of the ACVIII promoter by forskolin was potentiated by expression of a constitutively active form of CREB, CREB-VP16, whereas it was inhibited by expression of a dominant-negative form of CREB, A-CREB. Finally, over-expression of CREB in vivo, by viral-mediated gene transfer, induced ACVIII promoter activity in the brains of ACVIII-LacZ transgenic mice. These results suggest that the ACVIII gene is regulated by CREB in vitro and in vivo and that this regulation may contribute to CREB-dependent neural plasticity. [source] Mixed lineage leukemia histone methylases play critical roles in estrogen-mediated regulation of HOXC13FEBS JOURNAL, Issue 24 2009Khairul I. Ansari HOXC13, a homeobox-containing gene, is involved in hair development and human leukemia. The regulatory mechanism that drives HOXC13 expression is mostly unknown. Our studies have demonstrated that HOXC13 is transcriptionally activated by the steroid hormone estrogen (17,-estradiol; E2). The HOXC13 promoter contains several estrogen-response elements (EREs), including ERE1 and ERE2, which are close to the transcription start site, and are associated with E2-mediated activation of HOXC13. Knockdown of the estrogen receptors (ERs) ER, and ER, suppressed E2-mediated activation of HOXC13. Similarly, knockdown of mixed lineage leukemia histone methylase (MLL)3 suppressed E2-induced activation of HOXC13. MLLs (MLL1,MLL4) were bound to the HOXC13 promoter in an E2-dependent manner. Knockdown of either ER, or ER, affected the E2-dependent binding of MLLs (MLL1,MLL4) into HOXC13 EREs, suggesting critical roles of ERs in recruiting MLLs in the HOXC13 promoter. Overall, our studies have demonstrated that HOXC13 is transcriptionally regulated by E2 and MLLs, which, in coordination with ER, and ER,, play critical roles in this process. Although MLLs are known to regulate HOX genes, the roles of MLLs in hormone-mediated regulation of HOX genes are unknown. Herein, we have demonstrated that MLLs are critical players in E2-dependent regulation of the HOX gene. [source] LIN54 is an essential core subunit of the DREAM/LINC complex that binds to the cdc2 promoter in a sequence-specific mannerFEBS JOURNAL, Issue 19 2009Fabienne Schmit Recently, the conserved human LINC/DREAM complex has been described as an important regulator of cell cycle genes. LINC consists of a core module that dynamically associates with E2F transcription factors, p130 and the B-MYB transcription factor in a cell cycle-dependent manner. In this study, we analyzed the evolutionary conserved LIN54 subunit of LINC. We found that LIN54 is required for cell cycle progression. Protein interaction studies demonstrated that a predicted helix,coil,helix motif is required for the interaction of LIN54 with p130 and B-MYB. In addition, we found that the cysteine-rich CXC domain of LIN54 is a novel DNA-binding domain that binds to the cdc2 promoter in a sequence-specific manner. We identified two binding sites for LIN54 in the cdc2 promoter, one of which overlaps with the cell cycle homology region at the transcriptional start site. Gel shift assays suggested that, in quiescent cells, the binding of LIN54 at the cell cycle homology region is stabilized by the binding of E2F4 to the adjacent cell cycle-dependent element. Our data demonstrate that LIN54 is an important and integral subunit of LINC. Structured digital abstract ,,MINT-7239362: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with p130 (uniprotkb:Q08999) by anti tag coimmunoprecipitation (MI:0007) ,,MINT-7239376: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with B-Myb (uniprotkb:P10244) by anti tag coimmunoprecipitation (MI:0007) [source] Sp1 and Sp3 are involved in up-regulation of human deoxyribonuclease II transcription during differentiation of HL-60 cellsFEBS JOURNAL, Issue 8 2003San-Fang Chou Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate-13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis -regulatory elements and transcription factors involved in this process in HL-60 cells. cis -Regulatory elements in the DNase II promoter were located by 5, deletion and site-directed mutagenesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells. [source] Identification of a novel nuclear factor-kappaB sequence involved in expression of urokinase-type plasminogen activator receptorFEBS JOURNAL, Issue 11 2000Yao Wang We have previously defined the promoter of human urokinase-type plasminogen activator receptor (uPAR) gene in a 188-bp fragment between bases ,141 and +47 relative to the translation start site. Here, we report that a novel nuclear factor-kappaB (NF-,B)-like sequence (5,-GGGAGGAGTC-3,) at ,45 is located in the uPAR promoter and one of the two DNase I-protected regions, region I between bases ,51 and ,30. This NF-,B-like motif differs at positions 7,9 from the decameric consensus sequences of NF-,B (5,-GGGRNNYYCC-3, where R indicates A or G, Y indicates C or T, and N indicates any nucleotide) and at positions 1 and 7,9 from the ,B-like motifs (5,-HGGARNYYCC-3, where H indicates A, C, or T, R indicates A or G, Y indicates C or T, and N indicates any nucleotide). Nuclear extracts from HCT116 cells contain proteins that specifically bind to the NF-,B-like site at position ,45. Mutation of the NF-,B-like motif decreased the binding of transcription factor NF-,B and reduced the uPAR promoter activity in comparison with the wild-type sequences. Co-transfection with a dominant negative I-,B kinase-2 expression vector reduced uPAR promoter activity by 65,75%. These results demonstrate that a previously uncharacterized NF-,B motif is required for uPAR promoter activity. [source] Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifoliiFEBS JOURNAL, Issue 24 2000Hwan Young Lee A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv. trifolii. The matA, matB, and matC genes encode malonyl-CoA decarboxylase, malonyl-CoA synthetase, and a presumed malonate transporter, respectively. Together, these enzymes catalyze the uptake and conversion of malonate to acetyl-CoA. The deduced amino-acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N-terminal region encoding a helix-turn-helix domain. Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region. The addition of malonate or methylmalonate increased the association of MatR and DNA fragment. DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter. The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered ,46.5 relative to the transcription start site. Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR-mediated repression effect on the expression of Pmat -luc+ reporter fusion. [source] Characterization of the testis-specific promoter region in the human pituitary adenylate cyclase-activating polypeptide (PACAP) geneGENES TO CELLS, Issue 6 2010Aiko Tominaga Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5,-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP. [source] Single nucleotide polymorphisms 5, upstream the coding region of the NEIL2 gene influence gene transcription levels and alter levels of genetic damageGENES, CHROMOSOMES AND CANCER, Issue 11 2008Carla J. Kinslow NEIL2 (EC 4.2.99.18), a mammalian DNA glycosylase and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Because base excision repair (BER) proteins act collectively and in a progressive fashion, their proper balance is essential for optimal repair. Thus, inter-individual variability in transcription levels of NEIL2 may predispose to compromised DNA repair capacity and genomic instability by altering the balance of critical BER proteins. In a study of lymphocytes of 129 healthy subjects, using absolute quantitative reverse transcription PCR, we found that NEIL2 transcription varied significantly (up to 63 fold) and that this variability was influenced by certain single nucleotide polymorphisms (SNPs) located 5, of the start site. Using the mutagen sensitivity assay to characterize the biological significance of these SNPs, we observed a significant increase in mutagen-induced genetic damage associated with two SNPs in the promoter region of the NEIL2 gene. To characterize the functional significance of these SNPs, we engineered luciferase-reporter constructs of the NEIL2 promotor with mutations corresponding to these SNPs. We transfected these constructs into MRC-5 cells and evaluated their impact on NEIL2 expression levels. Our results indicate that NEIL2 expression was significantly reduced by over 50% (P < 0.01) in the presence of two SNPs, ss74800505 and rs8191518, located near the NEIL2 start site, which were in significant linkage disequilibrium (D, = 73%; P < 0.05). This first report on in vivo variability in NEIL2 expression in humans identifies SNPs in the NEIL2 promoter region that have functional effects. © 2008 Wiley-Liss, Inc. [source] Altered promoter usage characterizes monoallelic transcription arising with ERBB2 amplification in human breast cancersGENES, CHROMOSOMES AND CANCER, Issue 11 2006Christopher C. Benz Analysis of a collection of human breast cancers (n = 150), enriched in ERBB2-positive cases (n = 57) and involving tumor genotyping relative to population-matched blood genotyping (n = 749) for a common ERBB2 single nucleotide polymorphism Ala(G)1170Pro(C), revealed that ERBB2 amplification in breast cancer is invariably monoallelic. Analysis of paired breast cancer and blood samples from informative (G1170C heterozygotic) ERBB2-positive (n = 12) and ERBB2-negative (n = 17) cases not only confirmed monoallelic amplification and ERBB2 transcriptional overexpression but also revealed that most low ERBB2 expressing breast cancers (12/17) exhibit unbalanced allelic transcription, showing 3-fold to nearly 5,000-fold preferential expression from one of two inherited alleles. To explore cis-acting transcriptional mechanisms potentially selected during ERBB2 amplification, levels of four different ERBB2 transcript variants (5.2, 4.7, 2.1, and 1.4 kb) were correlated with total (4.6 kb) ERBB2 mRNA levels in ERBB2-positive (n = 14) versus ERBB2-negative (n = 43) primary breast cancers. Relative expression of only the 2.1 kb extracellular domain-encoding splice variant and a 4.7 kb mRNA variant that uses an alternative start site were significantly increased in association with ERBB2-positivity, implicating altered promoter usage and selective transcript regulation within the ERBB2 amplicon. Altogether, these findings provide new mechanistic insights into the development of ERBB2-positive breast cancer and strong rationale for delineating candidate cis-acting regulatory elements that may link allele-specific ERBB2 transcription in premalignant breast epithelia with subsequent development of breast cancers bearing monoallelic ERBB2 amplicons. © 2006 Wiley-Liss, Inc. [source] Densely methylated MLH1 promoter correlates with decreased mRNA expression in sporadic colorectal cancersGENES, CHROMOSOMES AND CANCER, Issue 1 2002Taiji Furukawa It has been reported that MLH1 is silenced by promoter methylation, and that this phenomenon is associated with microsatellite instability (MSI) in sporadic colorectal cancer (CRC). To clarify the significance of MLH1 promoter methylation in sporadic CRC, we examined the correlation between methylation status over the entire promoter region and mRNA expression in cases showing high-frequency MSI (MSI-H). MLH1 promoter methylation was analyzed using the bisulfite modification sequencing in 48 MSI-H cases. We also screened for somatic mutation, loss of heterozygosity, and immunohistochemical staining of MLH1. The results showed that methylation patterns could be subdivided into three types: methylation of more than 80% of the CpG sites analyzed (type 1 methylation), methylation of less than 20% (type 2 methylation), and methylation mainly in the region 500 to 921 bases upstream from the translation start site (type 3 methylation). Of the three types, only type 1 methylation correlated with decreased mRNA expression. The frequency of type 1 methylation was significantly higher in cases involving the proximal colon (66.7%, 18/27) compared to that of the distal colon and rectum (23.8%, 5/21, P = 0.004). Immunohistochemical staining of MSI-H cases showed that decreased MLH1 was found in 77.1% (37/48). Of the cases with decreased MLH1, type 1 methylation was present in 59.5% (22/37). Overall, our data suggested that the type 1 methylation pattern may affect MLH1 mRNA expression, such that the majority of MSI-H cases in sporadic CRC, especially proximal colon cancer, exhibited type 1 methylation. © 2002 Wiley-Liss, Inc. [source] An enhancer sequence directs LacZ expression to developing pharyngeal endoderm in transgenic miceGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2001Hema Parmar Abstract Summary: The murine Hoxc-6 homeobox gene comprises three exons with two distinct promoters (PRI and PRII) located 9 kb apart. To characterise the PRII promoter, a region 3 kb upstream of the transcription start site was sequenced, and an Antananapedia-like consensus binding sequence was found (Coletta et al., 1991). A LacZ reporter gene construct, containing three copies of this sequence, directs highly specific expression in cells forming pharyngeal endoderm in transgenic mice. Expression was first detected in a few individual anterior endoderm cells at E7.5, which increase in number up to E9.5, where expression was clearly visible in the pharyngeal endoderm. Expression of the endodermal genes HNF3,, Pax-9, Shh, and Nkx2.5 showed colocalization with the LacZ -positive cells in the foregut and pharyngeal endoderm. This novel enhancer provides a means of tracking the morphogenetic movement of endodermal cells fated to form the foregut. genesis 31:57,63, 2001. © 2001 Wiley-Liss, Inc. [source] A human phospholamban promoter polymorphism in dilated cardiomyopathy alters transcriptional regulation by glucocorticoids,HUMAN MUTATION, Issue 5 2008Kobra Haghighi Abstract Depressed calcium handling by the sarcoplasmic reticulum (SR) Ca-ATPase and its regulator phospholamban (PLN) is a key characteristic of human and experimental heart failure. Accumulating evidence indicates that increases in the relative levels of PLN to Ca-ATPase in failing hearts and resulting inhibition of Ca sequestration during diastole, impairs contractility. Here, we identified a genetic variant in the PLN promoter region, which increases its expression and may serve as a genetic modifier in dilated cardiomyopathy (DCM). The variant AF177763.1:g.203A>C (at position ,36,bp relative to the PLN transcriptional start site) was found only in the heterozygous form in 1 out of 296 normal subjects and in 22 out of 381 cardiomyopathy patients (heart failure at age of 18,44 years, ejection fraction=22±9%). In vitro analysis, using luciferase as a reporter gene in rat neonatal cardiomyocytes, indicated that the PLN-variant increased activity by 24% compared to the wild type. Furthermore, the g.203A>C substitution altered the specific sequence of the steroid receptor for the glucocorticoid nuclear receptor (GR)/transcription factor in the PLN promoter, resulting in enhanced binding to the mutated DNA site. These findings suggest that the g.203A>C genetic variant in the human PLN promoter may contribute to depressed contractility and accelerate functional deterioration in heart failure. Hum Mutat 29(5), 640,647, 2008. © 2008 Wiley-Liss, Inc. [source] Acholeplasma laidlawii up-regulates granulysin gene expression via transcription factor activator protein-1 in a human monocytic cell line, THP-1IMMUNOLOGY, Issue 3 2001Yutaka Kida Summary An antimicrobial protein granulysin is constitutively expressed in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. However, little is known about the precise regulatory mechanisms underlying granulysin gene expression. In this study, we examined the regulatory mechanisms underlying granulysin gene expression using a human monocytic cell line, THP-1, treated with Acholeplasma laidlawii. The level of granulysin mRNA expression in THP-1 cells was significantly augmented in response to stimulation with A. laidlawii. The transfection of reporter gene constructs into THP-1 cells indicated that DNA sequences between residues ,329 and ,239, relative to the transcriptional start site of the granulysin gene, are responsible for mediating gene induction. In addition, mutagenesis of a putative activator protein-1 (AP-1)-binding site between residues ,277 and ,271 in the granulysin promoter resulted in the reduction of granulysin promoter activity. Electrophoretic mobility shift assays (EMSA) demonstrated that nuclear extract prepared from A. laidlawii- treated THP-1 cells can generate specific binding to DNA oligonucleotides encompassing the AP-1-binding site, whereas unstimulated nuclear extract from the cells failed to do so. Furthermore, competition and supershift assays confirmed that A. laidlawii can induce the activation of AP-1. These results indicate that AP-1 dominantly participates in the regulation of inducible granulysin gene expression in THP-1 cells. Therefore, the finding of inducible granulysin gene expression by A. laidlawii suggests that inducible granulysin in macrophages may function as a protective weapon when microbial invasion occurs. [source] Functional analysis of Drosophila melanogaster hexokinase Hex-A locus: multiple Initiator-like elements enhance DPE containing promoter activityINSECT MOLECULAR BIOLOGY, Issue 1 2007P. C. Jayakumar Abstract Flight muscle Hexokinase-A (HEX-A) is the most conserved and essential hexokinase isoenzyme among Drosophila species. In this study, the Hex-A locus, encoding the HEX-A isoenzyme, has been analysed for the elements regulating its expression. By sequencing the 5, ends of Hex-A cDNA amplified by 5, RACE, we identified a transcription start site that overlapped the Initiator and downstream promoter elements. A 214 bp sequence, encompassing transcription start sites and promoter elements, was required for minimal promoter activity. DNA sequence to the 5, end of the minimal promoter element did not demonstrate any promoter activity; however, its inclusion with the basal promoter element enhanced the promoter activity. Oligonucleotide competition and site-directed mutagenesis identified the Initiator-like sequences, TCAWT, present in this region that were responsible for enhancing the promoter activity. The Hex-A locus is expressed as a single protein in Drosophila cell line, whereas in pupae, larvae and adult flies, it is expressed as two distinct types. [source] Evidence for heritable predisposition to epigenetic silencing of MLH1INTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Huiping Chen Abstract Epigenetic silencing of MLH1 is the most common cause of defective DNA mismatch repair in endometrial and colorectal cancers. We hypothesized that variation in the MLH1 gene might contribute to the risk for MLH1 methylation and epigenetic silencing. We undertook a case-control study to test for the association between MLH1 variants and abnormal MLH1 methylation. Eight MLH1 SNPs were typed in the normal DNA from women with endometrial carcinoma. For these studies, the cases were women whose cancers exhibited MLH1 methylation (N = 98) and the controls were women whose cancers had no MLH1 methylation (N = 219). One MLH1 SNP, rs1800734, located in the MLH1 CpG island at ,93 from the translation start site, was significantly associated with MLH1 methylation as were age at diagnosis and patient body mass index. In validation experiments, a similar-sized cohort of colorectal carcinoma patients (N = 387) showed a similar degree of association with the ,93 SNP; a smaller cohort of endometrial carcinomas (N = 181) showed no association. Combining all 3 cohorts showed an odds ratio of 1.61 (95% CI: 1.20,2.16) for the AA or AG vs. GG genotype at the ,93 SNP. Identification of risk alleles for MLH1 methylation could shed light on mechanisms of epigenetic silencing and may ultimately lead to new approaches to the prevention or treatment of malignancies associated with MLH1 inactivation. © 2007 Wiley-Liss, Inc. [source] Association between a common IL10 distal promoter haplotype and IgE production in individuals with atopic dermatitisINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 4 2009K. Lacy Summary Atopic dermatitis (AD) is a genetically determined inflammatory skin disease characterized by abnormal cytokine production, including increased production of interleukin 10 (IL10). Single nucleotide polymorphisms (SNP) and haplotypes in the IL10 gene promoter region on chromosome 1q31-32 have been implicated in several inflammatory diseases, but generally, only SNPs proximal to the transcription start site (TSS) have been investigated. The aim of this study was to identify proximal, distal and combined haplotype sets in the IL10 promoter region and to study their association with clinical phenotypes in atopic dermatitis. SNPs at positions ,3575, ,2849, ,2779, ,2763, ,1082, ,851, ,819 and ,592 in the IL10 promoter region were genotyped in individuals with atopic dermatitis (n= 47) and nonatopic control subjects (n= 40) using polymerase chain reaction-based techniques and induced heteroduplex generator (IHG) analysis. Pan-promoter, TSS-proximal and TSS-distal haplotypes were reconstructed using phase analysis. Fifteen haplotypes representing all eight SNPs were identified. Subgrouping identified four 4-locus and three 3-locus TSS-proximal haplotypes; and nine 4-locus TSS-distal haplotypes. No difference was found in haplotype or SNP frequencies between the AD and control groups, or between patients with mild or severe disease. However, a common 4-locus TSS-distal haplotype (TGAC) was significantly increased in patients with IgE levels over 1000 kIU L,1. This study is the first to analyse the association between haplotype groups in the IL10 promoter region and clinical phenotypes in AD. We have demonstrated a significant association between the TSS-distal haplotype TGAC, and IgE levels in AD patients. It remains to be shown if there is an association between the TGAC haplotype and IL10 production, which might account for the stimulation of IgE production. [source] Two novel somatic mutations in the human interleukin 6 promoter region in a patient with sporadic breast cancerINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 6 2003A. Saha Summary Two new single nucleotide mutations were observed within the promoter region of human interleukin-6 gene (IL-6) in the tumour sample of a patient with sporadic breast cancer, which was a somatic change. Both mutations, one at ,125 (C > G) and the other at position ,173 (G > T) from the translation start site, were transversions observed at new positions, not reported earlier. In addition to these two novel mutations in this patient, a known somatic polymorphism was also observed at position ,174 (G > C) (from the transcription initiation site, redesignated as ,236 from the translational initiation site as per the HUGO nomenclature). Further, a preliminary comparative analysis of the studied promoter region by the ,ConsInspector 3.0' program, where the mutated sequence (AF362378) was compared with the sequence existing in the database (Y00081), depicted the presence of the variations in putative binding sites for transcription factors such as glucocorticoid response element (GRE) and nuclear factor kappa-B (NF,-B), which could lead to differential expression of this gene. [source] Association of the VDR Translation Start Site Polymorphism and Fracture Risk in Older Women,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2007Susan P Moffett PhD Abstract We evaluated the association between the VDR translation start site polymorphism and osteoporotic phenotypes among 6698 older white women. Women with the C/C genotype had lower wrist BMD and an increased risk of wrist and all non-spine/low-trauma fractures. The high frequency of this variant confers a population attributable risk that is similar to several established risk factors for fracture. Introduction: The vitamin D receptor (VDR) is a nuclear receptor that regulates bone formation, bone resorption, and calcium homeostasis. A common C to T polymorphism in exon 2 of the VDR gene introduces a new translation start site and a protein that differs in length by three amino acids (T = 427aa, C = 424aa; rs10735810). Materials and Methods: We conducted genetic association analyses of this polymorphism, BMD, and fracture outcomes in a prospective cohort of 6698 white American women ,65 years of age. Incident fractures were confirmed by physician adjudication of radiology reports. There were 2532 incident nontraumatic/nonvertebral fractures during 13.6 yr of follow-up including 509 wrist and 703 hip fractures. Results: Women with the C/C genotype had somewhat lower distal radius BMD compared with those with the T/T genotype (CC = 0.358 g/cm2, CT = 0.361 g/cm2, TT = 0.369 g/cm2, p = 0.003). The C/C genotype was also associated with increased risk of non-spine, low traumatic fractures (HR: 1.18; 95% CI: 1.04, 1.33) and wrist fractures (HR: 1.33; 95% CI: 1.01, 1.75) compared with the T/T genotype in age-adjusted models. Further adjustments for distal radius BMD only slightly attenuated these associations. The VDR polymorphism was not associated with hip fracture. The population attributable risk (PAR) of the C/C genotype for incident fractures was 6.1%. The PAR for established risk factors for fracture were: low femoral neck BMD (PAR = 16.3%), maternal history of fracture (PAR = 5.1%), low body weight (PAR = 5.3%), corticosteroid use (PAR = 1.3%), and smoking (PAR = 1.6%). Similar PAR results were observed for wrist fractures. Conclusions: The common and potentially functional VDR translation start site polymorphism confers a modestly increased relative risk of fracture among older white women. However, the high frequency of this variant confers a population attributable risk that is similar to or greater than several established risk factors for fracture. [source] Craniosynostosis-Associated Gene Nell-1 Is Regulated by Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007Thien Truong Abstract We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. Introduction: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5, flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. Materials and Methods: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2,/, calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. Results: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. Conclusions: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis. [source] E2F1 represses ,-catenin/TCF activity by direct up-regulation of Siah1JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Wei Xie Abstract Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Its activity is strictly controlled by the pRB/E2F pathway. In the majority of cancer cells, however, this pathway is frequently found deregulated, and the underlying mechanism involving transcriptional control by E2F1 has not yet been fully elucidated. Here we report the identification of two putative E2F1-binding sites located upstream from Siah1 transcription start site (+1). Chromatin immunoprecipitation assay reveals that transcription factor E2F1 is capable of binding to the putative sites, and luciferase reporter assay shows that E2F1 can activate transcription from the Siah1 promoter. Ectopic expression of E2F1 elevates the Siah1 level, hence suppressing the ,-catenin/TCF activity. Consistently, knock-down of endogenous E2F1 by a shRNA strategy results in reduced expression of Siah1. Moreover, repression of ,-catenin/TCF activity by E2F1 can be attenuated by shRNA-based repression of endogenous Siah1, implying that Siah1 is a bona fide E2F1 target gene, which at least partly, mediates the suppression of ,-catenin/TCF signalling pathway. [source] SRrp37, a novel splicing regulator located in the nuclear speckles and nucleoli, interacts with SC35 and modulates alternative pre-mRNA splicing in vivoJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Pin Ouyang Abstract We report here the identification and characterization of a novel SR-related protein, referred to as SRrp37, based on its apparent molecular weight and subcellular location. SRrp37 was identified through a yeast two-hybrid screen during the course of searching for proteins interacting with pNO40, a ribosomal 60S core subunit. SRrp37 exhibited two alternative spliced isoforms generated by differential usage of the translation start site with the longer one, SRrp37, initiating at first exon and the shorter, SRrp37-2, starting from exon 2. Three distinct motifs can be discerned in the SRrp37 protein: (1) a serine,arginine (SR) dipeptide enriched domain, (2) a polyserine stretch, and (3) a potential nucleolar localization signal comprising a long array of basic amino acids. SRrp37's message was translated in tissue-specific patterns with both isoforms expressed at comparable levels in tissues showing expression. Indirect immunofluorescence analysis with an anti-SRrp37 antibody, as well as an experiment using myc-tagged proteins, demonstrated that SRrp37 was localized in nucleoli and nuclear speckles. GST pull-down assay showed that SRrp37 interacted physically with SC35. Using adenovirus E1A and chimeric calcitonin/dhfr constructs as splicing reporter minigenes, we found that SRrp37 modulated alternative 5, and 3, splicing in vivo. Together, SRrp37 may participate directly in splicing regulation or indirectly through interaction with SC35. Studies on this novel splicing regulator may provide new information on the intricate splicing machinery as related to the RNA metabolism involving processing of mRNA and rRNA. J. Cell. Biochem. 108: 304,314, 2009. © 2009 Wiley-Liss, Inc. [source] High glucose levels upregulate upstream stimulatory factor 2 gene transcription in mesangial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Lihua Shi Abstract Previously, we demonstrated that upstream stimulatory factor 2 (USF2) mediates high glucose-induced thrombospondin1 (TSP1) gene expression and TGF-, activity in glomerular mesangial cells and plays a role in diabetic renal complications. In the present studies, we further determined the molecular mechanisms by which high glucose levels regulate USF2 gene expression. In primary rat mesangial cells, we found that glucose treatment time and dose-dependently up-regulated USF2 expression (mRNA and protein). By using cycloheximide to block the de novo protein synthesis, similar rate of USF2 degradation was found under either normal glucose or high glucose conditions. USF2 mRNA stability was not altered by high glucose treatment. Furthermore, high glucose treatment stimulated USF2 gene promoter activity. By using the luciferase-promoter deletion assay, site-directed mutagenesis, and transactivation assay, we identified a glucose-responsive element in the USF2 gene promoter (,1,740 to ,1,620, relative to the transcription start site) and demonstrated that glucose-induced USF2 expression is mediated through a cAMP-response element-binding protein (CREB)-dependent transactivation of the USF2 promoter. Furthermore, siRNA-mediated CREB knock down abolished glucose-induced USF2 expression. Taken together, these data indicate that high glucose levels up-regulate USF2 gene transcription in mesangial cells through CREB-dependent transactivation of the USF2 promoter. J. Cell. Biochem. 103: 1952,1961, 2007. © 2007 Wiley-Liss, Inc. [source] Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response elementJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Arthur J. Cohen Abstract Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half-sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half-sites function as two overlapping RAREs that share the middle half-site. Ablation of the middle half-site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half-sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Characterization of the upstream mouse Cbfa1/Runx2 promoter,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2001Z. S. Xiao Abstract Cbfa1 (or Runx2/AML-3/PEPB2,) is a transcriptional activator of osteoblastic differentiation. To investigate the regulation of Cbfa1 expression, we isolated and characterized a portion of the 5,-flanking region of the Cbfa1 gene containing its "bone-related" or P1 promoter and exon 1. We identified additional coding sequence in exon 1 and splice donor sites that potentially give rise to a novel Cbfa1 isoform containing an 18 amino acid insert. In addition, primer extension mapping identified in the Cbfa1 promoter a minor mRNA start site located ,0.8 kb 5, upstream of the ATG encoding the MASN/p57 isoform and ,0.4 kb upstream of the previously reported start site. A luciferase reporter construct containing 1.4 kb of the mouse Cbfa1 promoter was analyzed in Ros 17/2.8 and MC3T3-E1 osteoblast cell lines that express high levels of Cbfa1 transcripts. The activity of this construct was also examined in non-osteoblastic Cos-7 and NIH3T3 cells that do not express Cbfa1 and mesenchymal-derived cell lines, including CH3T101/2, C2C12, and L929 cells, that express low levels of mature Cbfa1 transcripts. The 1.4 kb 5, flanking sequence of the Cbfa1 gene directed high levels of transcriptional activity in Ros 17/2.8 and MC3T3-E1 osteoblasts compared to non-osteoblasts Cos-7 cells, but this construct also exhibited high levels of expression in C310T1/2, L929, and C2C12 cells as well as NIH3T3 cells. In addition, Cbfa1 mRNA expression, but not the activity of the Cbfa1 promoter, was upregulated in a dose-dependent manner in pluripotent mesenchymal C2C12 by bone morphogenetic protein-2 (BMP-2). These data indicate that Cbfa1 is expressed in osteogenic as well as non-osteogenic cells and that the regulation of Cbfa1 expression is complex, possibly involving both transcriptional and post-transcriptional mechanisms. Additional studies are needed to further characterize important regulatory elements and to identify additional regions of the promoter and/or post-transcriptional events responsible for the cell-type restricted regulation of Cbfa1 expression. J. Cell. Biochem. 82: 647,659, 2001. © 2001 Wiley-Liss, Inc. [source] Pathogenic mutations inactivate parkin by distinct mechanismsJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Iris H. Henn Abstract Loss of parkin function is the major cause of autosomal recessive Parkinson's disease (ARPD). A wide variety of parkin mutations have been identified in patients; however, the pathophysiological mechanisms leading to the inactivation of mutant parkin are poorly understood. In this study we characterized pathogenic C- and N-terminal parkin mutants and found distinct pathways of parkin inactivation. Deletion of the C terminus abrogated the association of parkin with cellular membranes and induced rapid misfolding and aggregation. Four N-terminal missense mutations, located within the ubiquitin-like domain (UBL), decrease the stability of parkin; as a consequence, these mutants are rapidly degraded by the proteasome. Furthermore, we present evidence that a smaller parkin species of 42 kDa, which is present in extracts prepared from human brain and cultured cells, originates from an internal start site and lacks the N-terminal UBL domain. [source] Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Seon-Young Kim Abstract Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5,-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between ,57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position ,52/,39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-, reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level. [source] Nanoparticle formulation enhances the delivery and activity of a vascular endothelial growth factor antisense oligonucleotide in human retinal pigment epithelial cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2003Jithan V. Aukunuru ABSTRACT The objective of this study was to investigate the delivery and activity of a vascular endothelial growth factor (VEGF) antisense oligonucleotide in a human retinal pigment epithelial cell line (ARPE-19) using a biodegradable nanoparticulate delivery system. A 19-mer antisense phosphorothioate oligonucleotide (PS-ODN) complementary to bases 6,24 relative to the translational start site of the VEGF mRNA, a sense PS-ODN and a mismatch PS-ODN were examined for the inhibition of secretion and mRNA expression of VEGF using an enzyme-linked immunosorbent assay and reverse transcription,polymerase chain reaction, respectively. Nanoparticles of the antisense oligonucleotides were formulated using a poly(lactide-co-glycolide) (50:50) copolymer using a double emulsion solvent evaporation method. After preparing nanoparticles, drug loading, encapsulation efficiency and particle size were determined. The cells were exposed to either plain solution of oligonucleotide or nanoparticles of oligonucleotide from Day 3 through Day 6. Alternatively, the cells were incubated with PS-ODNs and lipofectin for 4h on Day 4. In all studies, VEGF secretion and mRNA expression were determined on Day 6. The particle size, drug loading and encapsulation efficiency were 252 nm, 5.5% and 16.5%, respectively. The antisense PS-ODN inhibited VEGF mRNA and protein secretion when delivered using nanoparticles or lipofectin but not in its free form. This was consistent with the ability of nanoparticles and lipofectin to elevate the cellular uptake of the oligonucleotide by 4-fold and 13-fold, respectively. Neither mismatch nor sense oligonucleotides inhibited VEGF secretion. In conclusion, biodegradable nanoparticles enhance cellular delivery of a VEGF antisense oligonucleotide and inhibit VEGF secretion and mRNA expression in a human retinal pigment epithelial cell line. [source] A single-nucleotide polymorphism in the 5,-untranslated region of the hPER2 gene is associated with diurnal preferenceJOURNAL OF SLEEP RESEARCH, Issue 3 2005JAYSHAN D. CARPEN Summary The PERIOD2 (PER2) gene is a key component of the molecular mechanism that generates circadian rhythms in mammals. A missense mutation in the human PER2 gene has previously been linked to advanced sleep phase syndrome (ASPS). We have investigated three other single-nucleotide polymorphisms in the hPER2 gene, one downstream of the transcription start site (C,1228T), one in exon 2 in the 5,-untranslated region (5,-UTR) (C111G), and one missense mutation (G3853A) causing a glycine to glutamine substitution in the predicted protein. Subjects selected from a group of 484 volunteers for extreme morning or evening preference, or intermediate diurnal preference were genotyped with regard to the three polymorphisms (n = 35 for each group). Whereas allele frequencies for the other two polymorphisms did not differ significantly between any of the groups, the 111G allele frequency was significantly higher in subjects with extreme morning preference (0.14) than in subjects with extreme evening preference (0.03) (Fisher's exact test, two-sided P value = 0.031, odds ratio = 5.67). No significant difference in 111G allele frequency was observed between either of these groups and subjects with intermediate diurnal preference. Computer prediction indicated that the C111G polymorphism, which occurs 12 bases upstream from the translation start codon, might alter the secondary structure of the transcript. The PER2 111G allele associates with morning preference and is a potential candidate allele for ASPS. [source] | |||||||||||||||||||||||||