1: Biochim Biophys Acta. 2005 Jun 30;1729(2):81-7. 

The 5' region of the human hSUV3 gene encoding mitochondrial DNA and RNA
helicase: promoter characterization and alternative pre-mRNA splicing.

Minczuk M, Lilpop J, Boros J, Stepien PP.

Department of Genetics, University of Warsaw, Pawinskiego 5A, 02-106 Warsaw,
Poland. mminczuk@ibb.waw.pl

The human nuclear hSUV3 gene encodes ATP-dependent RNA and DNA helicase, which
predominantly localizes in the mitochondria. In yeast, the Suv3 helicase is a
component of mitochondrial degradosome, a two-subunit complex, which degrades
aberrant mtRNAs. In contrast to the well-documented physiological role of the
yeast SUV3, the function of its human orthologue remains unknown. In this
report, we have analyzed the hSUV3 5' genomic region. Our data suggest that
hSUV3 is a housekeeping gene. Deletion analysis and in vitro mutagenesis
revealed the presence of an enhancer region and regulatory elements in basal
promoter including: (i) direct 10-bp-long repeats, which share significant
sequence similarity with the consensus for the NF-kappaB/Rel family
transcription factors, (ii) Sp1 general transcription factor binding site, and
(iii) NRF-1 transcription factor binding sites, the latter typical for
nuclear-encoded mitochondrial genes. Furthermore, we show that the 5' region of
the hSUV3 pre-mRNA can be alternatively spliced.

PMID: 15919122 [PubMed - indexed for MEDLINE]


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2: Clin Exp Immunol. 2004 Aug;137(2):329-40. 

Disruption of MAP kinase activation and nuclear factor binding to the IL-12 p40
promoter in HIV-infected myeloid cells.

Chambers KA, Parks RJ, Angel JB.

Molecular Medicine Program, Ottawa Health Research Institute, University of
Ottawa, Ottawa, Ontario, Canada.

Progressive immunodeficiency in HIV infection is paralleled by a decrease in
IL-12 production, a cytokine crucial for cellular immune function. Here we
examine the molecular mechanisms by which HIV infection suppresses IL-12 p40
expression. HIV infection of THP-1 myeloid cells resulted in decreased
LPS-induced nuclear factor binding to the NF-kappaB, AP-1, and Sp1 sites of the
IL-12 p40 promoter. By site-directed mutagenesis we determined that each of
these sites was necessary for transcriptional activation of the IL-12 p40
promoter. Binding of NF-kappaB p50, c-Rel, p65, Sp1, Sp3, c-Fos, and c-Jun
proteins to their cognate nuclear factor binding sites was somewhat impaired by
HV infection, although a role for other as yet unidentified factors cannot be
dismissed. The cellular levels of these transcription factors were unaffected by
HIV infection, with the exception of a decrease in expression of NF-kappaB p65,
consistent with the observed decrease in its binding to the IL-12 p40 promoter
following HIV infection. Analysis of regulation of upstream LPS-induced MAP
kinases demonstrated impaired phosphorylation of JNK and p38 MAPK, and
suppressed phosphorylation and degradation of IkappaBalpha following HIV
infection. These results suggest that alterations in nuclear factor binding to
numerous sites in the IL-12 p40 promoter, together may contribute to the
suppression in IL-12 p40 transcription previously reported. These effects on
nuclear factor binding may be a direct effect of HIV infection on the IL-12 p40
promoter, or may occur indirectly as a consequence of altered MAP kinase
activation.

PMID: 15270850 [PubMed - indexed for MEDLINE]


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3: J Neurosci. 2003 Oct 15;23(28):9403-8. 

Forebrain-specific neuronal inhibition of nuclear factor-kappaB activity leads
to loss of neuroprotection.

Fridmacher V, Kaltschmidt B, Goudeau B, Ndiaye D, Rossi FM, Pfeiffer J,
Kaltschmidt C, Israel A, Memet S.

Unite de Biologie Moleculaire de l'Expression Genique, Centre National de la
Recherche Scientifique 2582, Institut Pasteur, Paris Cedex 15, France.

The transcription factor Rel/nuclear factor (NF)-kappaB is known for its
fundamental role in regulating immune and inflammatory responses. In the brain,
constitutive NF-kappaB activity has been detected exclusively in neurons, and a
large diversity of stimuli have been reported to induce NF-kappaB activity. Yet
the function of this transcription factor in the nervous system remains unclear,
and its role in neuroprotection or neurodegeneration is open to debate. Recently
it was suggested that kappaB-driven gene expression in neurons is controlled by
Sp1-like factors. To clarify such controversy, we have characterized here a
novel mouse model in which the entire NF-kappaB-dependent transcriptional
response is abolished in the forebrain. Calcium-calmodulin-dependent kinase II
alpha promoter-driven tetracycline transactivator was used for regulated
expression of a transdominant negative mutant of inhibitor kappaBalpha
(super-repressor) together with a green fluorescent protein tracer. Inhibition
of expression of a kappaB-dependent lacZ transgene was shown in triple
transgenic mice, which correlated with the loss of kappaB-specific DNA binding.
In transgenic organotypic hippocampal slice cultures, expression of the
super-repressor led to strong cell death after neurotoxic insults. These data
demonstrate for the first time that neuron-restricted ablation of
NF-kappaB-driven gene expression increases neurodegeneration. This might lead to
the path for new treatments of neurodegenerative diseases.

PMID: 14561868 [PubMed - indexed for MEDLINE]


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4: Toxicol Appl Pharmacol. 2003 Mar 15;187(3):147-61. 

Kinetics of lipopolysaccharide-induced transcription factor
activation/inactivation and relation to proinflammatory gene expression in the
murine spleen.

Zhou HR, Islam Z, Pestka JJ.

Department of Food Science and Human Nutrition, Michigan State University, East
Lansing, MI 48824-1224, USA.

Bacterial lipopolysaccharide (LPS) elicits inflammation and endotoxic shock by
inducing proinflammatory cytokine gene expression. The purpose of this study was
to test the hypothesis that differential activation of transcription factor
binding in the spleen correlates with proinflammatory cytokine gene expression
in mice exposed to LPS. When proinflammatory cytokine expression in spleen was
evaluated in mice injected ip with 4 mg/kg LPS over an 8-h period, tumor
necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 mRNAs were
elevated up to 5-, 6-, and 300-fold, respectively, over vehicle controls. Both
TNF- alpha and IL-6 mRNA peaked at 2 h and begin to decline thereafter, whereas
IL-1beta mRNA remained elevated from 2 to 8 h. The capacities of splenic nuclear
proteins to bind to six different consensus transcriptional control motifs
associated with proinflammatory cytokine promoters were also measured over 8 h.
Electrophoretic mobility shift assay (EMSA) revealed that binding activity was
markedly increased at 0.5 to 8 h for activator protein-1 (AP-1) as were CCAAT
enhancer-binding protein (C/EBP) and nuclear factor kappaB (NF-kappaB) at 0.5 to
1.5 h. At 0.5 h, cyclic AMP response element (CRE)-binding protein (CREB) and
binding was slightly elevated, whereas activator protein- 2 (AP-2) and
specificity protein 1 (Sp1) binding were not affected. Antibody supershift EMSA
and Western blot analysis confirmed that increased binding of these factors
correlated with LPS-induced increases in nuclear concentrations of AP-1 (c-Jun,
phosphorylated c-Jun, Jun D, and Jun B), C/EBPbeta, NF-kappaB (p50, p65, and
c-Rel), CREB (CREB-1, CREB-2, and ATF-2), and AP-2alpha proteins. Remarkably,
after 8 h, C/EBP, CREB, AP-2, and Sp1 binding activities were greatly depleted
relative to both naive and corresponding vehicle controls. When mice were
exposed to a second dose of LPS, 8 h after a 4 mg/kg priming dose, TNF-alpha and
IL-6 mRNA responses were markedly impaired, suggesting that the mice were
endotoxin tolerant at this time point. Taken together, the quiescent, active,
and suppressive phases of transcription factor binding observed in this model
were highly consistent with the rapid transient nature of LPS-induced
proinflammatory cytokine expression in vivo as well as tolerance to secondary
LPS exposure.

PMID: 12662898 [PubMed - indexed for MEDLINE]


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5: Apoptosis. 2003 Jan;8(1):11-8. 

Roles of the stress-induced gene IEX-1 in regulation of cell death and
oncogenesis.

Wu MX.

Department of Molecular and Cell Biology, Boston University Goldman School of
Dental Medicine, Boston, MA 02118, USA. mxwu@bu.edu

In response to changes in the external environment cells must initiate a
coordinated program of gene expression for them to adapt. IEX-1 (immediate early
response gene X-1) is precisely regulated by multiple transcription factors
among which p53, NF-kappaB/rel, Sp1 and c-Myc play central roles, to ensure
rapid and transient expression of IEX-1 in cells under a variety of stress
conditions. Overexpression of IEX-1 renders some cells sensitive to apoptosis
and accelerates cell cycle progression, but reduces proliferation of other
cells, whereas disruption of IEX-1 expression is associated with decreases in
both apoptosis and cell cycle progression. In sharp contrast to in vitro
studies, in vivo constitutive expression of IEX-1 prevents activated T cells but
not B cells from apoptosis, as shown using IEX-1-transgenic mice that target
IEX-1 expression specifically to lymphocytes driven by the Emu enhancer. The
animals developed a lupus-like disease and subsequently a high incidence of T
cell lymphomas when they aged, due to insufficient apoptosis of T cells. These
varied effects of IEX-1 on cell death and cell cycle progression in a
cell-context dependent fashion implicate that IEX-1 is involved in more than one
signaling pathway, understanding of which will certainly improve our knowledge
with respect to cancer biology, cell death and cell cycle regulation.

Publication Types:
    Review
    Review, Tutorial

PMID: 12510147 [PubMed - indexed for MEDLINE]


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6: J Immunol. 2002 Sep 15;169(6):3120-30. 

Regulation of IFN regulatory factor 4 expression in human T cell leukemia
virus-I-transformed T cells.

Sharma S, Grandvaux N, Mamane Y, Genin P, Azimi N, Waldmann T, Hiscott J.

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research,
McGill University, Montreal, Canada.

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF
transcription factor family that plays an essential role in the homeostasis and
function of mature lymphocytes. IRF-4 expression is tightly regulated in resting
primary T cells and is transiently induced at the mRNA and protein levels after
activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with
phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is
constitutively upregulated in human T cell leukemia virus type I (HTLV-I)
infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this
study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T
lymphocytes is associated with a leukemic phenotype, and we examine the
mechanisms by which continuous production of IRF-4 is achieved in
HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven
through activation of the NF-kappaB and NF-AT pathways, resulting in the binding
of p50, p65, and c-Rel to the kappaB1 element and p50, c-Rel, and NF-ATp to the
CD28RE element within the -617 to -209 region of the IRF-4 promoter.
Furthermore, mutation of either the kappaB1 or CD28RE sites blocks Tax-mediated
transactivation of the human IRF-4 promoter in T cells. These experiments
constitute the first detailed analysis of human IRF-4 transcriptional regulation
within the context of HTLV-I infection and transformation of CD4(+) T
lymphocytes.

PMID: 12218129 [PubMed - indexed for MEDLINE]


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7: FASEB J. 2001 Mar;15(3):577-9. Epub 2001 Jan 5. 

Molecular and in silico characterization of a promoter module and C/EBP element
that mediate LPS-induced RANTES/CCL5 expression in monocytic cells.

Fessele S, Boehlk S, Mojaat A, Miyamoto NG, Werner T, Nelson EL, Schlondorff D,
Nelson PJ.

Medizinische Poliklinik, Klinikum Innenstadt, Ludwig-Maximilians-University of
Munich, Germany.

The chemokine RANTES/CCL5 is a proinflammatory agent produced by a variety of
tissues in response to specific stimuli. In human monocytes, RANTES/CCL5
transcription is up-regulated rapidly and transiently in response to LPS. We
describe here two regions that help control LPS-driven transcription from the
human RANTES/CCL5 promoter in monocytic cells. These sites were analyzed by
using DNase I footprinting, transient transfection assays, site-directed
mutagenesis, and EMSA. RANTES site E (R(E), -125/-99) constitutively binds C/EBP
proteins in monocytic Mono Mac 6 cells. Mutation of region R(E) led to a
significant (40%-50%) reduction in LPS-induced promoter reporter activity.
Region R(AB) is composed of tandem kB-like elements R(A) and R(B) (-73/-34).
These sites working in concert act as an LPS-responsive promoter module. R(A)
constitutively binds Sp1, and Rel p50/p65 following LPS stimulation. Either
factor can mediate transcriptional effects at R(A). Induced Rel p50/p50 binding
to site R(B) is required for LPS regulation of RANTES/CCL5 transcription. A
series of computer models based on the RANTES/CCL5 promoter were generated to
represent the organization of these functional elements. The models could
identify LPS-regulated promoters in human, other vertebrate, and viral sequences
in various databases.

PMID: 11259372 [PubMed - indexed for MEDLINE]


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8: Mech Dev. 2000 Oct;97(1-2):149-55. 

Dynamic expression and activity of NF-kappaB during post-natal mammary gland
morphogenesis.

Brantley DM, Yull FE, Muraoka RS, Hicks DJ, Cook CM, Kerr LD.

Department of Cell Biology, Vanderbilt University School of Medicine, Nashville,
TN 37232, USA.

The Rel/NF-kappaB family of transcription factors has been implicated in such
diverse cellular processes as proliferation, differentiation, and apoptosis. As
each of these processes occurs during post-natal mammary gland morphogenesis,
the expression and activity of NF-kappaB factors in the murine mammary gland
were examined. Immunohistochemical and immunoblot analyses revealed expression
of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor,
IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and
involution. Electrophoretic mobility shift assay (EMSA) demonstrated that
DNA-binding complexes containing p50 and RelA were abundant during pregnancy and
involution, but not during lactation. Activity of an NF-kappaB-dependent
luciferase reporter in transgenic mice was highest during pregnancy, decreased
to near undetectable levels during lactation, and was elevated during
involution. This highly regulated pattern of activity was consistent with the
modulated expression of p105/p50, RelA, and IkappaBalpha.

PMID: 11025216 [PubMed - indexed for MEDLINE]


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9: J Biol Chem. 2000 Jun 16;275(24):18432-40. 

Glucocorticoids suppress tumor necrosis factor-alpha expression by human
monocytic THP-1 cells by suppressing transactivation through adjacent NF-kappa B
and c-Jun-activating transcription factor-2 binding sites in the promoter.

Steer JH, Kroeger KM, Abraham LJ, Joyce DA.

Department of Pharmacology, University of Western Australia, Nedlands, Western
Australia, Australia 6907.

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis
by activated monocyte/macrophages, contributing to an anti-inflammatory action
in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells,
glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of
transcriptional activity that occurred between 90 min and 3 h after LPS
exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel
transcription factor complexes, which bound specifically to oligonucleotide
sequences from the -106 to -88 base pair (bp) region of the promoter. The
glucocorticoid, dexamethasone, suppressed nuclear binding activity of these
complexes prior to and during the critical phase of TNF-alpha transcription.
Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs
showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88
bp) binding sites each contributed to the LPS-stimulated expression. Mutating
both sites largely prevented dexamethasone from suppressing TNF-alpha
promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and
PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the
PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated
expression but not to glucocorticoid response. Dexamethasone suppressed the
abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no
direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52
bp region. Small contributions to glucocorticoid response were attributable to
promoter sequences outside the -172 to -88 bp region and to sequences in the
TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress
LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through
antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding
sites in the -106 to -88 bp region of the TNF-alpha promoter.

PMID: 10748079 [PubMed - indexed for MEDLINE]


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10: J Biol Chem. 2000 Feb 18;275(7):4719-25. 

Inhibition of the RelA(p65) NF-kappaB subunit by Egr-1.

Chapman NR, Perkins ND.

Department of Biochemistry, Division of Gene Expression and Regulation, MSI/WTB
Complex, Dow Street, University of Dundee, Dundee, DD1 5EH Scotland, United
Kingdom.

Induction of transcription from the human immunodeficiency virus 1 long terminal
repeat by the RelA (p65) NF-kappaB subunit has been shown to be dependent upon
an interaction with the zinc finger DNA-binding domain of Sp1. It was unknown,
however, whether NF-kappaB could also interact with other zinc finger-containing
transcription factors. In this study we demonstrate that the early growth
response transcription factor Egr-1, whose DNA-binding domain shares a high
degree of homology with that of Sp1, can also interact with RelA in vitro and
regulate NF-kappaB transcriptional activity in vivo. Similar to the interaction
with Sp1, the Rel homology domain of RelA interacts with the zinc finger domain
of Egr-1. Surprisingly, and in contrast to Sp1, Egr-1 specifically represses
RelA transcriptional activity through its zinc finger domain. Moreover, the
interaction between RelA and the Egr-1 zinc fingers is mutually exclusive with
DNA binding suggesting a model in which Egr-1 directly sequesters NF-kappaB from
its target promoters. Because Egr-1 is induced by many of the same stimuli that
activate NF-kappaB, this novel transcriptional regulatory mechanism has many
implications for the involvement of both factors in cellular processes such as
apoptosis and the response to stress and infection.

PMID: 10671503 [PubMed - indexed for MEDLINE]


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11: J Immunol. 2000 Feb 15;164(4):1940-51. 

A prominent role for Sp1 during lipopolysaccharide-mediated induction of the
IL-10 promoter in macrophages.

Brightbill HD, Plevy SE, Modlin RL, Smale ST.

Department of Microbiology, Division of Dermatology, University of California,
Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2
cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine
synthesis and the development of Th2 responses. In macrophages, proinflammatory
cytokines appear to be induced by similar mechanisms, but the IL-10 induction
mechanisms have not been examined. We have analyzed the murine IL-10 promoter in
the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis
revealed only one element upstream of the core promoter that was essential for
promoter induction. A refined mutant analysis localized this element to
nucleotides -89 to -78, and gel shift experiments revealed that it represents a
nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported
by the high affinity of the interaction, the close correlation between the
nucleotides required for Sp1 binding and promoter function, and the ability of
an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence.
Evidence that Sp1 may be a target of signaling pathways involved in IL-10
induction was provided by the exclusive requirement for the Sp1 binding site, by
the ability of the Sp1 site to confer induction to a heterologous promoter, and
by the delineation of an Sp1 domain that can mediate induction. No relevant
contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding
sites, which regulate most proinflammatory cytokine promoters, was observed.
Together, these results demonstrate that IL-10 gene regulation is distinct from
the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a
central mediator of IL-10 induction.

PMID: 10657644 [PubMed - indexed for MEDLINE]


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12: DNA Cell Biol. 1999 Oct;18(10):751-61. 

NF-kappaB inhibits expression of the alpha1(I) collagen gene.

Rippe RA, Schrum LW, Stefanovic B, Solis-Herruzo JA, Brenner DA.

Department of Medicine, The University of North Carolina, Chapel Hill
27955-7038, USA. rarippe@med.unc.edu

Fibrosis results from an increase in the synthesis and deposition of type I
collagen. Fibrosis is frequently associated with inflammation, which is
accompanied by increased levels of tumor necrosis factor-alpha (TNFalpha) and
activation of the transcription factor NF-kappaB. However, several agents known
to activate NF-kappaB, such as phorbol 12-myristate 13-acetate (PMA) and
TNFalpha, result in decreased expression of type I collagen. Therefore, we
directly examined the effects of NF-kappaB on alpha1(I) collagen gene expression
in two collagen-producing cells, NIH 3T3 fibroblasts and hepatic stellate cells
(HSCs). Transient transfections of NIH 3T3 cells or HSCs using NF-kappaB p50,
p65, and c-Rel expression plasmids with collagen reporter gene plasmids
demonstrated a strong inhibitory effect on transcription of the collagen gene
promoter. Dose-response curves showed that p65 was a stronger inhibitor of
collagen gene expression than was NF-kappaB p50 or c-Rel (maximum inhibition
90%). Transient transfections with reporter gene plasmids containing one or two
Spl binding sites demonstrated similar inhibitory effects of NF-kappaB p65 on
the activity of these reporter genes, suggesting that the inhibitory effects of
NF-kappaB p65 are mediated through the critical Spl binding sites in the
alpha1(I) collagen gene promoter. Cotransfection experiments using either a
super-repressor I[ke]B or Spl partially blocked the inhibitory effects of p65 on
collagen reporter gene activity. Coimmunoprecipitation experiments demonstrated
that NF-kappaB and Spl do interact in vivo. Nuclear run-on assays showed that
NF-kappaB p65 inhibited transcription of the endogenous alpha1(I) collagen gene.
Together, these results demonstrate that NF-kappaB decreases transcription of
the alpha1(I) collagen gene.

PMID: 10541434 [PubMed - indexed for MEDLINE]


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13: Mol Cell Biol. 1999 Sep;19(9):6140-53. 

Identification by in vivo genomic footprinting of a transcriptional switch
containing NF-kappaB and Sp1 that regulates the IkappaBalpha promoter.

Algarte M, Kwon H, Genin P, Hiscott J.

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research,
and Departments of Microbiology & Immunology, Medicine, and Oncology, McGill
University, Montreal, Canada H3T 1E2.

In unstimulated cells, NF-kappaB transcription factors are retained in the
cytoplasm by inhibitory IkappaB proteins. Upon stimulation by multiple inducers
including cytokines or viruses, IkappaBalpha is rapidly phosphorylated and
degraded, resulting in the release of NF-kappaB and the subsequent increase in
NF-kappaB-regulated gene expression. IkappaBalpha gene expression is also
regulated by an NF-kappaB autoregulatory mechanism, via NF-kappaB binding sites
in the IkappaBalpha promoter. In previous studies, tetracycline-inducible
expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha)
progressively decreased endogenous IkappaBalpha protein levels. In the present
study, we demonstrate that expression of TD-IkappaBalpha blocked phorbol
myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced
IkappaBalpha gene transcription and abolished NF-kappaB DNA binding activity,
due to the continued cytoplasmic sequestration of RelA(p65) by TD-IkappaBalpha.
In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding
not only to the -63 to -53 kappaB1 site but also to the adjacent -44 to -36 Sp1
site of the IkappaBalpha promoter. In vivo protection of both sites was
inhibited by tetracycline-inducible TD-IkappaBalpha expression. Prolonged
NF-kappaB binding and a temporal switch in the composition of NF-kappaB
complexes bound to the -63 to -53 kappaB1 site of the IkappaBalpha promoter were
also observed; with time after induction, decreased levels of transcriptionally
active p50-p65 and increased p50-c-Rel heterodimers were detected at the kappaB1
site. Mutation of either the kappaB1 site or the Sp1 site abolished
transcription factor binding to the respective sites and the inducibility of the
IkappaBalpha promoter in transient transfection studies. These observations
provide the first in vivo characterization of a promoter proximal
transcriptional switch involving NF-kappaB and Sp1 that is essential for
autoregulation of the IkappaBalpha promoter.

PMID: 10454561 [PubMed - indexed for MEDLINE]


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14: J Inflamm. 1998;48(2):67-83. 

Involvement of Ets, rel and Sp1-like proteins in lipopolysaccharide-mediated
activation of the HIV-1 LTR in macrophages.

Sweet MJ, Stacey KJ, Ross IL, Ostrowski MC, Hume DA.

Department of Microbiology, University of Queensland, Brisbane, Australia.

The HIV-1 promoter was used as a model to identify transcription factors
involved in LPS-dependent transcription in RAW 264 murine macrophages.
Expression plasmids for Ets-2 and PU.1 trans-activated the HIV-1 LTR and
recombinant PU.1 and an Ets-2 DNA binding domain/GST fusion protein bound to the
5' kappa B site of the HIV-1 enhancer. Ets-2 mRNA was LPS-inducible in RAW 264
cells and LPS stimulated phosphorylation of threonine 72 residue within the
Ets-2 pointed domain. Induction of Ets-2 and other LPS-responsive transcription
factors was also observed upon addition of plasmid DNA, which complicates
interpretation of transient transfections. The proximal promoter region,
containing two Sp1 sites, was also LPS-responsive. We propose that the kappa B
elements and the tandem Sp1 sites act as LPS response elements and that kappa
B-mediated LPS action involves Ets and rel factors.

PMID: 9656143 [PubMed - indexed for MEDLINE]


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15: Mol Cell Biol. 1998 Jun;18(6):3234-44. 

Involvement of TFIID and USA components in transcriptional activation of the
human immunodeficiency virus promoter by NF-kappaB and Sp1.

Guermah M, Malik S, Roeder RG.

Laboratory of Biochemistry and Molecular Biology, The Rockefeller University,
New York, New York 10021, USA.

The purified Rel/NF-kappaB (p50/p65) complex and Sp1 markedly activate
transcription from the human immunodeficiency virus type 1 (HIV-1) promoter in a
highly purified HeLa reconstituted transcription system. Transcriptional
activation by NF-kappaB and Sp1 requires both TFIID and the USA fraction. The
USA-derived coactivators PC2 and PC4 fully reconstitute the USA coactivator
activity, both by repressing the basal level of transcription and by
potentiating activator function to yield large increases in the levels of
transcription induction. Under limiting concentrations, PC2 and PC4 also show
synergistic effects. The C-terminal portion (amino acids 416 to 550) of the p65
subunit of NF-kappaB is a potent activator when assayed as a Gal fusion in the
reconstituted transcription system and interacts both with TATA-binding protein
(TBP) and with several human TBP-associated factors (TAFs) that include
TAFII250. The p65 activation domain mediates transcription activation in the
presence of partially reconstituted TFIID species that include a minimal complex
containing only TBP and TAFII250. These studies also show that, like USA
components, TAFs can serve both to repress TBP-mediated transcription and,
following activator interactions, to reverse the repression and effect a net
increase in activity. Taken together, these data underscore the importance of
both TAFs and specific USA-derived coactivators for optimal activation of the
HIV-1 promoter, as well as certain parallels in their overall mechanisms of
action.

PMID: 9584164 [PubMed - indexed for MEDLINE]


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16: Mol Cell Biol. 1998 Mar;18(3):1266-74. 

Functional interference of Sp1 and NF-kappaB through the same DNA binding site.

Hirano F, Tanaka H, Hirano Y, Hiramoto M, Handa H, Makino I, Scheidereit C.

Max Delbruck Center for Molecular Medicine MDC, Berlin, Germany.

Gene activation by NF-kappaB/Rel transcription factors is modulated by
synergistic or antagonistic interactions with other promoter-bound transcription
factors. For example, Sp1 sites are often found in NF-kappaB-regulated genes,
and Sp1 can activate certain promoters in synergism with NF-kappaB through
nonoverlapping binding sites. Here we report that Sp1 acts directly through a
subset of NF-kappaB binding sites. The DNA binding affinity of Sp1 to these
NF-kappaB sites, as determined by their relative dissociation constants and
their relative efficiencies as competitor DNAs or as binding site probes, is in
the order of that for a consensus GC box Sp1 site. In contrast, NF-kappaB does
not bind to a GC box Sp1 site. Sp1 can activate transcription through
immunoglobulin kappa-chain enhancer or P-selectin promoter NF-kappaB sites. p50
homodimers replace Sp1 from the P-selectin promoter by binding site competition
and thereby either inhibit basal Sp1-driven expression or, in concert with
Bcl-3, stimulate expression. The interaction of Sp1 with NF-kappaB sites thus
provides a means to keep an elevated basal expression of NF-kappaB-dependent
genes in the absence of activated nuclear NF-kappaB/Rel.

PMID: 9488441 [PubMed - indexed for MEDLINE]


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17: J Biol Chem. 1997 Oct 31;272(44):27957-65. 

Identification of an upstream enhancer within a functional promoter of the human
leukemia inhibitory factor receptor gene and its alternative promoter usage.

Wang Z, Melmed S.

Cedars Sinai Research Institute-UCLA School of Medicine, Los Angeles, California
90048, USA.

Knockout of the leukemia inhibitory factor receptor (LIFR) gene results in
disrupted placental architecture, imbalanced bone development, and losses of
functional neurons. We here report the identification of an enhancer in a
functional human LIFR gene promoter and alternative promoter usage by this gene.
A single transcription start site was identified in placental JEG-3 cells and a
genomic clone containing 4876-nucleotide upstream sequences was found to have
promoter activity in JEG-3 cells. However, in osteogenic sarcoma U-2 OS cell,
Northern blot using a probe of the first exon detected in JEG-3 cells failed to
detect LIFR transcripts. 5'-Rapid amplification of cDNA ends (RACE) revealed an
alternative first exon and a 0.6-kilobase pair (kb) 5'-flanking region possessed
promoter activity in U-2 OS cells. For the 4.8-kb promoter active in placental
cells, a minimal promoter was localized within -162 nucleotides. Three regions
increased and one inhibited promoter activity. Subcloning of an activation
region (-4876 to -3453 nucleotides) into SV40 promoter either upstream or
downstream in either orientation to the luciferase reporter resulted in
10-35-fold luciferase induction, demonstrating the characteristics of an
enhancer. Transfections into nine cell lines of different tissue origin
indicated that the cloned promoter and enhancer in the 4.8-kb fragment was
placental tissue-specific. A 226-base pair fragment (-4625 to -4400 nucleotides)
was further localized as the minimal enhancer region, in which deletion of
either element A (-4625 to -4581 nucleotides) or element B (-4418 to -4400
nucleotides) resulted in the loss of enhancer activity. Electrophoretic mobility
shift assay confirmed that these two elements bind to specific nuclear proteins
individually. In the middle region between element A and B, disruption of
enhancer integrity also led to a loss of enhancer activity, although two SP1 and
three NF-kappaB/c-Rel binding sites did not contribute to enhancer function.
These results demonstrate a complex regulation of the human LIFR gene, including
alternative promoter usage and tissue-specific elements at the transcription
level.

PMID: 9346946 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


18: J Virol. 1997 Jul;71(7):5692-5. 

Human cytomegalovirus induces interleukin-8 production by a human monocytic cell
line, THP-1, through acting concurrently on AP-1- and NF-kappaB-binding sites of
the interleukin-8 gene.

Murayama T, Ohara Y, Obuchi M, Khabar KS, Higashi H, Mukaida N, Matsushima K.

Department of Microbiology, Kanazawa Medical University, Uchinada, Ishikawa,
Japan.

Cytomegalovirus (CMV) infection induced interleukin-8 (IL-8) gene transcription
in a human monocytic cell line, THP-1 cells, leading to IL-8 secretion. The
functional analysis of the IL-8 gene revealed that both AP-1- and NF-kappaB
factor-binding elements were involved in conferring the responsiveness to CMV.
Moreover, electrophoretic mobility shift assays demonstrated that CMV induced
the formation of NF-kappaB and AP-1 complexes. These results suggest that CMV
activates these transcriptional factors, resulting in IL-8 gene expression.

PMID: 9188651 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


19: J Clin Invest. 1997 Jun 15;99(12):3000-8. 

Deoxyribonucleic acid triplex formation inhibits granulocyte macrophage
colony-stimulating factor gene expression and suppresses growth in juvenile
myelomonocytic leukemic cells.

Kochetkova M, Iversen PO, Lopez AF, Shannon MF.

Division of Human Immunology, Hanson Centre for Cancer Research, Institute of
Medical and Veterinary Science, Adelaide, 5000 South Australia, Australia.

Juvenile myelomonocytic leukemia (JMML) is a severe childhood malignancy. The
autocrine production of GMCSF is believed to be responsible for the spontaneous
proliferation of JMML cells. A nuclear factor-kappaB (NF-kappaB)/Rel binding
site within the GM-CSF gene promoter, termed the kappaB element, plays an
important role in controlling transcription from the GM-CSF gene. We
investigated the effect of an oligonucleotide GM3, directed to form a DNA triple
helix across this kappaB element, on growth and GM-CSF production by JMML cells.
Treatment of these cells, either unstimulated or induced by TNFalpha, with GM3
led to a significant and specific inhibition of both GM-CSF production and
spontaneous colony formation. This constitutes the first report linking specific
triplex-mediated inhibition of gene transcription with a functional outcome;
i.e., cell growth. We observed the constitutive presence of NF-kappaB/Rel
proteins in the nucleus of JMML cells. The constitutive and TNFalpha-induced
NF-kappaB/Rel complexes were identical and were composed mainly of p50 and c-Rel
proteins. Treatment of the cells with a neutralizing anti-TNFalpha monoclonal
antibody completely abrogated constitutive nuclear expression of NF-kappaB/Rel
proteins. These results indicate that the aberrant, constitutive GM-CSF gene
activation in JMML is maintained by TNFalpha-mediated activation of
NF-kappaB/Rel proteins. Our findings identify the molecular basis for the
autocrine TNFalpha activation of the GM-CSF gene in JMML and suggest potential
novel and specific approaches for the treatment of this aggressive childhood
leukemia.

PMID: 9185524 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


20: J Biol Chem. 1997 May 9;272(19):12642-9. 

SWI/SNF stimulates the formation of disparate activator-nucleosome complexes but
is partially redundant with cooperative binding.

Utley RT, Cote J, Owen-Hughes T, Workman JL.

Department of Biochemistry and Molecular Biology and The Center for Gene
Regulation, The Pennsylvania State University, University Park, Pennsylvania
16802-4500, USA.

To investigate the potential mechanisms by which the SWI/SNF complex
differentially regulates different genes we have tested whether transcription
factors with diverse DNA binding domains were able to exploit nucleosome
disruption by SWI/SNF. In addition to GAL4-VP16, the SWI/SNF complex stimulated
nucleosome binding by the Zn2+ fingers of Sp1, the basic helix-loop-helix domain
of USF, and the rel domain of NF-kappaB. In each case SWI/SNF action resulted in
the formation of a stable factor-nucleosome complex that persisted after
detachment of SWI/SNF from the nucleosome. Thus, stimulation of factor binding
by SWI/SNF appears to be universal. The degree of SWI/SNF stimulation of
nucleosome binding by a factor appears to be inversely related to the extent
that binding is inhibited by the histone octamer. Cooperative binding of 5
GAL4-VP16 dimers to a 5-site nucleosome enhanced GAL4 binding relative to a
single-site nucleosome, but this also reduced the degree of stimulation by
SWI/SNF. The SWI/SNF complex increased the affinity of 5 GAL4-VP16 dimers for
nucleosomes equal to that of DNA but no further. Similarly, multimerized
NF-kappaB sites enhanced nucleosome binding by NF-kappaB and reduced the
stimulatory effect of SWI/SNF. Thus, cooperative binding of factors to
nucleosomes is partially redundant with the function of the SWI/SNF complex.

PMID: 9139720 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


21: J Biol Chem. 1997 Mar 28;272(13):8172-8. 

Characterization of the promoter for the human long pentraxin PTX3. Role of
NF-kappaB in tumor necrosis factor-alpha and interleukin-1beta regulation.

Basile A, Sica A, d'Aniello E, Breviario F, Garrido G, Castellano M, Mantovani
A, Introna M.

Istituto di Ricerche Farmacologiche "Mario Negri," via Eritrea, 62, 20157 Milan,
Italy.

The "long pentraxins" are an emerging family of genes that have conserved in
their carboxy-terminal halves a pentraxin domain homologous to the prototypical
acute phase protein pentraxins (C-reactive protein and serum amyloid P
component) and acquired novel amino-terminal domains. In this report, a genomic
fragment of 1371 nucleotides from the human "long pentraxin" gene PTX3 is
characterized as a promoter on tumor necrosis factor-alpha (TNFalpha) and
interleukin (IL)-1beta exposure in transfected 8387 human fibroblasts by
chloramphenicol acetyltransferase and RNase protection assays. In the same
cells, the PTX3 promoter does not respond to IL-6 stimulation. Furthermore,
IL-1beta and TNFalpha responsiveness is not seen in the Hep 3B hepatoma cell
line. The minimal promoter contains one NF-kappaB element which is shown to be
necessary for induction and able to bind p50 homodimers and p65 heterodimers but
not c-Rel. Mutants in this site lose the ability to bind NF-kappaB proteins and
to respond to TNFalpha and IL-1beta in functional assays. Sp1- and AP-1 binding
sites lying in proximity to the NF-kappaB site do not seem to play a major role
for cytokine responsiveness. Finally, cotransfection experiments with expression
vectors validate that the natural promoter contains a functional NF-kappaB site.

PMID: 9079634 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


22: Arterioscler Thromb Vasc Biol. 1997 Feb;17(2):365-74. 

Regulation of the tissue factor gene in human monocytic cells. Role of AP-1,
NF-kappa B/Rel, and Sp1 proteins in uninduced and lipopolysaccharide-induced
expression.

Oeth P, Parry GC, Mackman N.

Department of Immunology, Scripps Research Institute, La Jolla, Calif. 92037,
USA.

Tissue factor (TF) expression by peripheral blood monocytes during sepsis
initiates intravascular thrombosis. Bacterial lipopolysaccharide (LPS) rapidly
induces TF gene transcription in monocytes. The human TF promoter contains
binding sites for the transcription factors AP-1, c-Rel/p65, Egr-1, and Sp1.
NF-kappa B/Rel proteins have been shown to physically interact with both AP-1
and Sp1 proteins. In this study, we investigated the role of these transcription
factors in uninduced and LPS-induced TF gene expression in human monocytic THP-1
cells. Deletional analysis indicated that five Sp1 sites mediated basal
expression in uninduced cells. The two AP-1 sites bound c-Fos/c-Jun heterodimers
in both unstimulated and LPS-stimulated cells. Maximal LPS induction of the TF
promoter required the two AP-1 sites and the kappa B site within the LPS
response element. Disruption of the conserved spacing between the proximal AP-1
site and the kappa B site abolished LPS induction. Replacement of the two AP-1
sites with intrinsically bent DNA partially restored LPS induction, suggesting
an additional structural role for the AP-1 sites. Synergistic transactivation of
the LPS response element in Drosophila Schneider cells by coexpression of c-Fos,
c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of
c-Jun and p65. These data indicated that c-Fos/c-Jun, c-Rel/p65, and Sp1
regulate TF gene expression in human monocytic cells.

PMID: 9081693 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


23: Brain Res. 1996 Jun 3;723(1-2):141-7. 

Cloning the 5' flanking region of neuron-specific Hel-N1: evidence for positive
regulatory elements governing cell-specific transcription.

King PH.

Department of Neurology, University of Alabama, Birmingham 35294-0007, USA.
P_king@email.neuro.uab.edu

A 5.4 kilobase-pair segment of DNA flanking the 5' end of Hel-N1 was isolated
and characterized. Primer extension studies with normal human brain and
neuroblastoma cells revealed a major and minor transcription-initiation site.
Sequence analysis of the initial 536 bp upstream to the major start site
revealed a core promoter (-1 to -181) which contained two CCAAT boxes, a
weakly-conserved TATA box, and an SP1 site. This region was also moderately
GC-rich (62%). Using a transient luciferase-reporter-gene assay, the core
promoter was found to be essential for basal transcription both in neural (PC12)
and non-neural (HeLa and glial) cell types. Two positive regulatory elements,
however, were identified in the initial 536 bp (-1 to -181 and -182 to -350)
which produced a five- to six-fold increase in transcriptional activity in PC12
cells vs. HeLa or glial cells. These elements, therefore, were sufficient to
confer cell-specific enhanced transcription and likely contribute to the
neuronal specificity of Hel-N1 mRNA expression.

PMID: 8813391 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


24: Gene. 1996 May 8;170(2):271-6. 

Cloning and transcription factor-binding sites of the human c-rel proto-oncogene
promoter.

Viswanathan M, Yu M, Mendoza L, Yunis JJ.

Department of Pathology, Thomas Jefferson University, Philadelphia, PA
19107-6799, USA.

We report here the cloning, sequencing, functional analysis and DNase I
footprinting of the human c-rel promoter region. The results revealed an 824-bp
BsaAI-StuI minimal promoter region with a large number of NF-kappa B, Ap2 and
Sp1-binding sites, some of them variants of known consensus sequences. This is
the first promoter in the Rel/NF-kappa B/I kappa B family to be subjected to a
detailed footprinting analysis for the binding of transcription activator
proteins. Our finding of 14 Ap2-binding sites may indicate why the human c-rel
promoter, unlike the chicken c-rel promoter, has a strong function and is highly
responsive to phorbol esters. The presence of five NF-kappa B and six
Sp1-binding sites in turn adds to growing evidence that, in mammals, the
promoter of the Rel/NF-kappa B/I kappa B family may utilize multiple NF-kappa B-
and Sp1-binding sites for their interactive regulation. Furthermore, there are
putative binding sites for the PU.1 and Oct 1/2 transcription activator
proteins, also present in the mouse c-rel promoter, which may help explain the
preferential transcription of the c-rel gene in B- and T-lymphoid cells.

PMID: 8666258 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


25: Anticancer Res. 1996 Jan-Feb;16(1):61-9. 

Transcription factor decoy approach to decipher the role of NF-kappa B in
oncogenesis.

Sharma HW, Perez JR, Higgins-Sochaski K, Hsiao R, Narayanan R.

Division of Oncology, Roche Research Center, Hoffman-La Roche Inc., Nutley, NJ
07110, USA.

Antisense inhibition of the RelA subunit of NF-kappa B transcription factor (but
not the NFKB1 subunit) causes pronounced inhibition of tumor cell growth in
vitro and in vivo. Inhibition of either subunit, however, results in inhibition
of the heterodimeric NF-kappa B complex in antisense-treated cells. Either of
the subunits of NF-kappa B can form homo- or heterodimers with other members of
the Rel oncogene family. In an effort to decipher the role of homo- vs
heterodimeric NNF-kappa B in regulating tumor cell growth, we have used a decoy
approach to trap these complexes in vivo. Using double-stranded
phosphorothioates as a direct in vivo competitor for homo- vs heterodimeric
NF-kappa B, we demonstrate that decoys more specific to RelA inhibit growth
tumor cell growth in vitro. We demonstrate that RelA, either as a homodimer or a
heterodimer with some other members of the Rel family and not the classical
NF-kappa B (RelA/NFKB1), is involved in the differential growth control of tumor
cells. Our results indicate that such transcription factor decoys can be a
non-antisense tool to study the function of DNA-binding transcription factors.

PMID: 8615671 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


26: Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10944-8. 

Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human
immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.

Granelli-Piperno A, Pope M, Inaba K, Steinman RM.

Laboratory of Cellular Physiology and Immunology, Rockefeller University, New
York, NY 10021, USA.

Productive infection of T cells with human immunodeficiency virus 1 (HIV-1)
typically requires that the T cells be stimulated with antigens or mitogens.
This requirement has been attributed to the activation of the transcription
factor NF-kappa B, which synergizes with the constitutive transcription factor
Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous
replication of HIV-1 takes place in nonactivated memory T cells after syncytium
formation with dendritic cells (DCs). These syncytia lack activated cells as
determined by an absence of staining for Ki-67 cell cycle antigen. The
expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in
isolated T cells and DCs from humans and mice. We have used immunolabeling,
Western blot analysis, and electrophoretic mobility shift and supershift assays.
T cells lack active NF-kappa B but express Sp1 as expected. DCs express high
levels of all known NF-kappa B and Rel proteins, with activity residing
primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain
the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B
and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1.
Therefore, HIV-1-induced cell fusion brings together factors that upregulate
virus transcription. Since DCs and memory T cells frequently traffic together in
situ, these unusual heterologous syncytia could develop in infected individuals
and lead to chronic HIV-1 replication without ostensible immune stimulation.

PMID: 7479915 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


27: J Biol Chem. 1995 Oct 20;270(42):24995-5000. 

Erratum in:
    J Biol Chem 1995 Dec 15;270(50):30235.

Activation of transcription factor NF-kappa B is suppressed by curcumin
(diferuloylmethane) [corrected]

Singh S, Aggarwal BB.

Department of Molecular Oncology, University of Texas M.D. Anderson Cancer
Center, Houston 77030, USA.

When activated, NF-kappa B, a ubiquitous transcription factor, binds DNA as a
heterodimeric complex composed of members of the Rel/NF-kappa B family of
polypeptides. Because of its intimate involvement in host defense against
disease, this transcription factor is an important target for therapeutic
intervention. In the present report we demonstrate that curcumin
(diferuloylmethane), a known anti-inflammatory and anticarcinogenic agent, is a
potent inhibitor of NF-kappa B activation. Treatment of human myeloid ML-1a
cells with tumor necrosis factor (TNF) rapidly activated NF-kappa B, which
consists of p50 and p65 subunits, and this activation was inhibited by curcumin.
AP-1 binding factors were also found to be down-modulated by curcumin, whereas
the Sp1 binding factor was unaffected. Besides TNF, curcumin also blocked
phorbol ester- and hydrogen peroxide-mediated activation of NF-kappa B. The
TNF-dependent phosphorylation and degradation of I kappa B alpha was not
observed in curcumin-treated cells; the translocation of p65 subunit to the
nucleus was inhibited at the same time. The mechanism of action of curcumin was
found to be different from that of protein tyrosine phosphatase inhibitors. Our
results indicate that curcumin inhibits NF-kappa B activation pathway at a step
before I kappa B alpha phosphorylation but after the convergence of various
stimuli.

PMID: 7559628 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


28: Anticancer Res. 1995 Sep-Oct;15(5B):1857-67. 

A DNA motif present in alpha V integrin promoter exhibits dual binding
preference to distinct transcription factors.

Sharma HW, Higgins-Sochaski K, Perez JR, Narayanan R.

Division of Oncology, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ
07110, USA.

Antisense inhibition of the RelA subunit but not the NFKB1 subunit of NK-kappa B
transcription factor results in a block of cellular adhesion and inhibition of
tumor cell growth in vitro and in vivo. Studies aimed at dissecting the
molecular mechanism of antisense relA action led to our identification of a
kappa B-like motif present in aV integrin promoter. The alpha V/kappa B motif is
closely related to RelA/c-Rel-binding sequences, such as 65-2 and TF-1. However,
unlike these two kappa Blike motifs, the alpha V/kappa B motif detected a
nuclear Sp1 activity distinct from kappa B activity, which was subsequently
confirmed to be derived from Sp1. In comparison to the conventional GC
box-containing Sp1 motif, the alpha V/kappa B motif also binds in vitro to c-Rel
and RelA but not to NFKB1. Antisense inhibition of RelA inhibited the alpha
V/kappa B activity. Direct in vivo competition of alpha V/kappa B-binding
activity by a decoy approach also resulted in inhibition of alpha V/kappa B
activity in intact cells. A variant of the alpha V/kappa B motif was found to
retain the dual ability to detect Sp1 and the NF-kappa B complex in the nuclear
and cytoplasmic extracts. Such dual interacting ability of a DNA motif offers
yet another way of gene regulation in vivo and hence can affect cellular growth.
Our results identify alpha V integrin as one of the molecular targets for
relA/NF-kappa B and may explain growth inhibition by antisense relA.

PMID: 8572570 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


29: FASEB J. 1995 Jul;9(10):883-9. 

Regulation of the tissue factor gene.

Mackman N.

Department of Immunology, Scripps Research Institute, La Jolla, California
92037, USA.

The tissue factor (TF) gene is expressed in a cell type-specific manner in vivo.
It is constitutively expressed by several extravascular cell types and inducibly
expressed within the vasculature by monocytes and endothelial cells. TF
expression initiates thrombotic episodes associated with various diseases,
including atherosclerosis, septic shock, and cancer. Regulatory elements within
the human TF promoter have been identified by functional analysis of TF
promoter-luciferase gene plasmids transiently transfected into various cell
types. Transcription factors that control expression of the TF gene were
identified using gel shift mobility assays. Induction of the TF gene in human
monocytic cells and endothelial cells exposed to bacterial lipopolysaccharide or
cytokines is mediated by a distal enhancer (-227 to -172 bp) containing two AP-1
sites and a kappa B site. Functional interactions between Fos-Jun heterodimers
and c-Rel-p65 heterodimers are required for transcriptional activation of the TF
gene. In contrast, serum and phorbol ester induction of the TF gene in human
epithelial cells is controlled by a proximal enhancer (-111 to +14 bp)
containing three overlapping Egr-1/Sp1 binding sites. Sp1 is constitutively
expressed whereas Egr-1 expression is induced by serum or phorbol ester
stimulation. Sp1 also mediates basal promoter activity. Thus, TF gene expression
is complex and is regulated by a number of transcription factors that bind to
distinct regions of the TF promoter.

Publication Types:
    Review
    Review, Tutorial

PMID: 7615158 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


30: J Biol Chem. 1995 Feb 24;270(8):3849-57. 

Regulation of the tissue factor promoter in endothelial cells. Binding of NF
kappa B-, AP-1-, and Sp1-like transcription factors.

Moll T, Czyz M, Holzmuller H, Hofer-Warbinek R, Wagner E, Winkler H, Bach FH,
Hofer E.

Department of Transplantation Immunology, Vienna International Research
Cooperation Center, Vienna, Austria.

Tissue factor is up-regulated on endothelial cells and monocytes in response to
cytokines and endotoxin and is the main trigger of the extrinsic pathway of the
coagulation cascade. We have isolated the porcine tissue factor gene and studied
the regulation of the promoter, which has not been investigated previously in
endothelial cells. Comparison of the promoter sequences with the respective
human and murine genes reveals short stretches of homology, which encompass
potential binding sites for AP-1, NF kappa B, and Sp1 transcription factors.
Using DNase I footprinting, we detect binding of nuclear factors to these
promoter elements. Transfection experiments demonstrate that a 300-base pair
fragment containing the conserved elements can mediate induced transcription and
that the NF kappa B-like element is essential. In accordance, electrophoretic
mobility shift assays show a strong increase in the binding of factors to the NF
kappa B-like site following induction. We further provide evidence that RelA
(p65), c-Rel, and possibly novel polypeptides bind to the tissue factor NF kappa
B element. In addition, we show constitutive binding of members of the Fos/Jun
and Sp1 families to the AP-1 and Sp1 sites, respectively. We propose a concerted
action of AP-1-, NF kappa B-, and Sp1-like factors in transcription from the
tissue factor promoter in endothelial cells.

PMID: 7876129 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


31: J Biol Chem. 1994 Dec 2;269(48):30616-9. 

In vitro study of functional involvement of Sp1, NF-kappa B/Rel, and AP1 in
phorbol 12-myristate 13-acetate-mediated HIV-1 long terminal repeat activation.

Li Y, Mak G, Franza BR Jr.

Cold Spring Harbor Laboratory, New York 11724.

We examined the cooperative activity between the Sp1 and NF-kappa B/Rel sites of
the human immunodeficiency virus type 1 long terminal repeat in response to
phorbol 12-myristate 13-acetate (PMA) stimulation in an in vitro transcription
assay. Sp1 sites alone do not account for the activation induced by PMA. When
mutations in Sp1 sites were combined with mutations in the NF-kappa B/Rel sites,
a dramatic reduction in PMA-induced transcriptional activity was observed. This
reduction was much greater than the reduction associated with mutations
involving only the NF-kappa B/Rel sites. This finding suggests that there is
functional cooperation between Sp1 and NF-kappa B/Rel and that this is one
possible mechanism for transcription activation by NF-kappa B/Rel. The three AP1
sites in the negative regulatory region of the long terminal repeat, however,
seem to be uninvolved in the earliest moments of transcriptional activation by
PMA.

PMID: 7982981 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


32: J Virol. 1994 Nov;68(11):7131-8. 

Interaction of the v-Rel oncoprotein with cellular transcription factor Sp1.

Sif S, Gilmore TD.

Department of Biology, Boston University, Massachusetts 02215.

We previously showed that v-Rel, the oncoprotein of the avian retrovirus Rev-T,
can increase expression from promoters containing binding sites for the cellular
transcription factor Sp1 in chicken embryo fibroblasts (S. Sif, A.J. Capobianco,
and T.D. Gilmore, Oncogene 8:2501-2509, 1993). In those experiments, v-Rel
appeared to increase the transactivating function of Sp1; that is, v-Rel
stimulated transactivation by a GAL4-Sp1 protein that lacked the Sp1 DNA-binding
domain. We have now shown that in vitro-synthesized v-Rel and GAL4-Sp1 form a
complex that can be immunoprecipitated with either anti-Sp1 or anti-v-Rel
antiserum. We have also shown that a glutathione S-transferase (GST)-Sp1 fusion
protein can specifically interact with in vitro-translated v-Rel and with in
vivo-synthesized v-Rel from transformed chicken spleen cells. In addition, we
have found that the abilities of wild-type and two mutant forms of v-Rel to
increase transactivation by Sp1 in vivo correlate with their abilities to
interact with Sp1 in vitro. The sequences important for the interaction of v-Rel
with Sp1 in vitro have been mapped to the first 147 amino acids of v-Rel. Other
Rel proteins, such as c-Rel, RelA, p52, and p50, were also able to form a
complex with Sp1 in vitro. These results suggest that v-Rel increases expression
from Sp1 site-containing promoters by functionally interacting with Sp1 and that
cellular Rel proteins and Sp1 are likely to interact to influence transcription
from natural promoters.

PMID: 7933095 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


33: Infect Immun. 1993 Oct;61(10):4427-33. 

Shigella flexneri invasion of HeLa cells induces NF-kappa B DNA-binding
activity.

Dyer RB, Collaco CR, Niesel DW, Herzog NK.

Department of Microbiology, University of Texas Medical Branch, Galveston
77555-0605.

Although information about the genetic basis and mechanisms of Shigella flexneri
cellular invasion is accumulating, little is known about changes in cell
signaling and their consequences following bacterium-host cell interactions. A
general result of signal transduction is alterations in the levels and/or
activities of transcription factors. Alterations in transcription factor binding
activities were observed following challenge with S. flexneri. Changes in the
DNA-binding activities of cellular transcription factors to AP1, AP2, cyclic AMP
response element, CTF1/NF1, NF-kappa B/Rel, OCT1, and SP1 DNA-binding sites were
investigated by electrophoretic mobility shift assays. NF-kappa B/Rel
DNA-binding activity was enhanced more than 11-fold by cellular invasion;
noninvasive S. flexneri strains induced low levels of kappa B DNA binding. Both
subunits of the NF-kappa B transcription factor, p50 and p65, but not c-Rel
(p85), are components of the kappa B DNA-binding activity. These data suggest
that changes in cellular transcription factor binding activity are a consequence
of S. flexneri invasion, and these changes could play a role in the initial host
response or in the pathogenesis of the disease.

PMID: 8406833 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


34: Oncogene. 1993 Sep;8(9):2501-9. 

The v-Rel oncoprotein increases expression from Sp1 site-containing promoters in
chicken embryo fibroblasts.

Sif S, Capobianco AJ, Gilmore TD.

Department of Biology, Boston University, Massachusetts 02215.

The v-Rel oncoprotein of the avian Rev-T retrovirus is a member of a family of
related transcription factors, which also includes the subunits of NF-kappa B
and several other interacting cellular proteins. We show here that v-Rel
specifically increased expression from a reporter plasmid containing multiple
Sp1 binding sites approximately sixfold in chicken embryo fibroblasts (CEFs),
even though v-Rel did not bind directly to these sites. v-Rel also increased
expression from a reporter plasmid containing a human immunodeficiency virus
type 1 (HIV-1) long terminal repeat (LTR) in which the kappa B binding sites
were mutated but which still contained intact Sp1 binding sites. The increase in
Sp1-site transactivation does not precisely correlate with transformation by
v-Rel since one non-transforming v-Rel mutant still induced expression from the
Sp1 site-containing promoter. v-Rel appears to increase expression from Sp1
site-containing promoters by affecting the transactivation domain of Sp1, since
v-Rel increased the activity of a Gal4-Sp1 fusion protein, which contains the
Sp1 transactivation domain but lacks the Sp1 DNA-binding domain. As compared
with v-Rel, c-Rel induced only a slight increase in expression from the reporter
plasmid containing Sp1 sites. However, v-Ras and v-Src (but not v-Myb) induced
increases in transcription from the reporter plasmid containing Sp1 sites to the
same extent as v-Rel, but through pathways that appear to be independent from
v-Rel. These results suggest that certain oncoproteins might increase
transcription from many genes that contain Sp1 binding sites, and that this
might be important for certain aspects of transformation by these proteins.

PMID: 8361761 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


35: Oncogene. 1992 Jan;7(1):163-70. 

Transcriptional activity of rel family proteins.

McDonnell PC, Kumar S, Rabson AB, Gelinas C.

Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey
08854-5638.

Our studies originally demonstrated that the v-rel oncoprotein repressed gene
expression in chicken lymphoid cells, while it activated transcription in rodent
fibroblasts. Here we report that the c-rel protein can activate expression of
genes linked to kappa B motifs when low levels of endogenous kappa B-binding
activity are present. In contrast v-rel, and to a lesser extent c-rel, inhibit
NF-kappa B-mediated activation of the human immunodeficiency virus long terminal
repeat (HIV LTR) in phorbol ester-stimulated HeLa cells. Competition assays show
that v-rel competitively inhibits both NF-kappa B and c-rel-mediated
transcriptional activation. Analysis of mutant HIV LTR-chloramphenicol
acetyltransferase (CAT) constructs in which all Sp1 or both NF-kappa B elements
have been deleted shows that NF-kappa B motifs are required for rel-mediated
effects on gene expression. Transforming v-rel mutants compete efficiently with
phorbol ester-activated kappa B factors, whereas a transformation-defective
mutant of v-rel is impaired in this activity. Taken together, these results
strengthen the hypothesis that v-rel functions as a dominant interfering member
of rel family proteins. These results also suggest that the ability of v- and
c-rel to activate or repress gene expression in specific cells may result from
their capacity to compete with endogenous rel family proteins whose expression
and/or activity are cell-specific.

PMID: 1741161 [PubMed - indexed for MEDLINE]


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