Input:
IL-4 down-regulates IL-2-, IL-3-, and GM-CSF-induced cytokine gene expression in peripheral blood monocytes.
IL-4, a product of the T-helper 0 (Th0) and 2 (Th2) subset, was originally described as a B-cell stimulatory factor and has subsequently been found to suppress IL-1 alpha, IL-1 beta, IL-6, IL-8, and TNF-alpha gene expression in monocytes stimulated with LPS, and to upregulate IL-1 receptor antagonist (IL1-RA) gene expression. In this study we investigated the effect of IL-4 on the expression of cytokine genes in monocytes evoked by other T-helper cell cytokines: IL-2, IL-3, and GM-CSF. IL-4 down-regulated mRNA accumulation of the proinflammatory cytokines IL-1 beta, IL-8, and TNF-alpha in monocytes stimulated with IL-2, IL-3, and GM-CSF. IL-4 also suppressed the IL-2-induced IL-6 mRNA expression. Temporal analysis of the IL-4 down-regulatory effect on the IL-2-, IL-3-, or GM-CSF-induced proinflammatory cytokine gene expression in monocytes provided evidence that IL-4 acts predominantly on the post-transcriptional level. This was supported by the observation that the down-regulatory capacity of IL-4 appeared to be dependent on de novo protein synthesis. IL-4 did not exert significant influence on the induction of expression of IL-1-RA or various CSFs by IL-2, IL-3, and GM-CSF. (ABSTRACT TRUNCATED AT 250 WORDS)

product

expression

IL-1 alpha

expression

IL-1 beta

expression

expression

expression

TNF-alpha

expression

expression

suppress

stimulated

down-regulated

mRNA accumulation

suppressed

induced

stimulated

expression

upregulate

expression

mRNA accumulation

IL-1 beta

mRNA accumulation

mRNA accumulation

TNF-alpha

induced

expression

induction

expression

induction

expression

induction

expression

GM-CSF

influence

induction

expression


Input:
Long-term inositol phosphate release, but not tyrosine kinase activity, correlates with IL-2 secretion and NF-AT induction in anti-CD3-activated peripheral human T lymphocytes.
The cascade of events within the first few minutes of T cell stimulation has been well characterized. Although many second messengers have been shown to be necessary and sufficient for T cell activation in a number of model systems, the rate-limiting step in peripheral T cells has not been demonstrated. To model effective versus ineffective CD3-mediated stimulation in peripheral T cells, we used two anti-CD3 mAbs that differ in their ability to stimulate purified T cells: OKT3, which causes early second messenger generation but is unable to activate T cells without a second signal, and 64.1, which stimulates T cell proliferation on its own. We found that tyrosine kinase activity was similar for both mAbs over a period of hours. However, the inositol phosphate response was stronger for 64.1 than for OKT3. To tie these events to gene activation, we measured NF-kappa B and NF-AT activity in the nucleus after anti-CD3 stimulation. Both stimuli induced the appearance of the NF-kappa B components (c-Rel, p65 (RelA), and p50 (NF-kappa B1)) and NF-kappa B DNA binding activity in the nucleus. However, only 64.1 induced NF-AT in the nucleus, correlating with its ability to activate T cells. Thus, NF-AT induction and IL-2 secretion were correlated with the levels of inositol phosphate release but not with gross levels of tyrosine kinase activity induced late following the response. On the other hand, NF-kappa B induction and IL-2 receptor expression occurred even with the smaller second messenger response generated by OKT3.

secretion

appearance

c-Rel

nucleus

appearance

nucleus

appearance

nucleus

secretion

induced

appearance

appearance

c-Rel


Input:
ZAP-70 tyrosine kinase, CD45, and T cell receptor involvement in UV- and H2O2-induced T cell signal transduction.
Several mammalian responses to UV irradiation, including the activation of NF-kappa B, are believed to involve tyrosine phosphorylation. UV irradiation and H2O2 treatment of T lymphocytes induce protein tyrosine phosphorylation and Ca2+ signals similar to those observed following biological stimulation. We have examined the role of cell surface molecules in these responses. Normal T lymphocytes whose surface expression of CD3 was depleted showed impaired UV-induced tyrosine phosphorylation and Ca2+ signals. Similarly, Jurkat T cell lines deficient in CD3 or CD45 expression also gave impaired UV responses. However, all these cell types still gave strong Ca2+ and tyrosine phosphorylation responses to H2O2. The T cell tyrosine kinase ZAP-70 was found to be highly responsive to UV and H2O2 treatment. ZAP-70 responsiveness to UV required expression of both CD3 and CD45, whereas only CD3 was required for the response to H2O2. UV-induced activation of NF-kappa B was blocked by CD3 depletion, indicating the importance of such cell surface molecules in biological responses to UV. In nonlymphoid cells, the epidermal growth factor receptor displayed increased tyrosine phosphorylation within seconds of UV irradiation. These results suggest that UV-induced signal transduction is mediated via cell surface receptors that normally respond to biological stimulation, whereas H2O2 is able to partially bypass this requirement.

expression

expression

deficient

expression

phosphorylation

epidermal growth factor receptor

tyrosine

increased

phosphorylation

responsive

responsive

responsiveness

response


Input:
Inhibition of NF-kappa B by sodium salicylate and aspirin [see comments]
The transcription factor nuclear factor-kappa B (NF-kappa B) is critical for the inducible expression of multiple cellular and viral genes involved in inflammation and infection including interleukin-1 (IL-1), IL-6, and adhesion molecules. The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs. This inhibition prevented the degradation of the NF-kappa B inhibitor, I kappa B, and therefore NF-kappa B was retained in the cytosol. Sodium salicylate and aspirin also inhibited NF-kappa B-dependent transcription from the Ig kappa enhancer and the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells.

inhibited

dependent

dependent

transcription

transcription

Ig kappa


Input:
Positive and negative regulation of IL-2 gene expression: role of multiple regulatory sites.
Interleukin 2 (IL-2) is an important lymphokine required in the process of T cell activation, proliferation, clonal expansion and differentiation. The IL-2 gene displays both T cell specific and inducible expression: it is only expressed in CD4+ T cells after antigenic or mitogenic stimulation. Several cis-acting regulatory sites are required for induction of the IL-2 gene after stimulation. In this study, we have analysed the function of these cis-acting regulatory sites in the context of the native IL-2 enhancer and promoter sequence. The results of this study suggest that the NFAT (-276 to -261), the distal octamer (-256 to -248) and the proximal octamer (-75 to -66) sites not only act as enhancers of IL-2 gene transcription in the presence of cellular stimulation, but also have a silencing effect on IL-2 gene expression in resting cells. Two other sites display disparate effects on IL-2 gene expression in different T leukemia cell lines: the distal purine box (-291 to -277) and the proximal purine box sites (-145 to -128). Finally, the AP-1 (-186 to -176) and the kappa B sites (-206 to -195) respond to different cellular activation in EL4 cells. The AP-1 site mediated the response to PMA stimulation while the kappa B site responded to IL-1 stimulation. These data suggest that the regulation of IL-2 gene expression is a complex process and multiple cis-acting regulatory sites interact to exert different effects in T cells representative of alternative stages of differentiation.

expression

expression

expression

expression

expression

negative regulation

expression

have a silencing effect

expression

Positive

expression

inducible

expression

expressed

required

induction

induction

act as enhancers

in the presence of

in the presence of

transcription

role

Positive

role

negative regulation

display disparate effects

expression

regulation

expression

interact to exert different effects

regulation

transcription


Input:
Superantigens activate HIV-1 gene expression in monocytic cells.
Binding of superantigens to MHC class II molecules results in transduction of biochemical signals leading to cellular activation and gene expression. We demonstrate that the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA) activate HIV-1-LTR-driven transcription of chloramphenicol acetyl transferase in the human monocytic cell line THP-1. Induction of HIV-1-LTR-driven transcription in THP-1 cells by superantigens was associated with the induction of nuclear factor-kappa B DNA-binding activity. Superantigens also increased viral protein secretion from the granulocyte-macrophage colony-stimulating factor-pretreated chronically infected human monocytic cell line U1. Induction of HIV-1 gene expression in monocytic cells by superantigens occurred via tumor necrosis factor-alpha-dependent and -independent mechanisms. Our results suggest that superantigens and other MHC class II ligands may activate HIV-1 gene expression in monocytes/macrophages.

activate

driven

activate

SEA

driven

driven

transcription

transcription

chloramphenicol acetyl transferase


Input:
Inhibition of activation of transcription factor AP-1 by CD28 signalling in human T-cells.
Co-stimulation of T-lymphocytes by T-cell receptor (TcR) occupancy and activation of the CD28 surface molecule results in enhanced proliferation and interleukin 2 (IL-2) production. The increase in IL-2 gene expression triggered by CD28 involves a kappa B-like sequence in the 5'-regulatory region of the IL-2 promoter, called CD28-responsive element. Stimulation of T-cells by agonistic anti-CD28 antibodies in conjunction with phorbol 12-myristate 13-acetate (PMA)- or TcR-derived signals induces the enhanced activation of the transcription factor NF-kappa B. Here we report that CD28 engagement, however, exerts opposite effects on the transcription factor AP-1. Whereas anti-CD28 together with PMA increased the DNA binding and trans-activation activity of NF-kappa B, PMA-induced activation of AP-1 was significantly suppressed. The inhibitory effect exerted by anti-CD28 was observed at the level of DNA binding as well as in functional reporter-gene assays. These results suggest that the two transcription factors are independently regulated and may perform different functions during T-cell activation.

engagement

production

expression

activation

increase

expression


Input:
Induction of IL-8 expression in T cells uses the CD28 costimulatory pathway.
IL-8, a potent chemotactic factor for neutrophil granulocytes and lymphocytes, is a proinflammatory cytokine secreted by a variety of cell types, including T cells. Stimulation of the CD28 cell surface molecule delivers costimulatory signals essential for lymphokine production in activated T cells via a conserved sequence element found in the promoter of several lymphokine genes. Anti-CD28-stimulated T cells produced significant amounts of IL-8; additionally, costimulation with anti-CD3 and anti-CD28 Abs resulted in a synergistic induction of IL-8 secretion. Sequence homology, single nucleotide mutations, and anti-CD28 Ab stimulation studies established that the NF-kappa B-like sequence in the promoter of the IL-8 gene functioned as a CD28 response element. Furthermore, cyclosporin A, but not rapamycin, blocked the synergistic induction of IL-8 expression achieved with anti-CD3 and anti-CD28 costimulation. The involvement of a CD28 response element in the induction of IL-8 expression in activated T cells may provide new insights into the pathogenesis and persistence of immune disorders characterized by increased levels of IL-8, such as psoriasis and rheumatoid arthritis.

expression

produced

expression

expression

levels

secreted

secretion

blocked

synergistic induction

Induction

expression

uses

Induction

Stimulation

resulted

synergistic induction

synergistic induction

secretion

synergistic induction

expression

involvement

induction

induction

expression

increased

levels

response

NF-kappa B-like sequence


Input:
The severe phenotype of females with tiny ring X chromosomes is associated with inability of these chromosomes to undergo X inactivation.
Mental retardation and a constellation of congenital malformations not usually associated with Turner syndrome are seen in some females with a mosaic 45,X/46,X,r(X) karyotype. Studies of these females show that the XIST locus on their tiny ring X chromosomes is either not present or not expressed. As XIST transcription is well correlated with inactivation of the X chromosome in female somatic cells and spermatogonia, nonexpression of the locus even when it is present suggests that these chromosomes are transcriptionally active. We examined the transcriptional activity of ring X chromosomes lacking XIST expression (XISTE-), from three females with severe phenotypes. The two tiny ring X chromosomes studied with an antibody specific for the acetylated isoforms of histone H4 marking transcribed chromatin domains were labeled at a level consistent with their being active. We also examined tow of the XISTE- ring chromosomes to determine whether genes that are normally silent on an inactive X are expressed from these chromosomes. Analyses of hybrid cells show that TIMP, ZXDA, and ZXDB loci on the proximal short arm, and AR and PHKA1 loci on the long arm, are well expressed from the tiny ring X chromosome lacking XIST DNA. Studies of the ring chromosome that has XIST DNA but does not transcribe it show that its AR allele is transcribed along with the one on the normal X allele. (ABSTRACT TRUNCATED AT 250 WORDS)

expressed

XIST

nonexpression

XIST

expression

XIST

expressed

ZXDA

expressed

ZXDB

expressed

present

XIST

lacking

XIST

transcription

XIST

transcribe

XIST


Input:
Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin-5 genes in helper T cells.
Mouse thymoma line EL-4 cells produce cytokines such as interleukin (IL)-2, IL-3, IL-4, IL-10, and granulocyte-macrophage colony-stimulating factor in response to phorbol 12-myristate 13-acetate (PMA). EL-4 cells also produce low levels of IL-5 when stimulated by PMA alone; however, cAMP greatly augments PMA-dependent IL-5 production. A transient transfection assay revealed that two signals, PMA and cAMP, are required for optimal activation of the IL-5 promoter. In contrast, cAMP almost completely inhibited the PMA-dependent activation of the endogenous IL-2 gene, as well as the transfected IL-2 promoter. These results indicate that the IL-5 gene is positively regulated by cAMP in a manner opposite to that for the IL-2 gene. One of the nuclear factors (NFs) that regulates the response of the IL-5 promoter to cAMP and PMA has properties similar to NF for activated t cell. The P sequence of the IL-4 gene, defined as a responsive element for PMA and calcium ionophore (A23187), shares sequence similarity with the NF kappa B and the NF-activated T cell binding sites. We attempted to determine whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and nuclear factor for activated T cell (NF-AT). In electromobility shift assays both NF-kappa B (P65 or P65/P50 heterodimer) and NF-AT bound to the P sequence. However, sequence specificity of NF-AT was more similar to that of NF(P), and only a small amount of P65 was detected in NF(P). These results indicate that a component or components of NF-AT have the potential to reconstitute NF(P), whereas NF-kappa B alone does not account for NF(P) in Jurkat crude extract. Taken together, these results suggest that NF-AT-like factors are involved in the regulation of IL-4 and IL-5 genes.

bound

bound

bound

expression

expression

produce

interleukin (IL)-2

produce

produce

produce

produce

granulocyte-macrophage colony-stimulating factor

produce

production

inhibited

activation

opposite

in response

produce

when

produce

augments

dependent

dependent

production

required

activation

activation

promoter

activation

promoter

activation

positively regulated

regulate

expression

regulates

response

response

promoter

defined as a responsive element

P sequence

regulation

regulation


Input:
Structure and expression of the human GATA3 gene.
GATA3, a member of the GATA family that is abundantly expressed in the T-lymphocyte lineage, is thought to participate in T-cell receptor gene activation through binding to enhancers. To understand GATA3 gene regulation, we cloned the human gene and the 5' end of the mouse GATA3 gene. We show that the human GATA3 gene contains six exons distributed over 17 kb of DNA. The two human GATA3 zinc fingers are encoded by two separate exons highly conserved with those of GATA1, but no other structural homologies between these two genes can be found. The human and mouse GATA3 transcription units start at a major initiation site. The promoter sequence analysis of these two genes revealed that they are embedded within a CpG island and share structural features often found in the promoters of housekeeping genes. Finally, we show that a DNA fragment containing the human GATA3 transcription unit, 3 kb upstream from the initiation site and 4 kb downstream from the polyadenylation site, displays T-cell specificity.

binding

expression

expressed

regulation


Input:
Characterization of NF(P), the nuclear factor that interacts with the regulatory P sequence (5'-CGAAAATTTCC-3') of the human interleukin-4 gene: relationship to NF-kappa B and NF-AT.
The P sequence of the human interleukin-4 (IL-4) gene, which was defined as a responsive element for phorbol 12-myristate 13-acetate and calcium ionophore (A23187) in Jurkat T cells, shares sequence similarity with the NF-kappa B and the NF-AT binding sites. We examined whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and NF-AT. NF-kappa B (P65 or P65/P50 heterodimer) bound to the P sequence in electrophoretic mobility shift assays (EMSA) and activated transcription through the P sequence when expression plasmids were cotransfected with P sequence-driven reporter plasmids in Jurkat T cells. In EMSAs, NF(P) binding was inhibited by the unlabeled NF-AT binding site but not by the unlabeled AP1 binding site and purified NF-AT contained an activity that bound to the P sequence. Both mobility shift and sequence specificity of NF-AT were similar to those of NF(P) and only a small amount of P65 was detected in NF(P) in crude nuclear extracts. These results indicate that the component(s) of NF-AT has the potential to reconstitute NF(P) whereas NF-kappa B alone cannot account for NF(P) in crude extracts. Unlike NF-AT, NF(P) does not contain AP1 as its DNA binding component.

interacts

P sequence

bound

bound

bound

detected

responsive element

P sequence


Input:
Hypoxia causes the activation of nuclear factor kappa B through the phosphorylation of I kappa B alpha on tyrosine residues.
The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor kappa B (NF-kappa B) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in I kappa B alpha degradation, increased NF-kappa B DNA binding activity, and transactivation of a reporter gene construct containing two NF-kappa B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf 301) inhibited I kappa B alpha degradation, NF-kappa B binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I kappa B alpha with NF-kappa B activation, we immunoprecipitated I kappa B alpha after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto I kappa B alpha prevented I kappa B alpha degradation and NF-kappa B binding. In comparison to other activators of NF-kappa B such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of I kappa B alpha following treatment with either of these agents. These results suggest that tyrosine phosphorylation of I kappa B alpha during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-kappa B.

inhibited

by

Inhibition

transfer of tyrosine phosphoryl groups

prevented

Inhibition

degradation

phosphorylation

I kappa B alpha

phosphorylation

I kappa B alpha

phosphorylation

I kappa B alpha

tyrosine

transfer of tyrosine phosphoryl groups

I kappa B alpha

phosphorylation

tyrosine

I kappa B alpha

phosphorylation

tyrosine

I kappa B alpha

causes

phosphorylation

results

degradation

by

degradation

increased

phosphorylation

during

phosphorylation

precedes

during

degradation

degradation

I kappa B alpha

degradation

I kappa B alpha

degradation

I kappa B alpha

degradation

I kappa B alpha

changes

phosphorylation


Input:
A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1.
Analysis of the chromatin organization of the integrated human immunodeficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high-resolution mapping of this site with DNase I and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four distinct nuclear protein complexes: a ubiquitous factor, a T-cell-specific factor, a B-cell-specific factor, and the monocyte/macrophage- and B-cell-specific transcription factor PU.1/Spi-1. In most HIV-1 isolates in which this PU box was not conserved, it was replaced by a binding site for the related factor Ets1. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors binding to site B, except for PU.1/Spi-1. A GC box containing a binding site for Sp1 was identified (nt 4623 to 4631). Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results identify a transcriptional regulatory element associated with a nuclease-hypersensitive site in the pol gene of HIV-1 and suggest that its activity may be controlled by a complex interplay of cis-regulatory elements.

bound

binding

containing a binding site


Input:
Expression of v-src in T cells correlates with nuclear expression of NF-kappa B.
NF-kappa B is a rapidly inducible transcriptional activator that responds to a variety of signals and influences the expression of many genes involved in the immune response. Protein tyrosine kinases transmit signals from cytokine and immune receptors. Very little information exists linking these two important classes of signaling molecules. We now demonstrate that v-src expression correlates with nuclear expression of a kappa B binding complex similar to that induced by phorbol ester and ionomycin, as detected by electrophoretic mobility shift assay using a variety of kappa B sites. This complex was blocked by the tyrosine kinase inhibitor, herbimycin A. The v-src-induced complex comprised the p50 and p65 components of NF-kappa B, as determined by supershift and immunoblot analysis. As a functional correlate of this finding, transient co-transfection of HIV-1 LTR reporter constructs in a different T cell line demonstrated that v-src activated this promoter in a kappa B-dependent manner. We found that transactivation of the HIV-1 LTR by v-src was more sensitive to mutations of the proximal, rather than the distal, kappa B element. The implications for T cell receptor signaling and HIV-1 gene expression are considered.

induced complex

Expression

v-src

expression

v-src

induced

v-src

induced complex


Input:
Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors NF-kappa B and AP-1 in human T cells in vitro after T-cell receptor stimulation.
Human immunodeficiency virus type 1 (HIV-1) negative factor (Nef) has been shown to down-regulate the transcription factors NF-kappa B and AP-1 in vitro. To define the mechanism of action of the Nef protein, the signal transduction pathways which may be affected in T cells by constitutive expression of the nef gene were examined. Stimulation of T cells with tumor necrosis factor, interleukin-1, or lipopolysaccharide resulted in the recruitment of transcriptional factors to a similar level whether or not the cells expressed the nef gene. On the other hand, stimulation of T cells by mitogens or antibodies to the T-cell receptor (TCR)-CD3 complex resulted in the down-regulation of transcriptional factors NF-kappa B and AP-1 in cells expressing the nef gene compared with cells not expressing the nef gene. Because the Nef protein does not affect the surface expression of the CD3-TCR complex, we conclude that the Nef protein down-regulates the transcriptional factors NF-kappa B and AP-1 in T cells in vitro through an effect on the TCR-dependent signal transduction pathway.

expression

nef

expressing

nef


Input:
Activation of nuclear factor kappa B in human neuroblastoma cell lines.
The nuclear factor kappa B (NF-kappa B) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF-kappa B translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor alpha (TNF alpha), phorbol ester, and other polyclonal signals. Using neuroblastoma cell lines as models, we have shown that in neural cells NF-kappa B was present in the cytosol and translocated into nuclei as a result of TNF alpha treatment. The TNF alpha-activated NF-kappa B was transcriptionally functional. NF-kappa B activation by TNF alpha was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12-myristate 13-acetate (PMA), which induce phenotypical differentiation of the SH-SY5Y neuroblastoma cell line, activated NF-kappa B, but only in that particular cell line. In a NGF-responsive rat pheochromocytoma cell line, PC12, PMA activated NF-kappa B, whereas NGF did not. In other neuroblastoma cell lines, such as SK-N-Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF-kappa B activation. We found, moreover, that in SK-N-Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF-kappa B was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF-kappa B activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNF alpha proved that NF-kappa B activation was not sufficient for induction of neuroblastoma differentiation.


Input:
Genes encoding general initiation factors for RNA polymerase II transcription are dispersed in the human genome.
General transcription factors are required for accurate initiation of transcription by RNA polymerase II. Human cDNAs encoding subunits of these factors have been cloned and sequenced. Using fluorescence in situ hybridization (FISH), we show here that the genes encoding the TATA-box binding protein (TBP), TFIIB, TFIIE alpha, TFIIE beta, RAP30, RAP74 and the 62 kDa subunit, of TFIIH are located at the human chromosomal bands 6q26-27, 1p21-22, 3q21-24, 8p12, 13q14, 19p13.3 and 11p14-15.1, respectively. This dispersed localization of a group of functionally related gene provides insights into the molecular mechanism of human genome evolution and their possible involvement in human diseases.


Input:
A novel heterodimerization partner for thyroid hormone receptor. Peroxisome proliferator-activated receptor.
Retinoid-like receptors play a central role in hormonal responses by forming heterodimers with other nuclear hormone receptors. In this study we have identified the peroxisome proliferator-activated receptor (PPAR) as a new thyroid hormone receptor (THR) auxiliary nuclear protein, heterodimerizing with THR in solution. Although these heterodimers do not recognize a classical thyroid hormone response element (TRE) characterized by direct repeat separated by four nucleotides (DR+4), PPAR behaves as a dominant negative regulator of thyroid hormone (TH) action. However, a TH-dependent positive effect is elicited by selective interaction of the THR beta-PPAR but not the THR alpha-PPAR heterodimer with a novel TRE (DR+2). The critical region of THR beta was mapped to 3 amino acids in the distal box of the DNA binding domain. Hence, PPAR can positively or negatively influence TH action depending on TRE structure and THR isotype.


Input:
Hypoxic induction of interleukin-8 gene expression in human endothelial cells.
Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis.

expression

release

induction

expression

led

release

Production

increased

increased

transcription

increased

macrophage chemotactic protein-1

induction

increased

increased

transcription


Input:
Function of NF-kappa B/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different NF-kappa B/Rel subunits.
The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-kappa B/Rel binding sites located at -109 to -118 (Ii kappa B-1) and -163 to -172 (Ii kappa B-2) from the transcription start site. We report here that the differential function of each of these NF-kappa B/Rel sites in several distinct cell types depends on cell-specific binding of NF-kappa B/Rel transcription factors. Ii kappa B-1 is a positive regulatory element in B-cell lines and in the Ii-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glia cell lines. In vivo protein-DNA contacts are detectable at Ii kappa B-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-kappa B/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii kappa B-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again, in vitro supershift studies indicate that the presence of p50, p52, p65, and cRel correlates with positive function whereas the presence of only p50 and p52 correlates with negative function. This differential binding of specific NF-kappa B/Rel subunits is likely to mediate the disparate functions of these two NF-kappa B/Rel binding sites.

binding complexes

cRel

presence

presence

cRel

presence

Function

major histocompatibility complex class II invariant chain

promoter

dependent

Function


Input:
Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells.
The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.

binds

expression

decreases

transcriptional activation

Positive regulators

maintained

expression

activate

transcriptional activation

induction

autoregulatory control

transcription

transcription


Input:
Patterns of Pan expression and role of Pan proteins in endocrine cell type-specific complex formation.
The Pan gene encodes at least two distinct transcripts, Pan-1 and Pan-2 (also known as E47 and E12, respectively), by the mechanism of alternative RNA splicing. Northern blot analyses performed on rat and mouse tissues have detected ubiquitously expressed Pan transcripts, but the abundance, distribution, and form of Pan proteins have not been clearly defined. Studies of cell lines representing endocrine, fibroblast, and lymphoid lineages using polyclonal antisera to detect E2A proteins have suggested that significant E2A protein expression is restricted to B-lymphocytes. We have developed a monoclonal antibody, Yae, which is specific for Pan/E2A proteins, and have used the Yae antibody to examine a variety of endocrine and nonendocrine cell lineages for differences in Pan/E2A protein expression, subcellular localization, and heteromeric complex formation. In contrast to previous results obtained using polyclonal antiseras to detect Pan/E2A proteins, we report comparable levels of Pan proteins in GH/PRL- and insulin-producing, B- and T-lymphocyte cells. IEF-1, a pancreatic beta-cell type-specific complex believed to regulate insulin expression, is demonstrated to consist of at least two distinct species, one of which does not contain Pan molecules. Although it has been postulated that pituitary endocrine cells and pancreatic endocrine beta-cells share identical Pan/E2A complexes, native-Western analyses of pituitary and endocrine beta-cells detect Pan proteins in distinct cell type-specific complexes.

heteromeric complex formation

Pan

expression

expression

Pan

comparable levels

Pan

producing

GH

producing

insulin

expression

insulin

detect

Pan

regulate

expression

expressed

Pan


Input:
Functional block for 1 alpha,25-dihydroxyvitamin D3-mediated gene regulation in human B lymphocytes.
Elements necessary for the steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) to induce a biological response include the presence of specific intracellular receptors (vitamin D3 receptors (VDR)) and modulation of gene expression via hormone-activated receptor binding to regulatory regions of target genes. These parameters were examined in normal and Epstein-Barr virus-immortalized human B cells and compared with 1 alpha,25-(OH)2D3-responsive cells of the T and monocytic lineages. Although resting tonsillar B cells did not express VDR mRNA, activation of these cells with interleukin-4 induced VDR in the absence of exogenously supplemented 1 alpha,25-(OH)2D3. As indicators of hormone-mediated gene regulation we analyzed modulation of CD23, a common B cell/monocyte surface antigen, and 24-hydroxylase. 1 alpha,25-(OH)2D3 inhibited CD23 expression in U937 cells, yet failed to modulate CD23 expression in B cells. Furthermore, 1 alpha,25-(OH)2D3 induced 24-hydroxylase mRNA expression and metabolic activity in both U937 cells and lectin-activated T cells, yet failed to induce 24-hydroxylase mRNA or its metabolic activity in B cells. These findings suggest that although human B lymphocytes can express VDR mRNA and protein, they exhibit a functional block for vitamin D-dependent gene regulation.

expression

expression

express

VDR

inhibited

expression

activated

VDR

induced

VDR

induced

expression

induce

modulation

modulation

modulate

expression

express

VDR

expression

express

VDR


Input:
Effects of alpha-lipoic acid and dihydrolipoic acid on expression of proto-oncogene c-fos.
The transcription factor AP-1 is an important human mediator of the cellular response to serum, growth factors, and phorbol esters such as 12-O-tetradecanoyl-phorbol-13 acetate (TPA). The AP-1 complex consists of distinct protein heterodimers encoded by the proto-oncogene c-fos and c-jun mRNA whose gene expression can be induced by TPA, cyclic AMP and growth factors. Recent findings suggest an involvement of reactive oxygen species in the pathway of TPA and protein kinase C leading to expression of c-fos and c-jun mRNA. To investigate the role of reactive oxygen species we studied the effects of alpha-lipoic acid and dihydrolipoic acid (natural thiol antioxidants) on the expression of c-fos mRNA in human Jurkat T cells. When cells were preincubated with dihydrolipoic acid (0.2 mM) the expression of c-fos mRNA was suppressed at 30 min after stimulation of TPA (0.5 microM) whereas in the case of preincubation of alpha-lipoic acid (0.2 microM), the expression was enhanced at 30 min. These studies support the idea that superoxide anion radical plays a role in the expression of c-fos mRNA.

expression

c-fos

suppressed

expression

induced

gene expression

leading

expression

When

suppressed

in the case of

enhanced

enhanced

expression

Effects

expression

involvement

leading

role

expression

effects

expression

plays a role

expression

gene expression

c-fos

gene expression

c-jun

expression

c-fos

expression

c-jun

expression

c-fos

expression

c-fos

expression

c-fos


Input:
Alternative splicing of RNA transcripts encoded by the murine p105 NF-kappa B gene generates I kappa B gamma isoforms with different inhibitory activities.
The gene encoding the 105-kDa protein (p105) precursor of the p50 subunit of transcription factor NF-kappa B also encodes a p70 I kappa B protein, I kappa B gamma, which is identical to the C-terminal 607 amino acids of p105. Here we show that alternative RNA splicing generates I kappa B gamma isoforms with properties different from those of p70. One 63-kDa isoform, termed I kappa B gamma-1, which lacks 59 amino acids C-terminal to ankyrin repeat 7, has a novel 35-amino acid C terminus encoded by an alternative reading frame of the p105 gene. A 55-kDa isoform, I kappa B gamma-2, lacks the 190 C-terminal amino acids of p70I kappa B gamma. In contrast to p70I kappa B gamma, which is a cytoplasmic protein, I kappa B gamma-1 is found in both the cytoplasm and nucleus, whereas I kappa B gamma-2 is predominantly nuclear. The I kappa B gamma isoforms also display differences in specificity and affinity for Rel/NF-kappa B proteins. While p70I kappa B gamma inhibits p50-, p65-, and c-Rel-mediated transactivation and/or DNA binding, both I kappa B gamma-1 and I kappa B gamma-2 are specific for p50 and have different affinities for this subunit. The absence in I kappa B gamma-1 and I kappa B gamma-2 of a protein kinase A site whose phosphorylation modulates p70I kappa B gamma inhibitory activity suggests that alternative RNA splicing may be used to generate I kappa B gamma isoforms that respond differently to intracellular signals.

specific

I kappa B gamma-1

specific

I kappa B gamma-2

generates

I kappa B gamma

generates

I kappa B gamma

found

I kappa B gamma-1

cytoplasm

found

I kappa B gamma-1

nucleus

found

I kappa B gamma-2

nuclear

generates

generates

generates

generates

generate

I kappa B gamma

respond

I kappa B gamma


Input:
Activation of nuclear factor kappa B in human lymphoblastoid cells by low-dose ionizing radiation.
Nuclear factor kappa B (NF-kappa B) is a pleiotropic transcription factor which is involved in the transcriptional regulation of several specific genes. Recent reports demonstrated that ionizing radiation in the dose range of 2-50 Gy results in expression of NF-kappa B in human KG-1 myeloid leukemia cells and human B-lymphocyte precursor cells; the precise mechanism involved and the significance are not yet known. The present report demonstrates that even lower doses of ionizing radiation, 0.25-2.0 Gy, are capable of inducing expression of NF-kappa B in EBV-transformed 244B human lymphoblastoid cells. These results are in a dose range where the viability of the cells remains very high. After exposure to 137Cs gamma rays at a dose rate of 1.17 Gy/min, a maximum in expression of NF-kappa B was seen at 8 h after a 0.5-Gy exposure. Time-course studies revealed a biphasic time-dependent expression after 0.5-, 1- and 2-Gy exposures. However, for each time examined, the expression of NF-kappa B was maximum after the 0.5-Gy exposure. The expression of the p50 and p65 NF-kappa B subunits was also shown to be regulated differentially after exposures to 1.0 and 2.0 Gy.

expression

expression

regulated

expression


Input:
Some antioxidants inhibit, in a co-ordinate fashion, the production of tumor necrosis factor-alpha, IL-beta, and IL-6 by human peripheral blood mononuclear cells.
Some antioxidants, including butylated hydroxyanisole (BHA), tetrahydropapaveroline (THP), nordihydroguiauretic acid, and 10,11-dihydroxyaporphine (DHA), were found to be potent inhibitors of the production of tumor necrosis factor (TNF)-alpha, IL-1 beta, and IL-6 by human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS) (IC50s in the low micromolar range). Inhibition of cytokine production was gene selective and not due to general effects on protein synthesis. Inhibition of cytokine production by PBMC was observed also when other inducers were used (staphylococci, silica, zymosan). Much higher concentrations of other antioxidants--including ascorbic acid, trolox, alpha-tocopherol, butylated hydroxytoluene, and the 5-lipoxygenase inhibitor zileuton--did not affect the production of these cytokines. The active compounds did not inhibit IL-1-induced production of IL-6 in fibroblasts, showing the cell selectivity of the effect. Antioxidant-mediated inhibition of cytokine production was correlated with low levels of the corresponding messenger RNAs. Nuclear run-on experiments showed that THP inhibited transcription of the IL-1 beta gene. THP decreased the concentration of the transcription factors NF-kappa B and AP-1 detected in nuclear extracts of PBMC cultured in the presence or absence of LPS. THP and DHA markedly decreased the levels of TNF-alpha and IL-1 beta in the circulation of mice following LPS injection. Thus antioxidants vary widely in potency as inhibitors of the activation of transcription factors and of the transcription of genes for pro-inflammatory cytokines. Coordinate inhibition of the transcription of genes for inflammatory cytokines could provide a strategy for therapy of diseases with inflammatory pathogenesis and for septic shock.

production

IL-beta

production

production

tumor necrosis factor-alpha

production

tumor necrosis factor (TNF)-alpha

production

IL-1 beta

production

production

tumor necrosis factor (TNF)-alpha

production

IL-1 beta

production

production

levels

TNF-alpha

levels

IL-1 beta

inhibit

production

inhibitors

production

inhibit

induced

inhibited

transcription

decreased

levels

induced

production

affect

production

transcription

IL-1 beta


Input:
Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine element 0.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.

associated

NF-CLE0 gamma

binding

NF-CLE0 gamma

produced

GM-CSF

produced

expression

GM-CSF

expression

expression

transfection

CN A

transfection

CN B

expression

GM-CSF

expression

inhibited

expression

recovers

transfection

inhibited

inhibited

transcription

inhibited

binding

potentiates

activation

activation

granulocyte-macrophage colony-stimulating factor

by stimulation with

produced

stimulate

transcription

transfection

transfection

augments

transfection

transcription

activate

transcription

to induce

expression

induced

binding

induction

GM-CSF

induction

transcription

transcription

GM-CSF

transcription

GM-CSF


Input:
Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat.
The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.

interaction

Tat


Input:
An intricate arrangement of binding sites for the Ets family of transcription factors regulates activity of the alpha 4 integrin gene promoter.
alpha 4 integrins mediate cell-cell and cell-extracellular matrix interactions that are critical for maturation and function of the immune system as well as differentiation of skeletal muscle. Here we examine molecular mechanisms controlling the pattern of alpha 4 expression. The activity of constructs containing 5' deletion mutants of the alpha 4 gene promoter was compared in transfection assays into cell lines that express alpha 4 and cell lines that do not. The sequence between position -42 and -76 base pairs (bp) was required for efficient transcription in cells that express alpha 4, but it showed no activity in HeLa cells, which do not express alpha 4. Three binding sites for the Ets family of transcription factors are found in this region: two adjacent sites at positions -50 and -54 bp and a more 5' site at position -67 bp. Using a series of constructs containing deletions and mutations in this region, we found that the 3'-most site alone was sufficient for binding GA-binding protein alpha (GABP alpha)/GABP beta and for a low level of transcriptional activation. When all three sites were present, a second complex "a" was detected, which contains an unknown member of the Ets family. Formation of complex a was cell-type specific and correlated with a high level of transcription. Deletion of the 5'-most Ets site had no effect on binding to GABP alpha/GABP beta, but it eliminated a. Concomitant with this loss of a, a new Ets-1-containing complex "c" appeared. Complex c substituted efficiently for complex a in transcriptional activation. We conclude that although neither of the two 5'-most Ets sites alone binds nuclear protein, they appear to act as modulators which control the pattern of Ets proteins that bind the alpha 4 gene promoter. This arrangement of Ets sites, coupled with the tissue- and developmental-specific expression of Ets members, likely play a key role in defining the pattern of alpha 4 integrin.

sufficient for binding

GA-binding protein alpha

binding

GABP alpha

bind

alpha 4

promoter

expression

alpha 4 integrins

express

alpha 4 integrins

express

alpha 4 integrins

express

alpha 4 integrins

pattern

alpha 4 integrin

required

transcription

activity

transcription

sufficient

transcriptional activation

transcriptional activation

alpha 4

promoter

high level

transcription

in transcriptional activation

alpha 4 integrins

regulates

alpha 4 integrin

promoter

controlling

expression

effect

binding

play a key role in defining

pattern

transcription

alpha 4 integrins

transcription

alpha 4 integrins


Input:
Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site.
Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.

binding

c-Rel

binding

bound

kappa B-like site

TF

bound

c-Rel

bound

bound

bound

bound

migrate

TF

migrate

TF

comigrated

TF

c-Rel

comigrated

TF

presence

c-Rel

TF

presence

TF

absence

TF

bound

c-Rel

bound

binding

c-Rel

binding

expression

TF

induction

TF

induction

TF

mediated

binding

induction

induction

expression


Input:
Stimulation of HIV replication in mononuclear phagocytes by leukemia inhibitory factor.
This study examined the effects of leukemia inhibitory factor (LIF) on human immunodeficiency virus (HIV) replication in mononuclear phagocytes (MNP). LIF induced a dose-dependent increase in p24 antigen production in the chronically infected promonocytic cell line U1. The magnitude and time kinetics of the LIF effects were similar to interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF), other cytokines known to induce HIV replication in this cell line. To characterize mechanisms responsible for these LIF effects, levels of HIV mRNA, activation of the DNA binding protein nuclear factor (NF)-kB, signal transduction pathways, and potential interactions with other cytokines were analyzed. LIF increased steady-state levels of HIV mRNA at 2.0, 4.3, and 9.2 kB. This was detectable by 24 h and persisted until 72 h. The DNA binding protein NF-kB is a central mediator in cytokine activation of HIV transcription. NF-kB levels were higher in unstimulated U1 cells as compared to the parent cell line U937. In both cell lines LIF increased NF-kB activity. Induction of NF-kB and HIV replication by cytokines are at least in part dependent on reactive oxygen intermediates. The oxygen radical scavenger N-acetyl-L-cysteine, but not an inhibitor of nitric oxide synthase, inhibited LIF-induced HIV replication. LIF induces the production of other cytokines in monocytes but its effects on HIV replication were not inhibited by antibodies to IL-1, TNF, or IL-6. These results identify LIF as a stimulus of HIV replication. (ABSTRACT TRUNCATED AT 250 WORDS)

production

p24 antigen

increase

LIF

production


Input:
Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells.
Dysregulation of cytokines secreted by T cells may play an important role in the pathogenesis of AIDS. To investigate the effects of human immunodeficiency virus type 1 (HIV-1) Tat on interleukin-2 (IL-2) expression, we used IL-2 promoter-chloramphenicol acetyltransferase constructs and IL-2-secreting Jurkat T cells as a model system. Transient expression of HIV-1 Tat induced a five- to eightfold increase in IL-2 promoter activity in Jurkat T cells stimulated with phytohemagglutinin and phorbol myristate acetate. IL-2 secretion was increased more than twofold in both Jurkat T cells and primary T cells stimulated by extracellular HIV-1 Tat protein. Analysis of mRNA suggested that Tat exerts its effect on IL-2 primarily at the transcriptional level. The NF-kappa B site at positions -206 to -195 of the IL-2 promoter was required but not sufficient for the Tat effect. The Tat-mediated increase in IL-2 promoter activity could selectively be blocked by antisense tat or-unlike the analogous effect of human T-cell lymphotropic virus type 1 Tax-by cyclosporin A. The observed increase in IL-2 levels might facilitate virus spread from or to T cells. Furthermore, it might contribute to the hypergammaglobulinemia or, together with other cytokines found to be dysregulated, the T-helper cell dysfunctions observed in AIDS patients.

expression

expression

Tat

secretion

secreting

secretion

upregulates

Tat

secretion

increased

secretion

effects

Tat

expression

effect

Tat

transcriptional

transcriptional


Input:
The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.
The macrophage colony-stimulating factor (M-CSF) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes/macrophages and the placenta. In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte/macrophage lineage, we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process. Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion. Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor, we investigated whether PU.1 binds and activates the M-CSF receptor promoter. Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site. Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only. Furthermore, PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells. These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor.

binds

M-CSF receptor

promoter

bind

M-CSF receptor

promoter

bind

M-CSF receptor

promoter

binding

specific site

M-CSF receptor

expression

macrophage colony-stimulating factor receptor

expressed

macrophage colony-stimulating factor (M-CSF) receptor

eliminate

binding

decrease

eliminate

M-CSF receptor

promoter

directs

expression

activate

M-CSF receptor

activates

M-CSF receptor

promoter

transactivates

M-CSF receptor

promoter

from

expressed


Input:
Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture.
All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.

expression

expression

inhibition

induction

inhibition

induction

induction

induction


Input:
Comparative analysis of NFAT (nuclear factor of activated T cells) complex in human T and B lymphocytes.
Nuclear factor of activated T cells (NFAT) is a transcriptional activator that binds to sequences in the interleukin-2 (IL-2) promoter and is thought to be largely responsible for the T cell-specific inducibility of IL-2 expression. Electrophoretic mobility shift assays (EMSA) showed that specific NFAT binding activity could also be induced in human B cells. The B cell NFAT complex, however, was not functional, since it failed to activate transcription from an NFAT-driven chloramphenicol acetyltransferase (CAT) construct. Competition with an AP-1 motif or with anti-Jun and anti-Fos antibodies abolished binding to the NFAT motif in both T and B cells, indicating that Jun and Fos are critical for NFAT complex formation in both cell types. Purified recombinant Jun and Fos proteins failed to bind directly to the NFAT motif. However, when combined with unstimulated B or T cell extracts, full-length, but not truncated, Jun/Fos heterodimers were able to form an NFAT complex, indicating the presence of a constitutively expressed nuclear factor(s) in B and T cells necessary for the formation of the NFAT complex in both cell types. An NFAT oligonucleotide carrying mutations in the 5' purine-rich part of the NFAT sequence failed to form a complex and to compete with the wild type motif for NFAT complex formation in both T and B cells. We therefore propose a model whereby a core NFAT complex consisting of Jun, Fos, and a constitutive nuclear factor is formed in both T and B cells, but an additional factor and/or post-translational modification of a factor, missing in B cells, might be required for transactivation by NFAT.

binds

promoter

binding activity

promoter

expression

responsible

inducibility

inducibility

expression

induced

binding activity

activate

transcription

transcription

chloramphenicol acetyltransferase


Input:
Differential autoregulation of glucocorticoid receptor expression in human T- and B-cell lines.
Regulation of glucocorticoid receptor (GR) expression by its cognate ligand was examined in the glucocorticoid-sensitive human leukemic T-cell line 6TG1.1 and in the human B-cell line IM-9. In contrast to the decrease in GR mRNA seen in IM-9 cells after treatment with 1 microM dexamethasone for 16-18 h, treatment of 6TG1.1 cells resulted in an 8-fold increase in GR mRNA, as determined by Northern blot and RNase protection analysis, with a corresponding 3- to 4-fold increase in GR protein. Half-maximal induction of GR mRNA and protein in 6TG1.1 cells was observed between 10-100 nM dexamethasone, and inclusion of 1 microM RU 38486 completely blocked the effects of 100 nM dexamethasone, demonstrating that positive autoregulation of GR expression in 6TG1.1 cells is a receptor-mediated response. Positive autoregulation of GR expression was also observed in glucocorticoid-resistant CEM-C1 cells, which contain functional GR, but whose growth is unaffected by glucocorticoids. Thus, positive autoregulation is neither a consequence nor the sole cause of growth arrest. The degree of negative autoregulation in IM-9 cells and positive autoregulation in 6TG1.1 cells was unaffected by inhibition of protein synthesis with cycloheximide. Measurement of GR mRNA turnover in 6TG1.1 cells treated with actinomycin-D revealed a half-life of 2.5 h, which was unaffected by dexamethasone treatment. A similar half-life was determined in IM-9 cells and was also unaffected by steroid treatment. These results are consistent with the interpretation that glucocorticoid-mediated autoregulation of GR expression is a tissue-specific primary transcriptional response.

expression

glucocorticoid receptor

expression

GR

expression

GR

expression

GR

decrease

GR

blocked

induction

negative autoregulation

expression

increase

GR

increase

GR

positive autoregulation

GR

expression

consequence

Positive autoregulation

positive autoregulation

expression

autoregulation

expression

Regulation

expression

Positive autoregulation

expression

unaffected

negative autoregulation

unaffected

positive autoregulation

unaffected

GR

unaffected

GR

autoregulation

transcriptional

induction

GR

transcriptional

GR


Input:
Occurrence of a silencer of the interleukin-2 gene in naive but not in memory resting T helper lymphocytes.
In the immune system the first activation of a naive T cell by antigen is a key step in the shaping of the peripheral T cell specificity repertoire and maintenance of self-tolerance. In the present study, analysis of the interleukin-2 (IL-2) gene activation shows that naive human helper T cells (cord blood CD4+ T cells, adult CD4+CD45RO- T cells) regulate IL-2 transcription by a mechanism involving both a silencer and an activator acting on the purine-rich IL-2 promoter elements (NF-AT binding sites). By contrast, memory cells, either in vitro activated helper T cells reverting to a resting state, or CD4+ T (memory) clones, or CD4+CD45RO+ T cells isolated ex vivo, no longer have a silencer. Their IL-2 transcription seems to be controlled solely by the transition from inactive to active functional state of a positive transcription factor binding to these promoter elements as well as its cytoplasmic or nuclear location: in resting memory T cells the activator is located in the cytoplasm and is inactive, whereas in stimulated cells it is functional in promoting transcription and now resides in the nucleus. Thus, the regulation of the gene coding for the main T cell growth factor changes irreversibly after the first encounter of T cells with antigen. It is most likely that the presence of a silencer contributes to the more stringent activation requirements of naive CD4+ T cells.

binding

NF-AT binding sites

activation

functional in promoting

transcription

regulate

transcription

acting

regulate

transcription

acting

NF-AT binding sites

controlled

transcription

transcription

transcription


Input:
Stimulation of interleukin-1 alpha and interleukin-1 beta production in human monocytes by protein phosphatase 1 and 2A inhibitors.
Protein phosphatases 1 and 2A are important in regulating cellular functions by controlling the phosphorylation state of their substrates. In human monocytes, the inhibitors of these phosphatases, okadaic acid and calyculin A, were found to increase the mRNA accumulation and cytokine production of interleukin-1 beta and interleukin-1 alpha. The increased mRNA accumulation was found to be primarily because of the increase in the transcription rate of the interleukin-1 genes. Stimulation of interleukin-1 gene transcription may be caused by the stimulation of transcription factor activities, including those of AP-1, by these protein phosphatase inhibitors. Okadaic acid increased the synthesis of the interleukin-1 beta precursor and mature forms and their secretion. This increased processing and secretion correlated with the stimulation of IL-1 beta convertase mRNA accumulation. The stimulation of interleukin-1 alpha production by okadaic acid was more modest than that of interleukin-1 beta. However, the phosphorylation of the precursor interleukin-1 alpha cytokine was increased. These results show that protein phosphatase 1 and 2A inhibitors exert multiple effects on cytokine production in human monocytes and suggest that these two phosphatases play important roles in regulating interleukin-1 production.

production

interleukin-1 alpha

production

interleukin-1 beta

cytokine production

interleukin-1 beta

cytokine production

interleukin-1 alpha

production

interleukin-1 alpha

production

interleukin-1 beta

secretion

interleukin-1 beta

Stimulation

production

Stimulation

production

protein phosphatase 1

inhibitors

inhibitors

protein phosphatase 1

inhibitors

Protein phosphatases 1

inhibitors

phosphorylation

interleukin-1 alpha

increase

mRNA accumulation

increase

cytokine production

mRNA accumulation

interleukin-1 beta

mRNA accumulation

interleukin-1 alpha

because of

increase

stimulation

increased

secretion

stimulation

accumulation

accumulation

IL-1 beta convertase

stimulation

production

increased

phosphorylation


Input:
Human CD3-CD16+ natural killer cells express the hGATA-3 T cell transcription factor and an unrearranged 2.3-kb TcR delta transcript.
In this study we analyzed the T cell receptor(TcR) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene (RAG)-1. Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer(NK) cell lines. Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA (2.3 and 1.3 kb) found in TcR gamma/delta+ T lymphocytes. Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment. The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence. The J delta 1 segment was correctly spliced to C delta; in addition the four C delta exons were found to be already assembled. Two polyadenylation sites were present in the fourth C delta exon. However, only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA. The expression of hGATA-3, a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA-3-specific mRNA, suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells. Finally, no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed.

express

expressed

expressed

recombination-activating gene (RAG)-1

expression

expression

TcR delta

involved in the regulation

transcription

involved in the regulation

expression

express

TcR delta

expressed

T cell receptor(TcR) delta

deriving

TcR delta

found

TcR delta

transcription

TcR delta

express

transcription


Input:
Involvement of Alu sequences in the cell-specific regulation of transcription of the gamma chain of Fc and T cell receptors.
The Fc epsilon RI-gamma chains are expressed in a variety of hematopoietic cells where they play a critical role in signal transduction. They are part of the high affinity IgE receptor in mast cells, basophils, Langerhans cells, and possibly other cells; a component of the low affinity receptor for IgG (Fc gamma RIIIA or CD16) in natural killer cells and macrophages; and part of the T cell antigen receptor in subsets of T cells. Here we have investigated the transcriptional regulation of the gamma chain gene by analyzing the 2.5-kilobase sequence upstream of the transcription start site. This sequence contains a promoter specific to cells of hematopoietic lineage. However, the tissue specificity of this promoter is only partial because it is active in all of the hematopoietic cells tested here, regardless of whether they constitutively express Fc epsilon RI- gamma chain transcripts. We have identified two adjacent cis-acting regulatory elements, both of which are part of an Alu repeat. The first (-445/-366) is a positive element active in both basophils and T cells. The second (-365/-264) binds to nuclear factors, which appear to be different in basophils and T cells, and acts as a negative element in basophils and as a positive one in T cells. Thus, this Alu repeat (90% identical to Alu consensus sequences) has evolved to become both a positive and negative regulator.

expressed

Fc epsilon RI-gamma chains

express

Fc epsilon RI- gamma chain


Input:
Interleukin-3 expression by activated T cells involves an inducible, T-cell-specific factor and an octamer binding protein.
Interleukin-3 (IL-3) is exclusively expressed by activated T and natural killer cells, a function that is tightly controlled both in a lineage-specific and in a stimulation-dependent manner. We have investigated the protein binding characteristics and functional importance of the ACT-1-activating region of the IL-3 promoter. This region binds an inducible, T-cell-specific factor over its 5' end, a site that is necessary for the expression of IL-3 in the absence of other upstream elements. Over its 3' end, it binds a factor that is ubiquitously and constitutively expressed. This factor is Oct-1 or an immunologically related octamer-binding protein, and it plays a role in coordinating the activity of several regulatory elements. These characteristics make the ACT-1 site analogous to the activating ARRE-1 site in the IL-2 promoter. Furthermore, and despite a lack of sequence homology, the promoters of IL-3 and IL-2 share an organizational pattern of regulatory elements that is likely to be important for the T-cell-specific expression of these genes.

binds

ACT-1-activating region

binds

ACT-1-activating region

expression

expressed

expression

expression

expression

necessary

expression

activating

ARRE-1 site

important

expression

controlled


Input:
ras protein activity is essential for T-cell antigen receptor signal transduction.
In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC.


Input:
Characterization of the nuclear and cytoplasmic components of the lymphoid-specific nuclear factor of activated T cells (NF-AT) complex.
The lymphoid-specific transcription complex, NF-AT, is involved in early gene activation in T cells and is assembled from a pre-existing, T cell restricted cytoplasmic factor and an inducible ubiquitous nuclear component within 30 min after activation through the antigen receptor. Recent studies have implicated the family of AP1 factors as components of the murine NF-AT complex. Evidence is provided here that the nuclear component of human NF-AT contains the phorbol ester-inducible transcription factor AP1 (Jun/Fos). We further characterize which AP1 family members can assume this role. Antisera to Fos inhibits NF-AT DNA binding as does an oligonucleotide containing a binding site for AP1. Constitutive expression in vivo of Fos, and to a lesser extent Fra-1, eliminates the requirement for phorbol 12-myristate 13-acetate (PMA) stimulation, leaving NF-AT-directed transcription responsive to calcium ionophore alone. Overexpression of cJun or JunD, but not JunB, also eliminates the requirement for PMA, indicating that many but not all Jun- and Fos-related proteins functionally activate NF-AT-dependent transcription in the presence of the cytoplasmic component. NF-AT DNA binding can be reconstituted in vitro using semi-purified AP1 proteins mixed with cytosol from T lymphocytes. Fos proteins are not needed for this reconstitution, and although JunB is not functional, it can participate in the NF-AT DNA binding complex. Finally, we have partially purified the cytoplasmic component of NF-AT and show by elution and renaturation from SDS-polyacrylamide gel electrophoresis gels that it has a molecular mass between 94 and 116 kDa and may have multiple differentially modified forms.

expression

Overexpression

JunB

expression

cJun

expression

JunD

Overexpression

Overexpression

Overexpression

expression


Input:
Protease treatment of nuclear extracts distinguishes between class II MHC X1 box DNA-binding proteins in wild-type and class II-deficient B cells.
The X box region is critical for directing the expression of class II major histocompatibility complex genes in B lymphocytes. Although several class II promoter-specific DNA binding factors have been described, only the X box region factor, RFX, shows a genetic correlation with class II expression, being deficient in some B cell lines derived from patients with class II-deficient congenital immunodeficiency. To further evaluate the role of X box DNA-binding proteins in class II gene expression, the role of the X box region was examined in both class II-positive and -negative lymphoid cells. In addition to the wild-type B cell line Raji, two class II transcriptional mutant cell lines, SJO and RJ2.2.5, and Jurkat, a class II negative T cell line, were examined. In contrast to wild-type B cells, neither of the class II mutant cell lines could use the X box region to direct the expression of a transiently transfected reporter gene, indicating that the X box-dependent transcriptional pathway is defective in these cells. The binding activity of the X1 box DNA-binding protein RFX was examined and found to be present in wild-type B cells and the mutant RJ2.2.5 but was absent in SJO and Jurkat. However, other X1 box-specific activities were detected in all these cell lines. To determine whether these different X1 box activities represented distinct DNA binding proteins or multimeric forms of the same factor(s), protease treatment of the crude nuclear extracts followed by DNA-binding assays were carried out and demonstrated that B cell extracts contain at least two X1-specific factors. One of these cleaved products (band 1 pk) correlates with RFX activity. A similar comparison with protease-treated extracts prepared from Jurkat cells demonstrated the presence of the band 1pk activity despite an absence of the native RFX activity. In contrast, protease treatment and analysis of SJO extracts showed no detectable levels of the band 1pk activity. These results demonstrate that multiple X1 box-specific DNA-binding activities exist in all lymphoid cells, but the presence of an actively binding RFX species correlates with class II transcription.


Input:
The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction.
In these studies, we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 1 beta (proIL-1 beta) gene. A cell-type-independent lipopolysaccharide (LPS)-responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site (cap site) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes. The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts. Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences, two of which appeared to be important for gene induction. One of the essential proteins which bound to the enhancer was similar or identical to members of the C/EBP family of transcription factors required for both IL-1- and LPS-specific induction of the IL-6 gene (i.e., the NF-IL6 proteins). When ligated to the proIL-1 beta cap site-proximal region (located between -131 to +12), both the proIL-1 beta and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells, which are not normally competent for IL-1 beta expression. When ligated to the murine c-fos promoter, however, the proIL-1 beta enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells, suggesting the existence of a proIL-1 beta promoter-proximal requirement for tissue specificity.

induction

proIL-1 beta

expression

IL-1 beta

requires

induction

induction

human prointerleukin 1 beta

necessary

transcriptional regulation

important

induction

required

induction

required

induction

induction

When

inducible

inducible

proIL-1 beta

enhancer

requirement

proIL-1 beta

enhancer

proIL-1 beta

promoter

transcriptional regulation

proIL-1 beta


Input:
Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells.
Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA). We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression. Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA. The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133. In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA. This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts. This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it.

Expression

PILOT

expression

expression

interferon-gamma

expression

PILOT

expression

PILOT

suppressed

expression

suppressed

Induction

requires

Expression

induced

induced

interferon-gamma

requirement

expression

Induction

PILOT

requires

expression

affected

expression


Input:
Suppression of a cellular differentiation program by phorbol esters coincides with inhibition of binding of a cell-specific transcription factor (NF-E2) to an enhancer element required for expression of an erythroid-specific gene.
Induction by hemin increases, while induction with 12-O-tetradecanoylphorbol-13-acetate (TPA) represses, erythroid-specific gene expression in the human cell line K562. We analyzed the effects of hemin or TPA induction on the binding and activity of transcription factors at a regulatory element found within the transcriptional regulatory sequences of many erythroid-specific genes. TPA induction increases the binding of ubiquitous AP-1 factors to this element. TPA induction inhibits the binding of the lineage limited transcription factor NF-E2 to this transcriptional control element. Hemin induction of K562 cells does not facilitate the binding of NF-E2 to its recognition site. Hemin induction appears to nonspecifically increase the expression of transiently transfected genes in K562 cells. Beyond this nonspecific increase in gene expression, hemin induction acts to increase the activity of the lineage limited transcription factor NF-E2. The divergent effects of hemin and TPA on gene expression in K562 cells are mediated, in part, by their contrasting effects on the transcription factor NF-E2.


Input:
Transcriptional regulation of the pyruvate kinase erythroid-specific promoter.
Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does not bind TFIID. Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites indicate that only the association of CCACC/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter. Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells.

binds

produces

L-PK

expression

R-PK

by the alternative use of

produces

activity

L

erythroid promoter

drive

expression

transactivation

R-PK

promoter


Input:
Transcriptional regulation of interleukin 3 (IL3) in primary human T lymphocytes. Role of AP-1- and octamer-binding proteins in control of IL3 gene expression.
We have investigated the molecular and biochemical basis for activation of interleukin 3 (IL3) gene expression in primary human T lymphocytes following CD3 and CD2 receptor stimulation or activation by phytohemagglutinin plus phorbol 12-myristate 13-acetate. Using transfection and reporter gene assays specifically designed for primary T lymphocytes in conjunction with gel retardation assays, Western blot analyses and UV cross-linking studies, we found that c-Jun, c-Fos, and octamer-binding proteins play a major role in transcriptional activation of the IL3 gene via their interaction with two specific regions contained within the IL3 5'-flanking sequence. Additionally, the region between bases -107 and -59 of the IL3 promoter containing putative AP-2 and Sp1 binding motifs appears necessary for basal level expression of the IL3 gene. The data also indicate that CD2 receptor activation and phytohemagglutinin plus phorbol 12-myristate 13-acetate stimulation augment T cell IL3 gene expression through the same cis- and trans-activating signals. These results should contribute to a better understanding of the regulation of IL3 gene expression in human T lymphocytes.

interaction

c-Jun

interaction

c-Fos

expression

expression

expression

expression

expression

activation

expression

stimulation

activation

expression

activation

activation

expression

stimulation

activation

phytohemagglutinin

play a major role

activation

interaction

play a major role

activation

activation

transcriptional

necessary

expression

activation

augment

expression

regulation

Transcriptional

Role

control

control

expression

regulation

expression

Transcriptional

transcriptional


Input:
Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes.
We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

expression

zeta 2 globin

Transcriptional activation

zeta 2 globin

promoter

behaved as an authentic enhancer

zeta 2 globin

promoter

dispensable

expression

required

mediated

mediated

activity

activation

zeta 2 globin

promoter

activation

transcriptional

mediated

activation

activity

zeta 2 globin

promoter

formed the functional core

activation

transcriptional

zeta 2 globin

transcriptional

alpha globin


Input:
Costimulation of peripheral blood T cell activation by human endothelial cells. Enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1.
Endothelial cells (EC) act as APC for resting PBL in vitro, and may have important roles in vivo in the pathogenesis of allograft rejection and delayed hypersensitivity. We previously reported that human umbilical vein EC provide costimulatory signals to PHA-stimulated PBL via CD2:lymphocyte function-associated Ag-3 and an unidentified ligand pair, resulting in a three- to eight-fold enhancement of IL-2 production. The physiologic relevance of this increase was demonstrated by the proliferative advantage provided by EC to PBL suboptimally stimulated with mAb OKT3. We now report that EC costimulation causes increased levels of IL-2 mRNA as a result of increased IL-2 transcription in PBL. We therefore examined the effects of EC on T cell nuclear factors known to regulate IL-2 transcription, including c-jun and c-fos-two components of the transcription factor AP-1, NFAT, and others. PBL constitutively express c-jun transcripts, and the level of c-jun mRNA is not altered by PHA activation in the absence or presence of EC. In contrast, c-fos mRNA is absent from resting T cells and is induced on PHA activation. EC alone do not induce c-fos mRNA but augment the level of c-fos mRNA in PHA-activated T cells by 3- to 10-fold. This effect is largely independent of the CD2:lymphocyte function-associated Ag-3 pathway. Gel-shift analysis reveals the constitutive presence of nuclear factors in resting PBL that bind to the proximal AP-1 site of the IL-2 promoter and that contain immunoreactive c-Jun but not c-Fos protein. In contrast, AP-1 from PHA-activated cells contains c-Jun and low levels of c-Fos. Strikingly, costimulation with EC results in a dramatic increase (up to 15-fold) in the c-Fos content of AP-1. Levels of other nuclear factors involved in IL-2 regulation were not altered by EC, although NFAT-DNA complexes migrated at a slightly different mobility. In summary, our data suggest that changes in the composition of transcription factor AP-1 is a key molecular mechanism for increasing IL-2 transcription and may underlie the phenomenon of costimulation by EC.

content of AP-1

lymphocyte function-associated Ag-3

lymphocyte function-associated Ag-3

pair

bind

proximal AP-1 site

production

express

c-jun

Enhanced

transcription

provide costimulatory signals

provide costimulatory signals

pair

enhancement

production

causes

increased

increased

increased

increased

transcription

induced

c-fos

induce

c-fos

augment

c-fos

increasing

transcription

effects

c-fos

effects

c-jun

regulate

transcription

c-fos

regulate

transcription

c-jun

altered

c-jun

involved

regulation

regulation

is a key molecular mechanism

increasing

transcription

transcription

transcription

transcription

absent

c-fos

transcription


Input:
Negative transcriptional regulation of human interleukin 2 (IL-2) gene by glucocorticoids through interference with nuclear transcription factors AP-1 and NF-AT.
IL-2 gene transcription is affected by several nuclear proteins. We asked whether dexamethasone (Dex) and cyclosporin A (CsA) inhibit IL-2 gene transcription by interfering with the activity of nuclear proteins that bind to the IL-2 promoter. Nuclear extracts from primary human T lymphocytes were analyzed by electrophoretic DNA mobility shift assays. Both Dex and CsA inhibited the binding of transcription factors AP-1 and NF-AT, but not of NF-kB and OCT-1/OAF, to their corresponding sites on the IL-2 gene promoter. To correlate changes in nuclear factor binding in vitro with transcriptional activity in vivo and define the structural requirements for IL-2 promoter repression, we used transient DNA transfections. Jurkat cells were transfected with plasmids containing either the intact IL-2 promoter or its AP-1, NF-AT, and NF-kB motifs. Dex inhibited the IL-2 promoter and the AP-1, but not the NF-AT and NF-kB plasmids. In contrast, CsA inhibited the IL-2 promoter and the NF-AT, but not the AP-1 and NF-kB plasmids. These results suggest that in human T lymphocytes both Dex and CsA inhibited IL-2 gene transcription through interference with transcription factors AP-1 and NF-AT. We propose that, while maximum inhibition may involve interaction with both transcription factors, AP-1 is the primary target of Dex.

bind

promoter

binding

their corresponding sites

Negative transcriptional regulation

inhibit

transcription

inhibited

binding

repression

promoter

inhibited

inhibited

inhibited

transcription

inhibition

transcription

requirements

repression

involve

inhibition

affected

transcription

transcription

transcription

transcription