Mechanism Of Transcriptional Activation Research Articles

Abstract 4978: FOXP3 up-regulates p21 expression in breast cancer: Evidence for tumor suppressor

Abstract FOXP3 is the first X-linked breast cancer suppressor gene that works at least in part by repression of oncogenes, including HER-2 and SKP2. Since FOXP3 is also known as a transcription activator, we investigated the possibility that FOXP3 may induce expression of tumor suppressor genes. p21 loss has been implicated in conferring oncogenic activity to known tumor suppressor gene KLF4 and cancer drug tamoxifen. Regulators of p21, therefore, play critical roles in tumorigenesis. Here, we report that X-linked tumor suppressor FOXP3 is essential for p21 expression in normal epithelia and that lack of FOXP3 is associated with p21 down-regulation in breast cancer samples. Among 62 primary breast cancer samples tested, a strong association between FOXP3 down-regulation and lack of p21 was observed. Moreover, siRNA silencing of FOXP3 in primary human mammary epithelial cells leads to a dramatic reduction in expression of p21, which demonstrates an important role of FOXP3 in maintaining expression of p21 in normal breast epithelial cells. In breast cancer cell line MCF-7, an inducible expression of FOXP3 is associated with induction of p21. Since silencing p21 reduced the efficacy of FOXP3-mediated growth inhibition, the induced expression of p21 contributes to growth inhibition by FOXP3. A specific FOXP3 binding site in the intron 1, which is associated with acetylated histone H3, is essential for p21 induction by FOXP3. FOXP3 specifically inhibited binding of histone deacetylase 2 (HDAC2) and HDAC4 to the site and increased local histone H3 acetylation. Short hairpin RNA silencing of either HDAC2 or HDAC4 is sufficient to induce p21 expression. Our data provides a novel mechanism for transcription activation by FOXP3 and a genetic mechanism for lack of p21 in a large proportion of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4978.

Abstract 1718: ErbB-2 acts as coactivator of Stat3 in heregulin-induced breast cancer growth

Abstract We have previously demonstrated that heregulin (HRG), a ligand for ErbB receptors, activates signal transducer and activator of transcription 3 (Stat3) in primary cultures of ErbB-2 overexpressing C4HD cells, from a murine progestin-dependent mammary tumor. ErbB-2 activity is an absolute requirement in the mechanisms of HRG stimulation of Stat3 activity. Recent findings have demonstrated ErbB-2 nuclear migration and its function as a transcription factor. In this work, we studied whether HRG induces ErbB-2 nuclear migration and its interaction with Stat3 in breast cancer cells. We demonstrated that HRG induces ErbB-2 nuclear migration and colocalization with Stat3 in C4HD and human T47D breast cancer cells. Cyclin D1 is a cancer-related gene that contains Stat3 binding sites (GAS sites) but lacks ErbB-2 response elements (HAS sites). By chromatin immunoprecipitation assays (ChIP) and sequential ChIP, we demonstrated that HRG induces in vivo binding of Stat3 and ErbB-2 to the GAS sites of the cyclin D1 promoter. This finding prompted us to evaluate the ability of HRG to regulate cyclin D1 expression. By inhibiting ErbB-2 and Stat3 activation or silencing their expression, we demonstrated that ErbB-2 and Stat3 participate in the mechanism of HRG-induced cyclin D1 protein expression. Next, we explored whether HRG induces cyclin D1 promoter directly via Stat3 binding to its response elements. C4HD and T47D cells transiently transfected with a cyclin D1 promoter luciferase construct showed an enhanced transcriptional activity with HRG treatment. Overexpression of increasing amounts of ErbB-2 wt resulted in a dose-dependent ErbB-2 capacity to enhance Stat3 transcriptional activity induced by HRG. On the other hand, transfection with increasing amounts of an ErbB-2 mutant that is defective in nuclear entry but retains its cell-surface location and functions (ErbB-2ΔNLS) resulted in abrogation of HRG-induced Stat3 activation of cyclin D1 promoter. Finally, we addressed the effect of targeting ErbB-2 in in vivo HRG-dependent growth of C4HD breast tumors. Transfection of C4HD cells with the ErbB-2ΔNLS expression vector significantly inhibited these cells’ ability to form tumors in syngeneic mice. Taken together, these results suggest a new role of ErbB-2 as a coactivator in the mechanism of HRG-induced transcriptional activation of Stat3. We also found nuclear ErbB-2 to be a requisite for HRG stimulation of in vitro and in vivo breast cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1718.

Abstract 4801: Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer

Abstract Background: It was recently shown that a large portion of the human transcriptome can originate from within repetitive elements, leading to ectopic expression of protein-coding genes. However the mechanism of transcriptional activation of repetitive elements has not been definitively elucidated. Principal findings: For the first time, we directly demonstrate that hypomethylation of retrotransposons causes altered gene expression in humans. We also reveal that active LINE-1s switch from a tetranucleosome to dinucleosome structure, acquiring H2A.Z and nucleosome free regions upstream of TSSs, previously shown only at active single copy genes. Hypomethylation of a specific LINE-1 promoter was also found to induce an alternate transcript of the MET oncogene in bladder tumors and across the entire urothelium of tumor-bearing bladders. Conclusions: These data show that, in addition to contributing to chromosomal instability, hypomethylation of LINE-1s can alter the functional transcriptome and plays a role not only in human disease but also in disease predisposition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4801.

RNA Memory Model: A RNA-Mediated Transcriptional Activation Mechanism Involved in Cell Identity

I propose a new model, called the "RNA memory" model, for the possible role of RNAs in the maintenance and establishment of cell identity. This is cytoplasmic memory obtained by the transmission of mother noncoding (nc) RNAs to daughter cells. These RNAs are able to activate transcription via sequence homology in daughter cells. Regulation of RNA memory is strictly linked to the regulation of ncRNAs with repressive features, such as the RNAs involved in RNA interference (RNAi). Misregulation of this system could lead to misidentity, and thus it could be involved in cancer transformation, progression of viral or genetic diseases, and progression of senescence.

Histone acetylation and steroid receptor coactivator expression during clofibrate‐induced rat hepatocarcinogenesis

Peroxisome proliferators (PPs), non-genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid beta-oxidation system, including bifunctional enzyme (BE) and 3-ketoacyl-CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague-Dawley rats, NC- and KS-type. The KS-type rats showed enhanced susceptibility to ethyl-alpha-chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS-type and preneoplastic foci developed after 6-8 weeks of treatment, whereas no foci developed in the NC-type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator-activated receptor (PPAR) alpha levels between them. Among various coactivators for PPARalpha, only steroid receptor coactivator (SRC)-3 level was higher in the KS-type. To investigate the association between PPARalpha and SRC-3 or other proteins, nuclear extracts from CF-treated livers were applied to a PPARalpha column. In the KS-type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC-3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARalpha associated to the BE enhancer in CF-treated KS-type, but not in the NC-type. Histone H3 acetylation was increased 11-fold in the KS-type by CF treatment but not in the NC-type. As BE and TH are responsible for acetyl-CoA production and SRC-3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS-type livers is due to acetylation-mediated transcriptional activation and epigenetic mechanisms might be involved in CF-induced rat hepatocarcinogenesis.

A novel two-step transcriptional activation system for gene therapy directed toward epithelial cells

The two-step transcriptional activation (TSTA) mechanism in gene therapy amplifies cell type-specific promoter activity, allowing for increased levels of gene expression in target tissues. In this system, the specific promoter drives expression of a strong transcriptional activator that binds to DNA target sequences located upstream from a second promoter controlling the expression of the therapeutic gene. The majority of previous studies have exploited a fusion between the DNA binding domain of the yeast transcriptional activator Gal4 fused to the VP16 activation domain of herpes simplex virus 1 as the transcriptional activator. In this report, an alternative to this system is described based on a fusion protein containing the DNA binding domain of the bovine papillomavirus 1 transcriptional activator E2 fused to VP16 that induces target gene expression following binding to a minimal bovine papillomavirus 4 promoter containing upstream E2 binding sites and only 3 bp of promoter sequence upstream from the TATA box. VP16-E2 is superior to Gal4-VP16 as the transcriptional activator in a TSTA system driven by either of the two potentially cancer-specific promoters telomerase RNA and telomerase reverse transcriptase in several cell lines. Results also suggest that this new system has an advantage in epithelial cells and is therefore ideal for potential targeting of carcinomas. By incorporating the TRAIL gene as a transgene in the VP16-E2 TSTA system, selective killing of telomerase-positive cells occurs. We propose that our new system should be considered in future TSTA, particularly when targeting epithelial-derived cells.

Genotoxic stress-induced nuclear localization of oncoprotein YB-1 in the absence of proteolytic processing

Y-box-binding protein 1 (YB-1) is an oncogenic transcription factor whose overexpression and nuclear localization is associated with tumor progression and drug resistance. Transcriptional activation of YB-1 in response to genotoxic stress is believed to occur in the cytoplasm through sequence-specific endoproteolytic cleavage by the 20S Proteasome, followed by nuclear translocation of cleaved YB-1. To study the proteolysis model, we developed a two-step affinity purification of endogenous YB-1 protein species and characterized the products using mass spectrometry. Whereas full-length YB-1 was readily identified, the smaller protein band thought to be activated YB-1 was identified as hnRNP A1. An antibody specific for YB-1 was generated, which revealed only one YB-1 species, even after genotoxic stress-induced nuclear YB-1 translocation. These findings warrant re-evaluation of the mechanism of YB-1 nuclear translocation and transcriptional activation. The relationship between nuclear YB-1 and tumor progression may also have to re-evaluated in some cases.

Glucose is a key regulator of VEGFR2/KDR in human epithelial ovarian carcinoma cells

Epithelial ovarian cancer (EOC) is a serious gynecological cancer and there may be an increased risk of developing EOC in women with metabolic disruptions such as diabetes-related hyperglycemia, obesity or high glycemic load. Upregulation of vascular endothelial growth factor (VEGF) in ischemic conditions (e.g. hypoxia, hypoglycemia) induces tumor angiogenesis. We previously showed that EOC cells employ an autocrine VEGF/VEGFR2 signaling loop. Here we demonstrate the influence of glucose levels on VEGF and its receptors in the human EOC lines OVCAR-3 and CAOV-3. Glucose (but not pyruvate) deprivation induced significant increase in VEGF transcription and secretion, but a rapid reduction in VEGFR2 protein synthesis and glycosylation, combined with a reduction in co-receptor neuropilin-1 (NRP-1) protein levels. In contrast, mRNA for KDR and NRP-1 was increased upon glucose depletion suggesting a mechanism of feed back upon protein reduction. The addition of the proteosome inhibitor epoxomycin restored VEGFR2 under glucose free conditions, suggesting degradation as the main mechanism of VEGFR2 reduction and transcriptional activation through the unfolded protein response (UPR) which was activated in glucose-starved cells through the upregulation of the Endoplasmic reticulum chaperon GRP-78. Our finding that glucose can regulate VEGF/VEGFR2 levels suggests that initiation and/or progression of ovarian surface epithelial cells towards a neoplastic phenotype might be modulated by dietary conditions, and that a patient’s metabolic status may alter the effectiveness of the known anti-angiogenic therapies. This information provides opportunities to explore the biology of EOC progression and improve our understanding of the mechanistic insight of this interesting regulatory effect.

The ups and downs of HIV-1 gene expression

Following infection, Human Immunodeficiency Virus type 1 (HIV-1) becomes stably integrated into the genome of the host cell. It depends, therefore, on host cell factors to regulate its transcriptional output, both positively and negatively. HIV-1 transcriptional silencing requires chromatin remodelling and transcriptional repressor complexes, as well as heterochromatin formation at the integrated provirus. An important feature of silenced proviruses is their ability to be re-activated by either the viral transcription factor, Tat, or environmental stimuli. Activation of transcription is also controlled by numerous host cell factors. We have identified novel mechanisms of transcriptional activation and repression that depend on cellular macromolecular complexes such as proteasome and nuclear exosome. I will discuss recent developments in understanding the mechanisms by which these complexes control HIV-1 transcription, and the implications for viral latency.

BRG1 directly regulates nucleosome structure and chromatin looping of the α globin locus to activate transcription

α globin expression must be regulated properly to prevent the occurrence of α-thalassemias, yet many questions remain unanswered regarding the mechanism of transcriptional activation. Identifying factors that regulate chromatin structure of the endogenous α globin locus in developing erythroblasts will provide important mechanistic insight. Here, we demonstrate that the BRG1 catalytic subunit of SWI/SNF-related complexes co-immunoprecipitates with GATA-1 and EKLF in murine fetal liver cells in vivo and is recruited to the far-upstream major-regulatory element (MRE) and α2 promoter. Furthermore, based on our analysis of Brg1null/ENU1 mutant mice, BRG1 regulates DNase I sensitivity, H3ac, and H3K4me2 but not CpG methylation at both sites. Most importantly, BRG1 is required for chromatin loop formation between the MRE and α2 promoter and for maximal RNA Polymerase II occupancy at the α2 promoter. Consequently, Brg1 mutants express α globin mRNA at only 5–10% of wild-type levels and die at mid-gestation. These data identify BRG1 as a chromatin-modifying factor required for nucleosome remodeling and transcriptional activation of the α globin locus. These data also demonstrate that chromatin looping between the MRE and α2 promoter is required as part of the transcriptional activation mechanism.

Genome-Wide Identification of Direct Target Genes Implicates Estrogen-Related Receptor α as a Determinant of Breast Cancer Heterogeneity

Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor, the expression of which correlates with negative prognosis in breast cancer. ERRalpha shares functional features with the estrogen receptor alpha (ERalpha) and its activity is modulated by the ERBB2 signaling pathway. Using genome-wide binding sites location analyses in ERalpha-positive and ERalpha-negative breast cancer cell lines, we show that ERRalpha and ERalpha display strict binding site specificity and maintain independent mechanisms of transcriptional activation. Nonetheless, ERRalpha and ERalpha coregulate a small subset of common target genes via binding either to a dual-specificity binding site or to distinct cognate binding sites located within the extended promoter region of the gene. Although ERRalpha signaling in breast cancer cells is mostly independent of ERalpha, the small fraction of common ERRalpha/ERalpha targets comprises genes with high relevance to breast tumor biology, including genes located within the ERBB2 amplicon and GATA3. Finally, unsupervised hierarchical clustering based on the expression profiling of ERRalpha direct target genes in human breast tumors revealed four main clusters that recapitulate established tumor subtypes. Taken together, the identification and functional characterization of the ERRalpha transcriptional network implicate ERRalpha signaling as a determinant of breast cancer heterogeneity.

Transcriptional activation of the γ-globin gene in baboons treated with decitabine and in cultured erythroid progenitor cells involves different mechanisms

To investigate the mechanism(s) responsible for increased gamma-globin expression in vivo in decitabine-treated baboons and in vitro in cultured erythroid progenitor cells (EPC) from adult baboon bone marrow (BM). Fetal liver, adult BM erythroid cells pre- and post-decitabine, and cultured EPCs were analyzed for distribution of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl throughout the gamma-globin gene complex by chromatin immunoprecipitation. DNA methylation of the gamma-globin promoter was determined by bisulfite sequencing. Expression of the baboon Igamma- and Vgamma-globin chains was determined by high performance liquid chromatography (HPLC). Expression of BCL11A, a recently identified repressor of gamma-globin expression, was analyzed by Western blot. Increased gamma-globin expression in decitabine-treated baboons and cultured EPC correlated with increased levels of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl associated with the gamma-globin gene consistent with a transcriptional activation mechanism. Cultured EPC expressed the Igamma- and Vgamma-globin chains in a pattern characteristic of fetal development. The level of DNA methylation of the gamma-globin gene promoter in EPC cultures was similar to BM erythroid cells from normal adult baboons. Different BCL11A isoforms were observed in BM erythroid cells and cultured EPC. The mechanism responsible for increased gamma-globin expression in cultured EPC was unexpectedly not associated with increased DNA hypomethylation of the gamma-globin gene promoter compared to normal BM erythroid cells, in contrast to BM erythroid cells of decitabine-treated baboons. Rather, increased fetal hemoglobin in EPC cultures was associated with a fetal Igamma/Vgamma chain ratio and a difference in the size of the BCL11A protein compared to normal BM erythroid cells.

Illumination stimulates cAMP receptor protein-dependent transcriptional activation from regulatory regions containing class I and class II promoter elements in Synechocystis sp. PCC 6803

The cAMP receptor protein (Crp) is a global transcriptional regulator that binds sequence-specific promoter elements when associated with cAMP. In the motile cyanobacterium Synechocystis sp. strain PCC 6803, intracellular cAMP increases when dark-adapted cells are illuminated. Previous work has established that Crp binds proposed Crp target sites upstream of slr1351 (murF), sll1874 (chlA(II)), sll1708 (narL), slr0442 and sll1268 in vitro, and that slr0442 is downregulated in a crp mutant during photoautotrophic growth. To identify additional Crp target genes in Synechocystis, 11 different Crp binding sites proposed during a previous computational survey were tested for in vitro sequence-specific binding and crp-dependent transcription. The results indicate that murF, chlA(II) and slr0442 can be added as 'target genes of Sycrp1' in Synechocystis. Promoter mapping of the targets revealed the same close association of RNA polymerase and Crp as that found in Escherichia coli class I and class II Crp-regulated promoters, thereby strongly suggesting similar mechanisms of transcriptional activation.

The role of YY1 in reduced HP1alpha gene expression in invasive human breast cancer cells.

IntroductionHeterochromatin protein 1 (HP1) associates with chromatin by binding to histone H3 and contributes to gene silencing. There are three isoforms of HP1 in mammals: HP1α, β, and γ. Studies have shown that the level of HP1α is reduced in invasive human breast cancer cell lines such as MDA-MB-231 and HS578T compared with non-invasive cell lines such as MCF7 and T47D. It is hypothesized that reduced HP1α expression may lead to impaired epigenetic silencing of genes that are important in the acquisition of an invasive phenotype. We set out to determine whether reduced expression of HP1α in invasive breast cancer cell lines occurs at the level of transcription.MethodsWe used transient transfection assays to investigate the mechanism of differential transcriptional activity of the human HP1α gene promoter in different cell lines. Mutational analysis of putative transcription factor binding sites in an HP1α gene reporter construct was performed to identify transcription factors responsible for the differential activity. SiRNA-mediated knockdown and chromatin immunoprecipitation experiments were performed to determine the role of a specific transcription factor in regulating the HP1α gene.ResultsThe transcription factor yin yang 1 (YY1) was found to play a role in differential transcriptional activity of the HP1α gene. Examination of the YY1 protein and mRNA levels revealed that both were reduced in the invasive cell line HS578T compared with MCF7 cells. YY1 knockdown in MCF7 cells resulted in a decreased level of HP1α mRNA, indicating that YY1 positively regulates HP1α expression. Chromatin immunoprecipitation experiments verified YY1 occupancy at the HP1α gene promoter in MCF7 cells but not HS578T cells. Overexpression of YY1 in HS578T cells decreased cell migration in a manner independent of HP1α overexpression.ConclusionsOur data suggests that a reduction of YY1 expression in breast cancer cells could contribute to the acquisition of an invasive phenotype through increased cell migration as well as by reduced expression of HP1α.

TIF1β/KAP-1 Is a Coactivator of the Orphan Nuclear Receptor NGFI-B/Nur77

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1beta by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1beta, also known as KAP-1 (KRAB domain-associated protein) or KRIP-1, acts as a transcriptional corepressor for many transcription factors, in particular for the Krüppel-associated box domain-containing zinc finger transcription factors. TIF1beta is also an intrinsic component of two chromatin remodeling and histone deacetylase complexes, the N-CoR1 and nucleosome remodeling and deacetylation complexes. In contrast to these activities, we report that TIF1beta is a coactivator of NGFI-B and that it is as potent as the SRC coactivators in this context. Using pull-down assays and immunoprecipitation, we showed that TIF1beta interacts directly with NGFI-B and with other Nur family members. NGFI-B is an important mediator of hypothalamic corticotropin-releasing hormone (CRH) activation of proopiomelanocortin (POMC) transcription, and TIF1beta enhances transcription mediated through the NGFI-B target, the Nur response element (NurRE). The NurRE binds Nur factor dimers and is responsive to signaling pathways. In keeping with the role of NGFI-B as mediator of CRH signaling, we found that TIF1beta is recruited to the POMC promoter following CRH stimulation and that TIF1beta potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1beta and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1beta is an important coactivator of NGFI-B-dependent transcription.

Molecular Basis of Methanol-Inducible Gene Expression and Its Application in the Methylotrophic YeastCandida boidinii

Methanol is a promising feedstock for biotechnological and chemical processes as well as a primary source of energy to replace coal and petroleum. Methylotrophic yeasts, that can utilize methanol as the sole source of carbon and energy, have been studied intensively in terms of both physiological activities and potential applications. During growth on methanol, the enzymes involved in methanol metabolism are massively produced in these yeasts, indicating that the gene promoters of these enzymes are strong methanol-inducible promoters. Using these promoters, high-level heterologous gene expression systems have been developed in several methylotrophic yeast strains, such as Pichia pastoris, Hansenula polymorpha, and Candida boidinii. To achieve efficient industrial use of methanol and efficient protein production by methylotrophic yeasts, it is important to elucidate the molecular basis of methanol-inducible gene expression in these yeasts. This review describes recent advances in understanding of the regulation of methanol-inducible gene expression and the molecular mechanism of transcriptional activation in the methylotrophic yeast C. boidinii. Application of this gained knowledge led to successful production of useful enzymes in this yeast, which is also reviewed.

Functional Domains of ExsA, the Transcriptional Activator of the Pseudomonas aeruginosa Type III Secretion System

The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to evade phagocytosis and damage eukaryotic cells. Transcription of the T3SS regulon is controlled by ExsA, a member of the AraC/XylS family of transcriptional regulators. These family members generally consist of an approximately 100-amino acid carboxy-terminal domain (CTD) with two helix-turn-helix DNA binding motifs and an approximately 200-amino acid amino-terminal domain (NTD) with known functions including oligomerization and ligand binding. In the present study, we show that the CTD of ExsA binds to ExsA-dependent promoters in vitro and activates transcription from ExsA-dependent promoters both in vitro and in vivo. Despite possessing these activities, the CTD lacks the cooperative binding properties observed for full-length ExsA at the P(exsC) promoter. In addition, the CTD is unaffected by the negative regulatory activity of ExsD, an inhibitor of ExsA activity. Binding studies confirm that ExsD interacts directly with the NTD of ExsA. Our data are consistent with a model in which a single ExsA molecule first binds to a high-affinity site on the P(exsC) promoter. Protein-protein interactions mediated by the NTD then recruit an additional ExsA molecule to a second site on the promoter to form a complex capable of stimulating wild-type levels of transcription. These findings provide important insight into the mechanisms of transcriptional activation by ExsA and inhibition of ExsA activity by ExsD.

FOXP3 Up-regulates p21 Expression by Site-Specific Inhibition of Histone Deacetylase 2/Histone Deacetylase 4 Association to the Locus

p21 loss has been implicated in conferring oncogenic activity to known tumor suppressor gene KLF4 and cancer drug tamoxifen. Regulators of p21, therefore, play critical roles in tumorigenesis. Here, we report that X-linked tumor suppressor FOXP3 is essential for p21 expression in normal epithelia and that lack of FOXP3 is associated with p21 down-regulation in breast cancer samples. A specific FOXP3 binding site in the intron 1 is essential for p21 induction by FOXP3. FOXP3 specifically inhibited binding of histone deacetylase 2 (HDAC2) and HDAC4 to the site and increased local histone H3 acetylation. Short hairpin RNA silencing of either HDAC2 or HDAC4 is sufficient to induce p21 expression. Our data provides a novel mechanism for transcription activation by FOXP3 and a genetic mechanism for lack of p21 in a large proportion of breast cancer.

Suppression of the Deubiquitinating Enzyme USP5 Causes the Accumulation of Unanchored Polyubiquitin and the Activation of p53

Both p53 and its repressor Mdm2 are subject to ubiquitination and proteasomal degradation. We show that knockdown of the deubiquitinating enzyme USP5 (isopeptidase T) results in an increase in the level and transcriptional activity of p53. Suppression of USP5 stabilizes p53, whereas it has little or no effect on the stability of Mdm2. This provides a mechanism for transcriptional activation of p53. USP5 knockdown interferes with the degradation of ubiquitinated p53 rather than attenuating p53 ubiquitination. In vitro studies have shown that a preferred substrate for USP5 is unanchored polyubiquitin. Consistent with this, we observed for the first time in a mammalian system that USP5 makes a major contribution to Lys-48-linked polyubiquitin disassembly and that suppression of USP5 results in the accumulation of unanchored polyubiquitin chains. Ectopic expression of a C-terminal mutant of ubiquitin (G75A/G76A), which also causes the accumulation of free polyubiquitin, recapitulates the effects of USP5 knockdown on the p53 pathway. We propose a model in which p53 is selectively stabilized because the unanchored polyubiquitin that accumulates after USP5 knockdown is able to compete with ubiquitinated p53 but not with Mdm2 for proteasomal recognition. This raises the possibility that there are significant differences in proteasomal recognition of p53 and Mdm2. These differences could be exploited therapeutically. Our study reveals a novel mechanism for regulation of p53 and identifies USP5 as a potential target for p53 activating therapeutic agents for the treatment of cancer.

Methylmethane Sulfonate Increases the Level of Superoxide Anions in Yeast Cells

ABSTRACTTy1 is a Saccharomyces cerevisiae retrotransposon with life cycle and structure very similar to the known oncoviruses. The transposition of the Ty1 mobile genetic element is induced by different stress conditions and chemical agents. Mutagens and carcinogens activate Ty1 elements for retrotransposition by certain types of genome injuries through DNA-damage signaling pathways but the mechanism of transcriptional activation by DNA damage has not been enough elucidated. Many of the activators of Ty1 mobility are powerful inducers of oxidative stress. Reactive oxygen species (ROS) can generate a variety of DNA lesions and can lead to a significant activation of Ty1 mobility. Recent data indicate that treatment of S. cerevisiae cells with the Ty1-mobility inducer methylmethane sulfonate (MMS) results in increased ROS levels. In response to MMS, both a direct DNA damaging effect and an enhanced ROS levels play role in the activation of Ty retrotransposition. The results obtained in this study provide evidence for a dependence of Ty1 retrotranspsoition on increased production of ROS. The increased ROS levels may have an independent and a key role in the induction of Ty1 retrotransposition by MMS. We suppose that MMS treatment of cells with functioning mitochondrial oxidative phosphorylation induces Ty1 retrotransposition by both, increased ROS levels and direct DNA damages, and the increased ROS levels seems to have the major role in the induction of Ty1 mobility.