In vivo analysis of the state of the human uPA enhancer following stimulation by TPA

Ibañez–Tallon, Inés; Caretti, Giuseppina; Blasi, Francesco; Crippa, Massimo P.

doi:10.1038/sj.onc.1202644

Cited by 15 publications

(11 citation statements)

References 25 publications

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“…5B). This is in agreement with our previous results showing the presence of a DNase I-hypersensitive site on the uPA MP in PC3 cells (36). We also found that DAF-B is associated with HMGN1, another hallmark of transcriptionally active chromatin.…”

Section: Daf-a -B and -Bx Amplicons Represent Discrete Chromatin Stsupporting

confidence: 82%

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

Ferrai

Munari

Luraghi

et al. 2007

Journal of Biological Chemistry

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We show the interaction between the enhancer and the minimal promoter of urokinase-type plasminogen activator gene during active transcription by coupling micrococcal nuclease digestion of cross-linked, sonicated chromatin, and chromatin immunoprecipitation. This approach allowed the precise identification of the interacting genomic fragments, one of which is resistant to micrococcal nuclease cleavage. The interacting fragments form a single transcriptional control unit, as indicated by their common protein content. Furthermore, we show that the enhancer-MP interaction persists during the early stages of transcription and is lost upon ␣-amanitin treatment, indicating the requirement for active transcription. Our results support a looping model of interaction between the enhancer and the MP of the urokinase-type plasminogen activator gene.Transcription regulation in eukaryotic cells is a multistep process that involves the assembly of multiprotein complexes on gene regulatory regions (1). These regulatory regions contain two types of sequences: enhancers/silencers, which recruit a complex array of transcription factors and chromatin-modifying activities, and core promoter elements to which the general transcriptional machinery, including RNAP II, 2 is recruited. Understanding the molecular mechanism(s) involved in transcriptional regulation over long distances is of fundamental importance, because in most cases the activities of remote control elements are essential in turning on or off specific subsets of genes in a temporally and spatially regulated manner (2). The main models to explain distal enhancer function invoke enhancer-promoter communication, either through proteinprotein interactions resulting in the formation of DNA/chromatin loops (looping model), the free sliding of proteins recruited on the enhancer along the DNA (scanning model), or the establishment of modified chromatin domains between the enhancer and the promoter by facilitator proteins, which generate a progressive chain of higher order complexes along the chromatin fiber (3-9). The looping model, however, is supported by recent results in a number of different experimental systems, showing that the close physical proximity of distant regulatory sequences is required for proper gene expression (10 -15). The interaction between enhancer elements and proximal promoter culminates in the transcription activation event. However it is not yet clarified if the activation of transcription stems from the transient interaction of regulatory elements or through a more stable structure working as a single control unit.The uPA gene codes for a serine protease involved in the degradation of the extracellular matrix and in cell motility (16). The expression of this gene is normally dependent on the presence of inducers, but in cancer cells it is often constitutive. The regulatory region of the uPA gene contains an MP and an enhancer located 2 kb upstream (17). In highly invasive human prostate adenocarcinoma (PC3) cells this gene is constitutively expresse...

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Section: Daf-a -B and -Bx Amplicons Represent Discrete Chromatin Stsupporting

confidence: 82%

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

Ferrai

Munari

Luraghi

et al. 2007

Journal of Biological Chemistry

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“…Interestingly, another hypersensitive site is present approximately 3.5 kb from the start of DNase I-digested and restricted DNA was fractionated on a 1% agarose gel in 0.5 ϫ TBE, transferred to positively charged nylon membrane, and probed as described. 29 (B) In PC3 cells the regulatory region of the uPA gene displays strong hypersensitivity to DNase I in proximity of the enhancer (ϳϪ1950) and the proximal promoter (ϳϪ86). The latter is completely absent in HeLa cells.…”

Section: Resultsmentioning

confidence: 99%

“…29 Nuclear extracts, EMSAs, and Western blots Nuclear extracts were obtained by the method of Dignam et al 30 Aliquots were frozen and kept at Ϫ80°C. EMSAs were performed as described 31 using approximately 10 g nuclear extract proteins/lane.…”

Section: Detection Of Dnase I Hypersensitive Sitesmentioning

confidence: 99%

Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells

Ibañez–Tallon

Ferrai

Longobardi

et al. 2002

Blood

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IntroductionUrokinase-type plasminogen activator (uPA) is a serine protease involved in normal tissue remodeling and also in pathological events such as tumor invasion and metastasis. 1 Several human tumors have been found to overexpress uPA, and inhibition of the expression or of the enzymatic activity of this protease reduces the invasive and metastatic phenotype. 1 A number of physiological, pathological, and chemical stimuli activate transcription of the uPA gene. 2 Their effect is mediated by the uPA enhancer, located approximately 2000 bp upstream of the transcriptional start site. 3 This element contains an Ets-2 site juxtaposed to an octameric AP-1 A site at the 5Ј end and an eptameric AP-1 B site at the 3Ј end. 4 The region between the AP-1 sites is defined as cooperativity mediator (COM) and is necessary for the combined action of the AP-1-binding transcription factors. 5 The COM region is subdivided, in turn, in an upstream COM (uCOM) and a downstream COM (dCOM). By electrophoretic mobility shift assays, it was shown that uCOM can form 4 different complexes 6-8 with individual proteins, such as Oct-1, 9 with heterodimers of the Prep-1 and Pbx proteins 10,11 and with an as-yet-unidentified UEF-1 protein.The dCOM region appears to bind specifically only UEF-1 (M.P. unpublished data, May 2000). Interestingly, although some of the factors binding to the uCOM region are known transcriptional activators (ie, Oct-1 12 and Prep-1/Pbx with HoxB1 11,13 ), they do not display any transactivating activity in the uPA enhancer context. 6 However, both uCOM and dCOM are required for full enhancer activity. 6 It is thus the proteins binding to the AP-1 sites that behave as transactivators in the uPA enhancer. AP-1 family transcription factors activate many genes in response to a large number of stimuli. 14 Since members of the c-Jun family can form homodimers or heterodimers with members of the c-Fos family and with ATF-2, 15 it is evident that the number of transcription factors potentially involved in transcriptional activation is rather large. In the case of the uPA enhancer, Cirillo et al 16 have shown that, besides the transcription factor ATF-2, at least 2 members of the c-Jun family and 2 of the c-Fos family are involved in the response of HepG2 cells to induction by interleukin 1 (IL-1) and tetradecanoyl phorbol acetate (TPA). Furthermore, it was shown that the phosphorylation state of these proteins also is relevant to transcriptional activation. 16 Traditionally, the proximal promoter of the human uPA gene has been considered as spanning approximately 86 bp upstream of the transcription start site. 3 This region contains 5 high-(3 ϫ GGGCGG) and low-(2 ϫ GGGAGG) affinity binding sites for the Sp1 family transcription factors, immediately upstream of the TATA box. This family is composed of 4 members, with a high degree of structural but not functional similarity. 17 Sp1 and Sp4 are transcriptional activators, whereas Sp3 represses Sp1-mediated transcription. [18][19][20][21][22] Sp3 contains a portable repr...

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“…1,[6][7][8][9][10][11][12][13][14][15][16][17][18][21][22][23][24]26,[35][36][37] The intervening sequence (Ϫ1879/Ϫ87) is not well characterized, although it is known to contain a putative negative regulatory element. 38 By comparing a uPA-nonproducing, primary tumor cell line (HeLa) with a constitutively high-level uPA-producing, metastatic cell line (PC3), we discovered that the uPA minimal promoter was active only in the latter.…”

Section: Discussionmentioning

confidence: 99%

MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription

Benasciutti

Pagès

Kenzior

et al. 2004

Blood

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IntroductionIn cancer cells, different pathways may be activated and/or different transcription factors may be expressed, resulting in the specific expression of a repertoire of genes in distinct cell types. A good example is the urokinase-type plasminogen activator (uPA) gene, which is expressed at high basal levels in PC3 cells but is not expressed in HeLa or LNCaP cells. [1][2][3] uPA is a serine protease involved in cell migration in nonpathological (eg, tissue remodeling) and pathological (eg, tumor invasion and metastases formation) events. 4,5 Many human cancers, in fact, overexpress uPA, and reduction of the invasive and metastatic phenotype has been obtained by down-regulating the expression of the gene or by inhibiting the enzymatic activity of this protease. 4,5 The minimal promoter (MP) of the human uPA gene extends approximately 86 bp upstream of the transcription start site 1,6 and contains 5 (high, 3 ϫ GGGCGG; low, 2 ϫ GGGAGG) affinity binding sites for the Sp1-family transcription factors immediately upstream of the TATA box. This cis-acting element binds the transcription factor Sp1 in vivo in PC3 cells, which constitutively express the uPA gene. In this cell line, a uPA minimal promoterdriven reporter construct is 10-fold transcriptionally more active than in HeLa cells. 1 This result correlates with the presence of the phosphorylated form of Sp1 in PC3 cells and with the absence of the phosphorylated transcription factor 1 and the lack of transcription of the endogenous uPA gene in HeLa cells. Thus, the uPA minimal promoter plays a very important role in uPA gene expression, invasion, and metastasis formation in cancer cells.The expression of the uPA gene is inducible in several cells by stimulating the enhancer, located about 2 kb upstream of the transcription start site, 6 which binds transcription factors belonging to different families. Despite its relevance in these cells, the uPA enhancer appears to have no role (or a very modest one) in uPA expression in PC3 cells, where, instead, transcriptional activity is dependent on the binding of the transcription factor Sp1 to the minimal promoter. 1 Phosphorylation of Sp1 seems to be an absolute requirement, because HeLa cells, which express but do not phosphorylate Sp1, are unable to activate the MP.Recently, it has been shown that in a CCL39 (Chinese hamster fibroblast)-derivative cell line, which expresses a chimeric, estrogeninducible Raf:ER chimera, vascular endothelial growth factor (VEGF) expression is under the control of the p42/p44 mitogenactivated protein (MAP) kinase pathway, 28 which targets the transcription factor Sp1 and promotes its phosphorylation at residues Thr453 and Thr739. 29 The phosphorylated form of Sp1, in turn, drives transcription from the VEGF minimal promoter, which contains 2 binding sites for this transcription factor, which bracket a binding site for the transcription factor AP-2. 28 The role of VEGF-MP in the overall expression of VEGF is not known. Interestingly, the constitutive levels of VEGF and uPA mRNA i...

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In vivo analysis of the state of the human uPA enhancer following stimulation by TPA

Cited by 15 publications

References 25 publications

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

A Transcription-dependent Micrococcal Nuclease-resistant Fragment of the Urokinase-type Plasminogen Activator Promoter Interacts with the Enhancer

Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells

MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription

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