Transcription Of Endogenous Genes Research Articles

Short chain fatty acids induce TH gene expression via ERK-dependent phosphorylation of CREB protein

Butyrate modulates specific gene expression through various second-messenger signal transduction systems including activation of the PKA/cAMP pathway (Decastro, M., Nankova, B.B., Shah, P., Patel, P., Mally, P.V., Mishra, R., La Gamma, E.F., 2005. Short chain fatty acids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signaling pathway, Brain Res. Mol. Brain Res. 142 28–38; Mally, P., Mishra, R., Gandhi, S., Decastro, M.H., Nankova, B.B., Lagamma, E.F., 2004. Stereospecific regulation of tyrosine hydroxylase and proenkephalin genes by short-chain fatty acids in rat PC12 cells, Pediatr. Res. 55 847–854). In the current report, we provide additional evidence that exposure to butyrate causes a rapid activation of the MAP kinase pathway, associated with increased phosphorylation of CREB. Under these conditions, no changes in relative amounts of CREB protein were observed by Western blot. Pre-treatment with the MAPK specific inhibitor (U0126) or the adenylate cyclase inhibitor dideoxyadenosine (ddA) abolished the butyrate-induced: (i) accumulation of TH mRNA, (ii) the phosphorylation of ERK1/2 as well as (iii) CREB phosphorylation. PC12 cells transfected with a TH promoter-luciferase reporter gene showed a robust induction in response to butyrate that was significantly reduced after co-transfection of either of two dominant-negative CREB expression vectors. Nuclear run-on assays demonstrated that butyrate increases endogenous TH gene transcription. We conclude that the initial steps of butyrate-induced gene activation are mediated through the CREB/CREB family of transcription factors which are coupled to both the MAP kinase and cAMP-dependent second messenger systems. Our data delineate a molecular mechanism through which short chain fatty acid's, their related drug-congeners (e.g., valproate) or even diet-derived butyrate (from fermentation of carbohydrates in the gut) can in principle, modulate brain catecholaminergic systems by modifying TH gene expression, dopaminergic levels and the corresponding animal behavior. These molecular relationships also offer a plausible explanation of how the well-recognized clinical effects of ketogenic diets can alter human behavior via the same central mechanisms.

STABILIZED1, a Stress-Upregulated Nuclear Protein, Is Required for Pre-mRNA Splicing, mRNA Turnover, and Stress Tolerance in Arabidopsis

In plants, many gene transcripts are very unstable, which is important for the tight control of their temporal and spatial expression patterns. To identify cellular factors controlling the stability of unstable mRNAs in plants, we used luciferase imaging in Arabidopsis thaliana to isolate a recessive mutant, stabilized1-1 (sta1-1), with enhanced stability of the normally unstable luciferase transcript. The sta1-1 mutation also causes the stabilization of some endogenous gene transcripts and has a range of developmental and stress response phenotypes. STA1 encodes a nuclear protein similar to the human U5 small ribonucleoprotein-associated 102-kD protein and to the yeast pre-mRNA splicing factors Prp1p and Prp6p. STA1 expression is upregulated by cold stress, and the sta1-1 mutant is defective in the splicing of the cold-induced COR15A gene. Our results show that STA1 is a pre-mRNA splicing factor required not only for splicing but also for the turnover of unstable transcripts and that it has an important role in plant responses to abiotic stresses.

Differential Use of Functional Domains by Coiled-coil Coactivator in Its Synergistic Coactivator Function with β-Catenin or GRIP1

beta-Catenin, a pivotal component of the Wnt-signaling pathway, binds to and serves as a transcriptional coactivator for the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcriptional activator proteins and for the androgen receptor (AR), a nuclear receptor. Three components of the p160 nuclear receptor coactivator complex, including CARM1, p300/CBP, and GRIP1 (one of the p160 coactivators), bind to and cooperate with beta-catenin to enhance transcriptional activation by TCF/LEF and AR. Here we report that another component of the p160 nuclear receptor coactivator complex, the coiled-coil coactivator (CoCoA), directly binds to and cooperates synergistically with beta-catenin as a coactivator for AR and TCF/LEF. CoCoA uses different domains to bind GRIP1 and beta-catenin, and it uses different domains to transmit the activating signal to the transcription machinery, depending on whether it is bound to GRIP1 or beta-catenin. CoCoA associated specifically with the promoters of transiently transfected and endogenous target genes of TCF/LEF, and reduction of the endogenous CoCoA level decreased the ability of TCF/LEF and beta-catenin to activate transcription of transient and endogenous target genes. Thus, CoCoA uses different combinations of functional domains to serve as a physiologically relevant component of the Wnt/beta-catenin signaling pathway and the androgen signaling pathway.

A histone deacetylation-dependent mechanism for transcriptional repression of the gap junction gene cx43 in prostate cancer cells

The connexin 43 gene (cx43, GJA1) mediates gap junctional intercellular communication (GJIC), which regulates tissue homeostasis. cx43 is frequently downregulated in prostate cancer. We investigated the role of a histone deacetylase (HDAC)-dependent mechanism in the transcriptional repression of cx43 in a panel of prostate cancer cells. The impact of Trichostatin A (TSA), an inhibitor of HDAC, on exogenous and endogenous cx43 gene transcription was examined by the luciferase assay, Northern blot, nuclear run-on, Western blot, and chromatin immunoprecipitation assays. Trichostatin A induces transcription of cx43 gene and GJIC. The co-activator p300/CBP synergizes with TSA for cx43 promoter activation. We identified a promoter region where cooperation between Ap1 and Sp1 elements was essential for TSA-induced cx43 transcription. TSA increased the level of hyperacetylated histones bound to cx43 promoter. Our results highlight the potential utility of inhibitors of HDAC to restore cx43 gene expression in prostate cancer.

A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria

Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1, encoded by the 60-member var gene family, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

NIR is a novel INHAT repressor that modulates the transcriptional activity of p53

Most transcriptional repression pathways depend on the targeted deacetylation of histone tails. In this report, we characterize NIR, a novel transcriptional corepressor with inhibitor of histone acetyltransferase (INHAT) activity. NIR (Novel INHAT Repressor) is ubiquitously expressed throughout embryonic development and adulthood. NIR is a potent transcriptional corepressor that is not blocked by histone deacetylase inhibitors and is capable of silencing both basal and activator-driven transcription. NIR directly binds to nucleosomes and core histones and prevents acetylation by histone acetyltransferases, thus acting as a bona fide INHAT. Using a tandem affinity purification approach, we identified the tumor suppressor p53 as a NIR-interacting partner. Association of p53 and NIR was verified in vitro and in vivo. Upon recruitment by p53, NIR represses transcription of both p53-dependent reporters and endogenous target genes. Knock-down of NIR by RNA interference significantly enhances histone acetylation at p53-regulated promoters. Moreover, p53-dependent apoptosis is robustly increased upon depletion of NIR. In summary, our findings describe NIR as a novel INHAT that plays an important role in the control of p53 function.

Estrogen Dendrimer Conjugates that Preferentially Activate Extranuclear, Nongenomic Versus Genomic Pathways of Estrogen Action

Estrogenic hormones are classically thought to exert their effects by binding to nuclear estrogen receptors and altering target gene transcription, but estrogens can also have nongenomic effects through rapid activation of membrane-initiated kinase cascades. The development of ligands that selectively activate only the nongenomic pathways would provide useful tools to investigate the significance of these pathways. We have prepared large, abiotic, nondegradable poly(amido)amine dendrimer macromolecules that are conjugated to multiple estrogen molecules through chemically robust linkages. Because of their charge and size, these estrogen-dendrimer conjugates (EDCs) remain outside the nucleus. They stimulate ERK, Shc, and Src phosphorylation in MCF-7 breast cancer cells at low concentrations, yet they are very ineffective in stimulating transcription of endogenous estrogen target genes, being approximately 10,000-fold less potent than estradiol in genomic actions. In contrast to estradiol, EDC was not effective in stimulating breast cancer cell proliferation. Because these EDC ligands activate nongenomic activity at concentrations at which they do not alter the transcription of estrogen target genes, they should be useful in studying extranuclear initiated pathways of estrogen action in a variety of target cells.

A Chromatin-associated and Transcriptionally Inactive p53-Mdm2 Complex Occurs in mdm2 SNP309 Homozygous Cells

In cancer cells, the function of the tumor suppressor protein p53 is usually blocked. Impairment of the p53 pathway results in tumor cells with endogenous overexpression of Mdm2 via a naturally occurring single nucleotide polymorphism (SNP) in the mdm2 gene at position 309. Here we report that in mdm2 SNP309 cells, inactivation of p53 results in a chromatin-associated Mdm2-p53 complex without clearance of p53 by protein degradation. Nuclear accumulation of p53 protein in mdm2 SNP309 cells results after 6 h of camptothecin, etoposide, or mitomycin C treatment, with the p53 protein phosphorylated at Ser15. Chromatin immunoprecipitation demonstrated p53 and Mdm2 bound to p53 responsive elements. Interestingly, although the p53 protein was able to bind to DNA, quantitative PCR showed compromised transcription of endogenous target genes. Additionally, exogenously introduced p53 was incapable of activating transcription from p53 responsive elements in SNP309 cells, confirming the trans-acting nature of the inhibitor. Inhibition of Mdm2 by siRNA resulted in transcriptional activation of these p53 targets. Our data suggest that overproduction of Mdm2, resulting from a naturally occurring SNP, inhibits chromatin-bound p53 from activating the transcription of its target genes.

Calcium-sensing Receptor Gene Transcription Is Up-regulated by the Proinflammatory Cytokine, Interleukin-1β: ROLE OF THE NF-κB PATHWAY AND κB ELEMENTS

The calcium-sensing receptor (CASR) in parathyroid, thyroid, and kidney is essential for calcium homeostasis. Hypocalcemia is common in critically ill patients having increased circulating proinflammatory cytokines, although the causes are unknown. We hypothesized that the cytokines increase CASR expression and reduce the set point for parathyroid hormone suppression by extracellular calcium, leading to hypocalcemia and hypoparathyroidism. Here, we show in vivo in the rat that parathyroid, thyroid, and kidney CASR mRNA and protein increased after injection of interleukin-1beta. This was associated with decreased circulating parathyroid hormone, calcium, and 1,25-dihydroxyvitamin D levels. Interleukin-1beta stimulated endogenous CASR gene transcripts and transfected promoter reporter activity in human thyroid C-cells (TT cells) and kidney proximal tubule (HKC) cells. Cotransfection of NF-kappaB proteins enhanced activity of the reporter constructs, whereas cotransfection with inhibitor-kappaB or application of an NF-kappaB nuclear localization sequence peptide abrogated responsiveness to cytokine or NF-kappaB proteins. Mutagenesis of some, but not all, of the potential kappaB elements in the 5' part of the CASR gene led to loss of responsiveness to cytokine. These elements conferred cytokine responsiveness to a heterologous promoter, and in electrophoretic mobility shift assays, NF-kappaB complexes formed on the same three kappaB elements. In summary, the CASR gene has several functional kappaB elements that mediate its upregulation by proinflammatory cytokines and probably contribute to altered extracellular calcium homeostasis in the critically ill.

Daxx Mediates the Small Ubiquitin-like Modifier-dependent Transcriptional Repression of Smad4

Daxx has been shown to function as an apoptosis regulator and transcriptional repressor via its interaction with various cytoplasmic and nuclear proteins. Here, we showed that Daxx interacts with Smad4 and represses its transcriptional activity via the C-terminal domain of Daxx. In vitro and in vivo interaction studies indicated that the binding of Smad4 to Daxx depends on Smad4 sumoylation. Substitution of Smad4 SUMO conjugation residue lysine 159, but not 113, to arginine not only disrupted Smad4-Daxx interaction but also relieved Daxx-elicited repression of Smad4 transcriptional activity. Furthermore, chromatin immunoprecipitation analyses revealed the recruitment of Daxx to an endogenous, Smad4-targeted promoter in a Lys(159) sumoylation-dependent manner. Finally, down-regulation of Daxx expression by RNA interference enhanced transforming growth factor beta-induced transcription of reporter and endogenous genes through a Smad4-dependent, but not K159R-Smad4-dependent, manner. Together, these results indicate that Daxx suppresses Smad4-mediated transcriptional activity by direct interaction with the sumoylated Smad4 and identify a novel role of Daxx in regulating transforming growth factor beta signaling.

P21WAF1/CIP1 Selectively Controls the Transcriptional Activity of Estrogen Receptor α

Estrogen receptors (ER) are ligand-dependent transcription factors that regulate growth, differentiation, and maintenance of cellular functions in a wide variety of tissues. We report here that p21WAF1/CIP1, a cyclin-dependent kinase (Cdk) inhibitor, cooperates with CBP to regulate the ERalpha-mediated transcription of endogenous target genes in a promoter-specific manner. The estrogen-induced expression of the progesterone receptor and WISP-2 mRNA transcripts in MCF-7 cells was enhanced by p21WAF1/CIP1, whereas that of the cyclin D1 mRNA was reduced and the pS2 mRNA was not affected. Chromatin immunoprecipitation assays revealed that p21WAF1/CIP1 was recruited simultaneously with ERalpha and CBP to the endogenous progesterone receptor gene promoter in an estrogen-dependent manner. Experiments in which the p21WAF1/CIP1 protein was knocked down by RNA interference showed that the induction of the expression of the gene encoding the progesterone receptor required p21WAF1/CIP1, in contrast with that of the cyclin D1 and pS2 genes. p21WAF1/CIP1 induced not only cell cycle arrest in breast cancer cells but also milk fat globule protein and lipid droplets, indicators of the differentiated phenotype, as well as cell flattening and increase of the volume of the cytoplasm. These results indicate that p21WAF1/CIP1, in addition to its Cdk-regulatory role, behaves as a transcriptional coactivator in a gene-specific manner implicated in cell differentiation.

A Proteomic Microarray Approach for Exploring Ligand-initiated Nuclear Hormone Receptor Pharmacology, Receptor Selectivity, and Heterodimer Functionality

Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist versus antagonist character of the ligand. Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo- and heterodimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally different coactivator proteins can be functionally classified and their affinity for NHRs can be quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore heterodimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.

Transcriptional Regulation of Mouse μ Opioid Receptor Gene: Sp3 Isoforms (M1, M2) Function as Repressors in Neuronal Cells to Regulate the μ Opioid Receptor Gene

The 5'-flanking region of the mouse mu opioid receptor (MOR) gene has two promoters, referred to as distal and proximal. MOR mRNA is predominantly initiated by the proximal promoter. Previously, several important cis-elements and trans-factors have been shown to play a functional role in the proximal promoter of the MOR gene. In this study, we defined another functional, negative regulatory element located in the -219- to -189-base pair (translational start site designed as +1) region of the proximal promoter. It is designated as the Sp binding sequence for its sequence homology to the consensus Sp binding element. Mutation of the Sp binding element led to a 100% increase of MOR promoter activity in MOR-positive cells (NMB cells), confirming the negative role of the Sp binding sequence. Surprisingly, electrophoretic mobility shift analysis and chromatin immunoprecipitation assays revealed that Sp3 and its isoforms (M1 and M2) were specifically bound to the Sp binding sequence. In cotransfection assays of Drosophila melanogaster SL2 cells using cDNA encoding Sp1, Sp3, and the M1 and M2 isoforms of Sp3, the M1 and M2 isoforms trans-repressed the MOR promoter, whereas Sp1 and Sp3 trans-activated the MOR promoter. Significantly, ectopic expression of the M1 and M2 isoforms of Sp3 led to repression of the endogenous MOR gene transcripts in NMB cells. These results suggest that the binding of the M1 and M2 isoforms of the Sp3 transcription factor to the Sp binding sequence may play a role in mouse MOR gene expression.

Analysis of an approach to oviduct-specific expression of modified chicken lysozyme genes

The -2.7 kb enhancer (E) element of the chicken lysozyme gene domain appears to govern expression of the gene in macrophages but not in oviduct tubular gland cells, the only other site of lysozyme expression. The ultimate goal of our research was to determine whether lysozyme domain variants could be developed that would mainly be expressed in the oviduct so that transgenic birds could be produced that would deposit exogenous protein in the egg white. Accordingly, precise mutations were made by poxvirus-mediated gene targeting in FEF/PU.1 and CCAAT/enhancer-binding protein (C/EBP) transcription factor binding sites in the -2.7 kb E of cloned copies of a specific lysozyme gene variant that includes a hydrophobic pentapeptide tail encoding sequence inserted immediately prior to the stop codon. This variant contains the entire lysozyme domain and is cloned in a lambda bacteriophage vector (lambdaDIILys-HT); the novel tail sequence enables distinction in cell-based expression systems between transcripts of the variant and those of the endogenous gene. These various lysozyme domain mutants, in bacteriophage vector form, were tested for expression in cultured chicken blastodermal cells cotransfected with plasmids encoding the transcription factors C/EBP and v-Myb. In the absence of these plasmids, barely detectable levels of endogenous lysozyme gene transcription resulted in the blastodermal cells. In the presence of the plasmids, however, transcripts of the endogenous gene could be detected as well as varying levels (as evaluated by quantitative real-time PCR) of transcripts of all of the lysozyme domain mutants. These results are discussed in the context of the known role and occurrence of various transcription factors involved in gene expression in differentiating macrophage cells. The ultimate test of expression of the variants in macrophages vs. oviduct cells will be to use them to produce transgenic birds.

Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins.

We describe a facile method to activate or repress transcription of endogenous genes in a quantitative and specific manner by treatment with designed regulatory proteins (DRPs), in which artificial transcription factors (ATFs) are fused to cell-penetrating peptides (CPPs). Penetration of DRPs into cells is mediated by an N-terminal CPP fused to a nuclear localization signal; a DNA-binding domain and a transactivation domain follow. The DNA-binding domain was targeted to the vascular endothelial growth factor (VEGF)-A gene. An agonist DRP was rapidly taken up by cells and transported to the nucleus; soon after, the cells began transcribing the gene and secreting VEGF-A protein in a dose-dependent manner. Multiple copies of a short oligopeptide derived from a minimal transactivation domain of human beta-catenin was stronger than VP-16. The SRDX domain from the plant transcription factor, SUPERMAN, changed the DRP to a hypoxia-induced antagonist of VEGF-A. DRPs combine many of the potential benefits of transgenes with those of recombinant proteins.

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

AbstractTwo upstream regions of the human urokinase (uPA) gene regulate its transcription: the minimal promoter (MP) and the enhancer element. The activity of the minimal promoter is essential for basal uPA transcription in prostate adenocarcinoma PC3 cells. Binding of a phosphorylated Sp1 transcription factor is, in turn, essential for the activity of the MP. Here we report that the Jun kinase (JNK) pathway is required for the basal activity of the MP and for the expression of the endogenous uPA gene in PC3 cells and for activated transcription in LNCaP cells. On the other hand, the p42/p44 mitogen-activated protein kinase (MAPK) pathway activates uPA gene expression through Sp1 phosphorylation in HeLa, LNCaP, and CCL39-derivative cells that do not typically express uPA in basal conditions. In HeLa cells the dominant-negative form of JNK interferes with the p42/p44 MAPK activation of the uPA-MP. The results suggest that the stress-activated protein kinase (SAPK)/JNK pathway plays an important role in the phosphorylation of Sp1, which, in turn, leads to basal or activated transcription from the uPA-MP element.

Differential action of polycyclic aromatic hydrocarbons on endogenous estrogen-responsive genes and on a transfected estrogen-responsive reporter in MCF-7 cells

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that have been extensively studied for multiple toxicological endpoints in both laboratory animals and humans. The purpose of this study was to investigate the estrogenicity of PAHs in the human breast cancer cell line MCF-7. We investigated 14 PAHs for their ability to bind either the estrogen receptor (ER) or the aryl hydrocarbon receptor (AhR) and to activate target gene expression. PAHs were tested in a human recombinant estrogen receptor (hrER) competitive binding assay, and in both an estrogen response element (ERE)- and xenobiotic response element (XRE)-mediated reporter gene assay. We used quantitative RT-PCR to examine selected PAHs that showed activity in the ERE reporter gene assay for their ability to upregulate estrogen-responsive genes HEM45, progesterone receptor, and pS2, and the aryl hydrocarbon-responsive CYP1A1 gene. None of the 14 PAHs bound the hrER, but five of the PAHs (anthracene, B[ a]A, chrysene, B[ b]F, and B[ a]P) induced ER-reporter activity. This activity was dependent on the metabolism of PAHs in MCF-7 cells via the AhR pathway, which resulted in the formation of metabolites that bound the ER. None of the five PAHs that induced the ER-reporter were found to upregulate estrogen-responsive genes, yet four of the five PAHs induced AhR-dependent CYP1A1 gene expression. In contrast, a metabolite of B[ a]P, 3′OH-B[ a]P, and a PCB metabolite, 4′OH-2,4,6-BP, did weakly upregulate all three estrogen-responsive genes. Data from these studies indicate that induction of ER-reporter activity alone does not necessarily parallel endogenous gene transcription, and that the reporter gene assay may detect interactions that are not functional in vivo.

Upregulation of the Catalytic Telomerase Subunit by the Transcription Factor ER81 and Oncogenic HER2/Neu, Ras, or Raf.

One hallmark of tumor formation is the transcriptional upregulation of human telomerase reverse transcriptase, hTERT, and the resultant induction of telomerase activity. However, little is presently understood about how hTERT is differentially activated in tumor cells versus normal somatic cells. Specifically, it is unclear if oncoproteins can directly elicit hTERT expression. To this end, we now show that three oncoproteins, HER2/Neu, Ras, and Raf, stimulate hTERT promoter activity via the ETS transcription factor ER81 and ERK mitogen-activated protein (MAP) kinases. Mutating ER81 binding sites in the hTERT promoter or suppression of ERK MAP kinase-dependent phosphorylation of ER81 rendered the hTERT promoter unresponsive to HER2/Neu. Further, expression of dominant-negative ER81 or inhibition of HER2/Neu significantly attenuated telomerase activity in HER2/Neu-overexpressing SKBR3 breast cancer cells. Moreover, HER2/Neu, Ras, and Raf collaborated with ER81 to enhance endogenous hTERT gene transcription and telomerase activity in hTERT-negative, nonimmortalized BJ foreskin fibroblasts. Accordingly, hTERT expression was increased in HER2/Neu-positive breast tumors and breast tumor cell lines relative to their HER2/Neu-negative counterparts. Collectively, our data elucidated a mechanism whereby three prominent oncoproteins, HER2/Neu, Ras, and Raf, may facilitate tumor formation by inducing hTERT expression in nonimmortalized cells via the transcription factor ER81.

Role of AP1/NFE2 binding sites in endogenous α-globin gene transcription

AbstractHigh-level α-globin expression depends on cis-acting regulatory sequences located far upstream of the α-globin cluster. Sequences that contain the α-globin positive regulatory element (PRE) activate α-globin expression in transgenic mice. The α-globin PRE contains a pair of composite binding sites for the transcription factors activating protein 1 and nuclear factor erythroid 2 (AP1/NFE2). To determine the role of these binding sites in α-globin gene transcription, we mutated the AP1/NFE2 sites in the α-globin PRE in mice. We replaced the AP1/NFE2 sites with a neomycin resistance gene (neo) that is flanked by LoxP sites (floxed). Mice with this mutation exhibited increased embryonic death and α-thalassemia intermedia. Next, we removed theneo gene by Cre-mediated recombination, leaving a single LoxP site in place of the AP1/NFE2 sites. These mice were phenotypically normal. However, α-globin expression, measured by allele-specific RNA polymerase chain reaction (PCR), was decreased 25%. We examined the role of the hematopoietic-restricted transcription factor p45Nfe2 in activating expression through these sites and found that it is not required. Thus, we have demonstrated that AP1/NFE2 binding sites in the murine α-globin PRE contribute to long-range α-globin gene activation. The proteins that mediate this effect remain to be determined. (Blood. 2003;102:4223-4228)

Liver Receptor Homolog 1 Controls the Expression of Carboxyl Ester Lipase

The orphan nuclear receptor liver receptor homolog 1 (LRH-1) plays a central role in cholesterol homeostasis by regulating a number of hepatic and intestinal genes critical for reverse cholesterol transport and bile acid homeostasis. Herein, we describe the identification of carboxyl ester lipase (CEL) as a novel target of LRH-1 in pancreas, a tissue in which LRH-1 is abundantly expressed. In situ hybridization and gene expression studies demonstrate that both LRH-1 and CEL are co-expressed and confined to the exocrine pancreas. LRH-1 interacts with a consensus LRH-1 response element in the human CEL promoter, which is perfectly conserved in the rat gene, and induces CEL promoter activity in cotransfection assays. As reported for other LRH-1 target genes, the nuclear receptor short heterodimer partner represses LRH-1-induced CEL promoter activity. Chromatin immunoprecipitation demonstrates that binding of LRH-1 to the CEL promoter increases histone H4 acetylation corresponding with the activation of endogenous CEL gene transcription. Our data, identifying CEL as the first pancreatic LRH-1 target gene, indicate that LRH-1 is an important player in enterohepatic cholesterol homeostasis.