Direct Gene Activation Research Articles

Sex hormones, anti-hormones and adenocarcinoma of the endometrium

Endometrioid adenocarcinoma of the endometrium is a hormone-dependent disease. Estrogens act, after binding to the estrogen receptor, as transcription factors with direct activation of various cell regulatory genes that induce mitosis and thus proliferation. Unopposed, uninterrupted estrogen stimulation results clinically in endometrial proliferation, increased ultrasonographic Double-Layer Endothelial Thickness (DET), hyperplasia and bleeding. An incessant high mitotic activity leads to accumulation of DNA replication errors, which, when not repaired, could result in a malignant phenotype, atypical hyperplasia and invasive cancer. Therefore, in postmenopausal women endometrial neoplasia should be prevented by adequate opposition through the addition of a progestagen, daily or at least 12 days each month. In premenopausal women, with a desire for future childbearing, progestogen based reversion of hyperplasia, and even early cancer, can be obtained in a substantial proportion of patients. In the future, new anti-estrogens like fulvestrant could play a role in the non-surgical treatment of endometrial cancer, in those situations where surgery is precluded.

Direct and indirect transcriptional activation of virulence genes by an AraC‐like protein, PerA from enteropathogenic Escherichia coli

The plasmid-encoded Per regulatory locus of enteropathogenic Escherichia coli (EPEC) is generally considered to consist of three genes, perA, perB and perC. PerA, a member of the AraC-like family of transcriptional regulators, is known to be an activator of its own promoter (autoactivation) as well as of the plasmid-located bfp operon encoding bundle-forming pili, but its role in activation of the chromosomal locus of enterocyte effacement (LEE) pathogenicity island, which confers the property of intimate adherence on EPEC, requires clarification. Here, we show that PerA is also required for activation of the master regulatory LEE operon, LEE1, but that this activation is indirect, being achieved via autoactivation of the per promoter which ensures sufficient production of the PerC protein to activate LEE1. In contrast, PerA-dependent activation of the per and bfp promoters is direct and does not require the other Per proteins, but is modulated by the nucleoid-associated protein H-NS. The closely related VirF regulator from Shigella flexneri cannot substitute for PerA to activate these promoters, despite being able to bind their upstream regions in vitro. PerA can bind the per and bfp promoter fragments to form multiple complexes, while VirF forms only a single complex. Site-directed mutagenesis of the PerA protein suggests that, like VirF, it may use both of its carboxy-terminal helix-turn-helix motifs for DNA interaction, and may also make direct contacts with RNA polymerase. In addition, we have isolated mutations in the poorly characterized amino-terminal domain of PerA which affect its ability to activate gene expression.

Activation of REST/NRSF target genes in neural stem cells is sufficient to cause neuronal differentiation.

REST/NRSF is a transcriptional repressor that acts at the terminal stage of the neuronal differentiation pathway and blocks the transcription of several differentiation genes. REST/NRSF is generally downregulated during induction of neuronal differentiation. The recombinant transcription factor REST-VP16 binds to the same DNA binding site as does REST/NRSF but functions as an activator instead of a repressor and can directly activate the transcription of REST/NRSF target genes. However, it is not known whether REST-VP16 expression is sufficient to cause formation of functional neurons from neural stem cells (NSCs). Here we show that regulated expression of REST-VP16 in a physiologically relevant NSC line growing under cycling conditions converted the cells rapidly to the mature neuronal phenotype. Furthermore, when grown in the presence of retinoic acid, REST-VP16-expressing NSCs activated their target, as well as other differentiation genes that are not their direct target, converting them to the mature neuronal phenotype and enabling them to survive in the presence of mitotic inhibitors, which is a characteristic of mature neurons. In addition, these neuronal cells were physiologically active. These results showed that direct activation of REST/NRSF target genes in NSCs with a single transgene, REST-VP16, is sufficient to cause neuronal differentiation, and the findings suggested that direct activation of genes involved in the terminal stage of differentiation may cause neuronal differentiation of NSCs.

Perp Is a Mediator of p53-Dependent Apoptosis in Diverse Cell Types

The induction of apoptosis by the p53 protein is critical for its activity as a tumor suppressor [1]. Although it is clear that p53 induces apoptosis at least in part by direct transcriptional activation of target genes, the set of p53 target genes that mediate p53 function in apoptosis in vivo remains to be well defined. The Perp (p53 apoptosis effector related to PMP-22) gene is highly expressed in cells undergoing p53-dependent apoptosis as compared to cells undergoing p53-dependent G1 arrest [2]. Perp is a direct p53 target, and its overexpression is sufficient to induce cell death in fibroblasts, implicating it as an important component of p53 apoptotic function. Here, through the generation of Perp-deficient mice, we analyze the role of Perp in the p53 apoptosis pathway in multiple primary cell types by comparing the cell death responses of Perp null cells to those of wild-type and p53 null cells. These experiments demonstrate the involvement of Perp in p53-mediated cell death in thymocytes and neurons but not in E1A-expressing MEFs, indicating a cell type-specific role for Perp in the p53 cell death pathway. In addition, we show that Perp is not required for proliferation-associated functions of p53. Thus, Perp selectively mediates the p53 apoptotic response, and the requirement for Perp is dictated by cellular context.

The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB.

RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNA(Leu) genes in cells decreases when ERK is inactivated. Furthermore, mutation of an ERK docking domain or phosphoacceptor site in BRF1 prevents serum induction of pol III transcription. These data identify a novel target for ERK, and suggest that its ability to stimulate biosynthetic capacity and growth involves direct transcriptional activation of tRNA and 5S rRNA genes.

MTA3, a Mi-2/NuRD Complex Subunit, Regulates an Invasive Growth Pathway in Breast Cancer

Estrogen receptor is a key regulator of proliferation and differentiation in mammary epithelia and represents a crucial prognostic indicator and therapeutic target in breast cancer. Mechanistically, estrogen receptor induces changes in gene expression through direct gene activation and also through the biological functions of target loci. Here, we identify the product of human MTA3 as an estrogen-dependent component of the Mi-2/NuRD transcriptional corepressor in breast epithelial cells and demonstrate that MTA3 constitutes a key component of an estrogen-dependent pathway regulating growth and differentiation. The absence of estrogen receptor or of MTA3 leads to aberrant expression of the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transitions. Aberrant Snail expression results in loss of expression of the cell adhesion molecule E-cadherin, an event associated with changes in epithelial architecture and invasive growth. These results establish a mechanistic link between estrogen receptor status and invasive growth of breast cancers.

Regulation of APETALA3 floral homeotic gene expression by meristem identity genes.

The Arabidopsis APETALA3 (AP3) floral homeotic gene is required for specifying petal and stamen identities, and is expressed in a spatially limited domain of cells in the floral meristem that will give rise to these organs. Here we show that the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) are required for the activation of AP3. The LFY transcription factor binds to a sequence, with dyad symmetry, that lies within a region of the AP3 promoter required for early expression of AP3. Mutation of this region abolishes LFY binding in vitro and in yeast one hybrid assays, but has no obvious effect on AP3 expression in planta. Experiments using a steroid-inducible form of LFY show that, in contrast to its direct transcriptional activation of other floral homeotic genes, LFY acts in both a direct and an indirect manner to regulate AP3 expression. This LFY-induced expression of AP3 depends in part on the function of the APETALA1 (AP1) floral homeotic gene, since mutations in AP1 reduce LFY-dependent induction of AP3 expression. LFY therefore appears to act through several pathways, one of which is dependent on AP1 activity, to regulate AP3 expression.

Regulation of Neuronal Survival and Death by E2F-Dependent Gene Repression and Derepression

Neuronal death induced by a variety of means requires participation of the E2F family of transcription factors. Here, we show that E2F acts as a gene silencer in neurons and that repression of E2F-responsive genes is required for neuronal survival. Moreover, neuronal death evoked by DNA damaging agents or trophic factor withdrawal is characterized by derepression of E2F-responsive genes. Such derepression, rather than direct E2F-promoted gene activation, is required for death. Among the genes that are derepressed in neurons subjected to DNA damage or trophic factor withdrawal are the transcription factors B- and C-myb. Overexpression of B- and C-myb is sufficient to evoke neuronal death. These findings support a model in which E2F-dependent gene repression and derepression play pivotal roles in neuronal survival and death, respectively.

The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis.

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. We find that during embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk; shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. We find that Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo.

Targeting the targets of Type III effector proteins secreted by phytopathogenic bacteria

Targeting the targets of Type III effector proteins secreted by phytopathogenic bacteria

A model Notch response element detects Suppressor of Hairless-dependent molecular switch.

Cell-cell signaling mediated by Notch is critical during many different developmental processes for the specification or restriction of cell fates. Currently, the only known transduction pathway involves a DNA binding protein, Suppressor of Hairless [Su(H)] in Drosophila and CBF1 in mammals, and results in the direct activation of target genes. It has been proposed that in the absence of Notch, Su(H)/CBF1 acts as a repressor and is converted into an activator through interactions with the Notch intracellular domain [1--4]. Recently, we have also suggested that the activation of specific target genes requires synergy between Su(H) and other transcriptional activators [5]. Here we have designed an assay that allows us to directly test these hypotheses in vivo. Our results clearly demonstrate that Su(H) is able to function as the core of a molecular switch, repressing transcription in the absence of Notch and activating in the presence of Notch. In its capacity as an activator, Su(H) can cooperate synergistically with a DNA-bound transcription factor, Grainyhead. These interactions indicate a simple model for Notch target-gene regulation that could explain the precision of gene activation elicited by Notch signaling in different developmental fate decisions.

Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.

Neurological disorders develop in most people infected with human immunodeficiency virus type 1 (HIV-1). However, the underlying mechanisms remain largely unknown. Here we report that binding of HIV-1 transactivator (Tat) protein to low-density lipoprotein receptor-related protein (LRP) promoted efficient uptake of Tat into neurons. LRP-mediated uptake of Tat was followed by translocation to the neuronal nucleus. Furthermore, the binding of Tat to LRP resulted in substantial inhibition of neuronal binding, uptake and degradation of physiological ligands for LRP, including alpha2-macroglobulin, apolipoprotein E4, amyloid precursor protein and amyloid beta-protein. In a model of macaques infected with a chimeric strain of simian-human immunodeficiency virus, increased staining of amyloid precursor protein was associated with Tat expression in the brains of simian-human immunodeficiency virus-infected macaques with encephalitis. These results indicate that HIV-1 Tat may mediate HIV-1-induced neuropathology through a pathway involving disruption of the metabolic balance of LRP ligands and direct activation of neuronal genes.

Looping versus linking: toward a model for long-distance gene activation.

Locus control regions (LCRs) are defined by their ability, in transgenic assays, to direct high-level, tissue-specific expression of linked genes at all sites of integration examined and at moderately constant levels per gene copy. The most extensively examined LCR is that associated with the b-globin locus in mammals, where the b-globin genes reside in a linear array and are usually arranged in order of their developmental expression (Fig. 1). The b-globin LCR consists of several DNase I hypersensitive sites (HSs) spread over a region of 20–30 kb; the DNA sequence associated with each of the HSs contains numerous binding sites for erythroid-specific and ubiquitous transcription factors (for review, see Grosveld et al. 1993; Orkin 1995; Martin et al. 1996; Hardison et al. 1997). Expression of stably integrated b-globin transgenes in the absence of the LCR occurs only at low levels and varies depending upon the site of integration, and so is said to be subject to position effects. The high level of expression driven by the LCR at ectopic sites appears to be the product of at least two separable activities, namely the establishment of an ‘open’ chromatin domain and direct gene activation. Gene activation is also a property of enhancers, which are defined by their ability to direct high-level expression of linked genes in transient transfection assays. Although enhancers typically also improve the expression of transgenes integrated into the genome, such expression varies among integration sites due to negative position effects. Thus, unlike LCRs, enhancers are only capable of gene activation at a subset of genomic loci. Presumably, LCRs subsume the function of enhancers along with a more dominant chromatin ‘opening’ activity that can override negative effects from neighboring regions. In the case of the b-globin LCR, 58 HS2 is known to act as an enhancer in transient transfection assays, but elements in addition to HS2 are required for LCR activity in single-copy transgenes (Ellis et al. 1993, 1996). Evidence from one study indicates that an otherwise intact human b-globin locus translocated near centromeric heterochromatin may be subject to a position effect (Rees et al. 1994). Thus, the distinction between enhancers and LCRs may only be one of degree, but in most transgenic assays is still measurable. Given the apparent importance of LCRs in the regulation of many tissue-specific genes, the mechanism by which these elements function has been the subject of a great deal of debate. The currently predominant model for LCR function involves the establishment of an open chromatin domain encompassing the regulated genes, with subsequent gene activation accomplished by direct interactions between elements of the LCR and gene promoters by DNA looping. In this review, we will summarize models for the establishment of active chromatin domains by LCRs and describe in detail the evidence for the looping model of long-distance gene activation. Recent data, especially relating to the function of boundary elements and evidence for facilitators of enhancer and LCR function, indicate that simple LCR–promoter interactions are unlikely. Based on these data, we propose an alternative to the looping model, which we term the linking model.

DPP on the brinker

Decapentaplegic (DPP) is a member of the TGFβ family of signalling molecules that is implicated in patterning many structures, including the wing of Drosophila. In the wing, three target genes of DPP have been identified. These are expressed at different positions relative to the source of DPP, consistent with regulation in a concentration-dependent manner. Target-gene expression depends on the receptor, Thick veins (TKV), and the positively acting Mad/Medea transcription factors. Two recent papers add another wrinkle to the story in the form of a putative transcriptional repressor, Brinker, that is negatively regulated by DPP. brinker was identified by Jazwinski et al.1 Jazwinska A. et al. The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell. 1999; 96: 563-573 Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar in a screen for embryonic lethal mutations because it caused a phenotype similar to ectopic activation of DPP signalling. Campbell and Tomlinson 2 Campbell G. Tomlinson A. Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell. 1999; 96: 553-562 Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar identified the same gene through an enhancer-trap line that was expressed in a DPP-responsive manner in the developing wing. Both groups observed that clones of cells mutant for brinker resulted in phenotypes characteristic of ectopic DPP signalling. The phenotypes were autonomous to the mutant clone, showing that brinker did not affect production of DPP. Furthermore, ectopic expression of DPP target-genes was induced even when the brinker mutation was combined with mutations that prevented transduction of the DPP signal. This suggested that brinker functions to inhibit the DPP target genes directly. As brinker expression is negatively regulated by DPP signalling, one model that emerges is that DPP induces expression of its target-genes by blocking transcription of brinker. The fact that the phenotypes associated with brinker mutant cells are characteristic of those produced by low, rather than high, levels of DPP activity suggests that repression of brinker is only sufficient to confer effects of intermediate DPP levels. Direct activation of target genes via Mad/Medea is probably required for maximal effects. The amino-acid sequence of Brinker does not reveal homology to known repressors so future experiments will need to determine whether it indeed functions as a DNA-binding transcriptional repressor. In the meantime, a search for vertebrate homologues will almost certainly begin: given the conservation between DPP and TGFβ signalling in other organisms it seems highly likely that brinker relatives will operate elsewhere.

A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors

Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain of multiple nuclear receptors and with the p160 nuclear receptor coactivators, which upon cloning have proven to be variants of the SRC-1 protein. Because CBP represents a common factor, required in addition to distinct coactivators for function of nuclear receptors, CREB, and AP-1, we suggest that CBP/p300 serves as an integrator of multiple signal transduction pathways within the nucleus.

Overexpression of estrogen receptor in HTB 96 human osteosarcoma cells results in estrogen-induced growth inhibition and receptor cross talk

Estrogenic effects on the proliferation and differentiated cellular functions of bone cells have been described in vivo and in vitro. In particular, stimulatory effects on the growth rate of osteoblasts have been observed, although these are generally small. In an attempt to produce a more sensitive model for the study of estrogen action in bone, HTB 96 human osteoblast-like osteosarcoma cells, which lack endogenous estrogen receptor (ER), were stably transfected with an expression vector coding for the human ER gene. Several HTB 96 sublines expressing ER protein, detected by ligand binding and immunoassay, were isolated. The ability of 17 beta-estradiol (E2) to induce chloramphenicol acetyltransferase (CAT) activity from a cotransfected reporter vector containing the CAT gene linked to the Xenopus vitellogenin A2 gene estrogen response element demonstrated that the expressed ER was functional. ER continued to be expressed over a 30 week culture period. E2 but not other steroids significantly reduced growth rates and produced an altered morphology in HTB 96 sublines expressing higher levels of ER. The antiestrogen 4-hydroxytamoxifen partially reversed the E2 effect on growth rate. Transient transfection of cells expressing ER with a vector containing the CAT gene linked to the mouse mammary tumor virus long terminal repeat sequence, which contains response elements for the glucocorticoid receptor but not the ER, showed that E2 was able to inhibit CAT induction by dexamethasone. This result suggest that in ER-transfected HTB 9 cells the effects of E2 may result not from direct activation of endogenous genes but instead by transcriptional interference.(ABSTRACT TRUNCATED AT 250 WORDS)

Decidual cell biosynthesis of interleukin-6: regulation by inflammatory cytokines

Intrauterine infection is an important cause of preterm labor and delivery and is characterized by increased production of inflammatory cytokines by gestational tissues. We have evaluated the biosynthesis of the inflammatory cytokine, interleukin-6 (IL-6), by human decidua and its regulation by other cytokines essential to the inflammatory process. We found that decidual cells secrete small amounts of IL-6 in the presence of growth medium supplemented only with 10% fetal calf serum. Interleukin 1 (alpha and beta) and tumor necrosis factor (TNF) all induced a significant concentration-dependent stimulation of IL-6 production by decidual cells. Treatment of decidual cells with actinomycin D or cycloheximide abrogated the increase in IL-6 production induced by IL-1 beta. Northern blot analysis of cultured decidual cells revealed an increase in IL-6 messenger RNA (mRNA) over time in response to IL-1 beta. These data indicate that IL-1 beta stimulates an increase in IL-6 mRNA and protein production, reflecting either direct gene activation or stabilization of IL-6 mRNA. The concentration range tested (0.1 to 10 ng/mL) of each cytokine is within the range of values found in the amniotic fluid of women destined to deliver preterm due to infection of gestational tissues. Our data suggest that IL-6 is produced by human decidua in response to inflammation and, in conjunction with other inflammatory mediators, may play a role in the pathophysiology of preterm labor due to infection.

Decidual cell biosynthesis of interleukin-6: regulation by inflammatory cytokines.

Intrauterine infection is an important cause of preterm labor and delivery and is characterized by increased production of inflammatory cytokines by gestational tissues. We have evaluated the biosynthesis of the inflammatory cytokine, interleukin-6 (IL-6), by human decidua and its regulation by other cytokines essential to the inflammatory process. We found that decidual cells secrete small amounts of IL-6 in the presence of growth medium supplemented only with 10% fetal calf serum. Interleukin 1 (alpha and beta) and tumor necrosis factor (TNF) all induced a significant concentration-dependent stimulation of IL-6 production by decidual cells. Treatment of decidual cells with actinomycin D or cycloheximide abrogated the increase in IL-6 production induced by IL-1 beta. Northern blot analysis of cultured decidual cells revealed an increase in IL-6 messenger RNA (mRNA) over time in response to IL-1 beta. These data indicate that IL-1 beta stimulates an increase in IL-6 mRNA and protein production, reflecting either direct gene activation or stabilization of IL-6 mRNA. The concentration range tested (0.1 to 10 ng/mL) of each cytokine is within the range of values found in the amniotic fluid of women destined to deliver preterm due to infection of gestational tissues. Our data suggest that IL-6 is produced by human decidua in response to inflammation and, in conjunction with other inflammatory mediators, may play a role in the pathophysiology of preterm labor due to infection.

Studies in the in vivo expression of the influenza resistance gene Mx by in-situ hybridisation

The inbred laboratory mouse strain A2G carries a functional, interferon type 1 inducible gene, Mx which upon expression confers specific resistance to an otherwise lethal dose of influenza virus. We investigated in vivo Mx gene expression by performing Northern hybridisation and in-situ hybridisation on A2G (Mx+) and (CBA/J x C57)F1 (Mx-) mice that were induced either with human, natural interferon; human, recombinant interferon or double stranded poly(I):(C). All 3 inducers were able to stimulate Mx expression in all organs examined in the A2G strain. However, contrary to previous reports, Mx expression was confined to a small number of cell types; the main contributor was most probably mononuclear cells. Specialised cells such as hepatocyte, nephron, ovarian follicle and seminiferous tubules did not show detectable Mx level. There was also constitutive Mx expression in the epithelia of uterus and duodenum which suggested direct gene activation independent of blood-bourne interferon.

Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone.

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.